The present disclosure is in the field of cell reprogramming and provides a method for reprogramming differentiated cells into pluripotent stem cells and optionally subsequently differentiating them. The invention also provides a closed microfluidic system for reprogramming differentiated cells into pluripotent stem cells and optionally subsequently differentiating them under clinical grade conditions. The present application claims priority to the US provisional patent application 62/323,391 filed on April 15, 2016, the contents of which are incorporated herein by reference in their entirety. The present application is further in relation with the disclosure of Luni, Camilla et al. "High-efficiency cellular reprogramming with microfluidics" Nature Methods 13(5) (April 2016), the contents of which are incorporated herein by reference in their entirety.

BACKGROUND

Human-induced pluripotent stem cells (hiPSCs) are pluripotent stem cells that hold great promise especially for in vitro studying embryogenesis and for the derivation of in vitro models of human tissues. They are characterized by key molecular signatures at genetic network and signaling pathway levels. NANOG, OCT4 and SOX2 genes constitute the essential pluripotency master transcriptional network that in a feedforward manner supports secondary transcription factors (e.g., MYC family members, ZFX) and stabilizes pluripotency. These regulatory hubs are dependent on FGF and TGF-β signaling pathways to sustain self-renewal (Figure 1 a).

The pluripotency in somatic cells is normally obtained through molecular changes induced by forced expression of four transcription factors: OCT4, SOX2, KLF4 and MYC (OSKM). However, there are several publications that have highlighted other factor sets suitable for cell reprogramming (see, by way of example, Thomson's factors OCT4, SOX2, NANOG, and LIN28, and other scientific and patent literature). In the initial stage of reprogramming, the exogenous expression of these transcription factors perturbs the transcriptional network of the somatic cells. In response, the cells integrate intrinsic and extrinsic cues to remodel chromatin and reach a new epigenetic state. The latter stages of reprogramming, maturation and stabilization, bring to the endogenous expression of the core circuitry of pluripotency, comprising OCT4, SOX2 and NANOG, and the activation of the same signaling networks that govern pluripotency in embryonic stem cells.

In order to increase reprogramming efficiency and improve overall quality of the obtained hiPSCs, different strategies of transcription factors delivery have been developed. Moreover, a better understanding of epigenetic reorganization and rewiring of the pluripotency genetic network during reprogramming has been achieved. Particularly interesting methods are the ones that use non-integrating vectors to exogenously induce the expression of the critical transcription factors, such as episomal vectors (Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801 2009) and modified mRNAs (mmRNA) (Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618-630 (2010) and Yoshioka, N. et al. Efficient generation of human iPSCs by a synthetic self- replicative RNA. Cell Stem Cell 13, 246-254 (2013)) which ensure the obtainment of transgene-free hiPSCs. On the other hand, assisted by available high-throughput technologies, multiple studies screened epigenetic and transcriptional barriers during reprogramming. Fewer works reported the role of signaling pathways at different stages of reprogramming: for example, TGF-β pathway activation could be a negative regulator during acquisition of epithelial phenotype, whereas has a positive effect on reprogramming during the very first steps and at later stages, when it acts as a promoter of pluripotency.

Currently, reprogramming is performed in laboratories within standard devices for cell culture, like Petri dishes and multi-well plates, which however show multiple limitations. The process is, even at best, highly inefficient. For instance, using mmRNA - the most efficient technology so far - the maximum yield is ~3 hiPSC colonies per 100 somatic cells seeded (Schlaeger, T. M. et al. A comparison of non-integrating reprogramming methods. Nat. Biotechnol. 33, 58-63 2015).

Thus, it is likely that the reprogramming process requires a defined sequential activation of intrinsic and extrinsic cues signals acting on specific signaling pathways. This complexity together with the intrinsic low efficiency of the current reprogramming methods, and the apparent stochasticity of the process, limits the ability to further optimize the reprogramming process of human somatic cells by acting only on exogenous perturbations.

Current processes that have been developed for the manufacture of cellular products for cellular therapies have until now been carried out manually or at best by using semi- automated procedures. Compliant - Good Manifacturing Practice (GMP) reprogramming methods are performed with the following steps: i) biopsy acquisition; ii) cell isolation; iii) cell reprogramming; iv) hiPSCs isolation, expansion, banking and characterization. These steps required batch record and Standard Operating Procedure (SOP) preparation, compliant GMP production team training and cGMP facility preparation, supply chain establishing a complete list of raw materials as well as establishing relevant quality control and bioassay tests, primary and secondary vendor qualifications, raw material part setup, and logistics management (including storage conditions, tracking expiration, reorder points, and material flow to and within GMP manufacturing sites).

Stefano Giulitti: "Hiqh-throughput Human Cell Reprogramming through Substrate and Microfluidics Integration"! 1" discloses a platform for cell reprogramming which, however, does not provide optimized efficiency. In fact, Giulitti does disclose an open- loop system which provides efficiency of 16%.

The processes of cell reprogramming, reprogramming to progenitors and lineage reprogramming under GMP requires: i) specific training of the involved staff prior to execution of the process; ii) adequate facilities to maintain a clean and hygienic manufacturing area and to prevent cross contamination from adulterants; iii) records made, manually or by instruments, during manufacture that demonstrate that all the steps required by the defined procedures and instructions were in fact taken and that the quantity and quality of the product was as expected; iv) the use of xeno-free, feeder-free raw materials for clinical-grade manufacturing (e.g., chemically defined and non-enzymatic passaging and coating solutions).

For these reasons the cGMP process for cell reprogramming is currently time consuming, expensive, and has low efficiency.

SUMMARY OF THE INVENTION

New methods that directly act on the molecular mechanisms of human somatic cell reprogramming to pluripotency are crucial for the derivation of high-quality pluripotent stem cells and for their therapeutic applications. Somatic cells are induced to acquire a pluripotent stem cell (hiPSC) state through forced exogenous expression of key transcription factors, or exogenous perturbations that modulate signaling pathways. Here the inventors report that reprogramming process is amenable to extreme efficiency improvement emphasizing extrinsic endogenous signaling. The present inventors successfully downscaled cell reprogramming to microliter volume in microfluidics. Small-volume confinement with efficient delivery of exogenous factors with episomal or non-integrating vectors resulted in 50-fold increase of reprogramming efficiency. The high hiPSC quality and purity (85% NANOG+/TRA-1-60+ cells) achieved by the inventors allow direct differentiation of freshly obtained hiPSC in microfluidics (μ-hiPSC) into functional cells. More preferably, purity of high hiPSC is at least of 80%.

The present inventors have found that downscaling mmRNA reprogramming process to a confined microliter volume generates a favorable environment for the reacquisition of pluripotency. As previously reported by the present inventors (Giobbe, G. G. et al. Functional differentiation of human pluripotent stem cells on a chip. Nat. Methods 12, 637-640 2015), a confined cell microenvironment has a strong impact on the self- regulated autocrine and paracrine signaling, including for pluripotency maintenance. Taking advantage of large-scale microfluidic technology, the inventors aimed at developing an improved and optionally remotely controlled generation of hiPSC in feeder-free, xeno-free conditions and within a closed-loop process, which required a very small amount of medium during a reprogramming lasting about 14 days. The findings of the inventors make the process suitable for hiPSC therapeutic application as well as scientific investigation in properly defined conditions.

The present disclosure shows that the method of cell reprogramming of the invention can be translated into an integrated process within a close network of microfluidic units that complies with current technical requirements for manufacturing products for therapies.

The method herein disclosed has several advantages, both technological and scientific. As for the first aspect, the microfluidic system requires only few microliters of medium per day for each independent culture channel, with an overall 100-fold reagent savings compared to a well in a 6-well plate. Cost reduction coupled with system automation makes feasible the implementation of the process to hundreds of parallel reprogramming experiments. By way of example 32 parallel experiments can be carried out in a single chip.

Microfluidics is well suited to comply with GMP requirements, because the system allows carrying out the reprogramming method of the invention in a closed and remotely controlled condition, maximizing robustness and strongly decreasing the probability of contaminations.

According to a first aspect, a methodology that greatly improves the reprogramming efficiency under clinical grade conditions is disclosed. In particular the specification provides a method of reprogramming adult cells to iPSC the method comprising -seeding adult cells in a microfluidic cell culture chamber

-submitting the seeded cells to reprogramming by delivering into said cells suitable reprogramming factors and/or inducing into said cells the synthesis of suitable reprogramming factors, with suitable vectors

-culturing said cells until iPSC are obtained wherein the height of the culture medium comprising said suitable reprogramming factors and/or vectors in the microfluidic cell culture chamber with respect to the bottom of the chamber is in the range from 500 to 50 μηι.

In other words, according to one aspect of the present invention, reprogramming takes place in a microfluidic cell culture chamber, wherein the height of the culture medium with respect to the bottom of the chamber is in the range from 500 to 50 μηι. This is an optimized height for the reprogramming step.

The height in the range from 500 to 50 μηι allows to obtain efficiency of 150%.

According to a second aspect, a closed microfluidic system for cellular reprogramming is disclosed. In particular the specification provides a microfluidic system configured to perform cell reprogramming, the microfluidic system including

at least a stock station configured to include a reprogramming solution,

at least a culturing device including one or more microfluidic culture cell chambers configured to allow cell adhesion and a delivery device to deliver solutions from the stock station to the culturing device;

the system being configured to keep the stock station or the reprogramming solution of the stock station at a cryo temperature condition to obtain a cryo-preserved ready-to- use reprogramming solution.

In other words, according to further aspects of the present invention, a microfluidic system configured to perform cell reprogramming is disclosed. The system is configured to keep the ready-to-use reprogramming solution in a frozen condition. The cryo temperature condition allows the use of pre-prepared reprogramming solutions, such as for example transfection solutions, and keep such solutions for a long time period. The time period can be more than 1 day, preferably for more than 5 days, and more preferably more than 10 days, such as for example 14 days or 16 days. In particular, one or more reprogramming solutions can be conserved as frozen solutions, and heated time by time to room temperature or heated at higher temperature suitable for cell culturing (such as for example at 37°C). The heating can take place just before a culturing phase. In other words, the reprogramming solutions are not freshly prepared immediately before culturing, but pre-prepared as stock solutions and frozen. In particular, according to one embodiment of the invention, the working solution is prepared in advance. The amount of working solution is an amount that can be used for the mentioned-above long time period. Then the working solution can be divided in a plurality of aliquots or doses, i.e. a plurality of fractions of working solution. Each fraction is contained in vial or holder. The plurality of working solutions are frozen at the cryo-temperature condition. After freezing at cryo-temperature condition, the solutions are housed and located in a container or rack under cold condition and kept in a frozen status. Every aliquot is then heated and used time by time for culturing the cells.

In other words, the aliquot holder can include each a fraction or smaller quantity of the overall reprogramming solution. Each holder is heated to provide the ready-to-use solution only at the occurrence during the reprogramming solution. The holders are heated after one other according to a defined timing schedule.

The use of frozen pre-prepared solutions provides the advantage that the aliquot holders can be sealed reservoirs, not accessible from an external environment. Moreover, the one or more culture chambers can be independent from each other, each, and preferably every, connected with inlet and outlet microfluidic channels. When inlets and outlets are directly or indirectly connected to sealed reservoirs, a closed system can be obtained. The system can be protected from various types of contaminations at device inlets and outlets.

The closed system can further ensure that every step of the reprogramming process can occur independently from any external action with respect to the closed system, for example from an action of any operator. For example, it follows that change of medium occurs autonomously without any external action.

The microfluidic system can be used to perform the above-mentioned method of reprogramming adult cells to iPSC.

In relation to cryo-temperature, it should be noted that reprogramming can require mmRNA as vectors for the expression of the suitable reprogramming factors. These factors are instable at higher temperatures. Therefore, cryo-temperature for a reprogramming process can ensure stability of these transcription factors.

Further and specific embodiments forming the subject of the present disclosure are defined in the corresponding dependent claims and in the following examples.

GLOSSARY The term "reprogramming" in the present disclosure indicates the process that is used to de-differentiate adult, differentiated cells to cells that have an immature and pluripotent phenotype such as induced pluripotent stem cells (iPSC).

The term "differentiation" indicates the process used to convert immature and pluripotent cells into cells derived from the three germ layers.

iPSCs stands for induced pluripotent stem cells and hiPSCs stands for human-induced pluripotent stem cells that are a type of pluripotent stem cell that can be generated directly from adult cells.

Microfluidic culture chambers are available on the market and, according to the invention and to the state of the art they are chambers that have a bottom surface comparable to that of a standard 96-well plate, whereas a micrometric medium height, in particular, according to the invention, the microfluidic culture chamber will have an optimal medium height of about 50 to 500 μηι, preferably of about 200 μηι. Said optimal medium heights ensure significant accumulation of endogenous factors.

According to the present disclosure a "reprogramming working solution" is intended as a cell culture medium suitable for the culturing of the selected cells, comprising suitable reprogramming factors in suitable vectors and/or suitable vectors inducing into said cells the synthesis of suitable reprogramming factors according to the reprogramming known protocol selected. The working solution can be divided in fractions and each fraction can be housed in a respective holder or vial. The fractions can be then frozen. In any part of the description the expression "medium comprising said suitable reprogramming factors and/or vectors" can be substituted with the expression "reprogramming working solution".

The term "cryo temperature condition" as used herein with reference to temperature of the programming solution indicates that the solution can be temporarily kept or for a predetermined time period in a cryo condition and therefore saved in that condition. The cryo working solution temperature can broadly range from -30° to -220°C or more, preferably from -60°C and -200°C, preferably from -70°C and -190°C, more preferably from -75°C and -188°C, or from -78°C and -187°C, or at a temperature of -80°C and/or -186°C. The term "stock station" indicates a unit configured to house one or more working solutions. The stock station can act as a housing for the working solutions and/or for a plurality of holders or vials. The one or more working solutions can be first frozen out of the stock station and then kept in the frozen status in the stock station. The single vials or holders can therefore be heated after one other following a predefined timing and according to a culturing program.

In any part of the present disclosure the terms comprising/comprises or including/includes are intended as being possibly replaceable with the terms "consisting of" or "consists of.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 a Volume downscale is performed in a microfluidic cell culture system: each channel-shaped culture chamber is independent and contains cells adherent to the bottom surface. Medium handling in the system is automatically controlled via software, actuating an integrated pneumatic circuit. A two-week workflow is performed to convert fibroblasts into hiPSC by programmed daily transfections of reprogramming factors. On the right, an automated microfluidic chip, the main unit of this culture system, is shown.

Figure 1 Efficiency of transfection of nGFP in BJ fibroblasts, in terms of percentage of nGFP ⁺ cells, as a function of mmRNA quantity, derived from images taken 24 h after a single transfection. The gray arrows compare the typical mmRNA amount used in well to the optimized one in microfluidics with ceils at confluence. Error bars, meanis.d. (n>4). Ceils transfected in microfluidics show higher efficiency with much lower mmRNA consumption.

Figure 1 c Dependence of transfection efficiency on the duration of the incubation period. Experimental data (circles) obtained in microfluidics are compared to simulated ones (dots) derived from a stochastic mathematical model of the transfection process. Error bars, meanis.d. (n>4). Figure 1 d Western blot analysis of the indicated proteins expressed from exogenous mmRNAs in single microfluidic channels and wells, performed 24 h after two daily mmRNA transfections in BJ fibroblasts. Error bar, mean±s.d. (n ^~ 4). Figure 1e Epithelial morphology and transfection progression, controlled by nGFP expression, at day 8 of reprogramming in microfiuidics. Scale bar, 500 and 150 m (macro).

Figure 2a hiPSC are obtained by reprogramming at the micro-scale. Time course of a representative hiPSC colony generated and expanded inside a microfluidic channel. Scale bar (same for all Figures), 750 μηη.

Figure 2b-c BJ~derived hiPSC reprogrammed in microfiuidics and extracted for characterization. hiPSCs show pluripotency markers and, after defined monolayer differentiation, specific germ-layer markers by immunofluorescence. Scale bar (same for ail Figures), 100 m.

Figure 2d Upon spontaneous differentiation in vitro and in vivo, cells were able to generate embryoid bodies (EB), randomly expressing markers of the three germ layers (Supplementary Fig. 7), and teratomas with structures typical of the three germ layers, such as neural rosettes, cartilage, and gut-like structures. Scale bars, 100 μιη, except EB, 200 Mm.

Figure 2e μ-hiPSCs obtained as in (b) and expanded for 12 passages are karyotypicaily normal.

Figure 2f In situ characterization by immunofluorescence of early reprogramming stages (day 7) and hiPSC formation within a microfluidic channel. E-CAD antibody was used to detect BJ ceils that underwent mesenchymal-epithelial transition. At day 7 most of the cells in the channel are E-CAD ⁺ (white arrows indicate the lower pneumatic layer of automated chip).

Figure 2g A representative μ-hiPSC shows expression of pluripotency markers by immunofluorescence within an automated chip (day 21). Scale bars, 250 μη .

Figure 3a Micro-scale reprogramming occurs with high efficiency and purity. Improvement of reprogramming yield by protocol optimization. Reprogramming efficiency index, number of colonies expressing both NANOG and TRA-1-60 per 100 cells seeded; well, conventional reprogramming in 6-weil plate; μΡ feeder, microfiuidic non-optimized reprogramming with feeder layer; μΡ feeder incremental, similar to the previous one but adapting mmRNA dose to cell density during the first reprogramming days; Ρ feeder-free and μΡ feeder-free incremental, similar to the previous two but without feeder layer. Figure 3b TRA-1-60 ⁺ colonies at the end of the reprogramming protocol with feeder layer. Left, bright field image of a microfiuidic culture channel with superimposed TRA- 1-60-iabeled cells; right, 10-channel microfiuidic chip area showing the spatial distribution of TRA-1-60 ⁺ colonies in black. Figure 3c immunofluorescence of NANOG ⁺ /TRA-1-60 ^÷ μ-hiPSC derived in feeder-free conditions in microfluidics. Scale bars, 100 μιτι. Dot plot of hiPSC purity, measured as the ratio of NANGG* cells to the total number of cells in culture (day 5, 72 h after the last mmRNA transfection). Figure 3d Morphology of μ-hiPSCs derived from renal epithelial (RE) cells isolated from urine samples, at day 15 (top) and 19 (bottom). Scale bars, 200 μιτι. Dot plot represents the fold-change in reprogramming yield of RE cells in microfluidics compared to wells of a female (XX) and two male patients (XY). Figure 3e Reprogramming efficiencies of various primary cell samples, RE cells were isolated from anti-trypsin-deficiency (ATD) patients. Skeletal muscle fibroblasts (SkMF) were isolated from dystrophic muscles, (a, c, d, e) Each dot represents an independent mierof!uidie channel. Black bar, mean of respective data points shown. Figure 4a Enhanced endogenous signaling in microfluidics promotes reprogramming. Experimental design for microarray analysis. Freshly derived colonies (passage 0, pO) obtained in microfluidics or in wells were dissected into two halves (-4000 cells each). One was immediately processed for total RNA extraction, and the other one was seeded in a new well and expanded for three passages (p3). Both microfluidics- and well-derived hiPSC were expanded in conventional well plates. Colonies of -8000 cells from each condition were dissected again at p3 to extract total RNA. RNA samples, obtained both from pO and p3 of microfluidic- and well-generated hiPSC, were then processed for microarray analysis. Figure 4b Principal component analysis of freshly-derived and expanded hiPSC from microfluidics (/?=4) and wells (n=4), grouped according to results from hierarchical clustering. Each clone was isolated from reprogrammed samples in different microfluidic chips or well plates. Figure 4c Expression level of selected piuripotency markers in the 4 conditions (pO and p3 in the two culture systems). Color bar, relative median-centered gene expression presented as a green-biack-magenta-colored heatmap (iog ₂ ).

Figure 4d icroarray-derived expression profiles of genes differentially expressed between the 4 conditions (pO and p3 in the two culture systems) belonging to the intersection category shown in Figure 1 b. Color bar, same as in (c).

Figure 4e Perturbation of medium change frequency. Additional changes every 4 h were performed compared to standard daily medium management to promote the washout of endogenous factors. Doubling the number of medium changes per day (6 d ^{" Ί} ) compared to the standard experiment in microfiuidics (3 d ^"1 ) significantly reduced reprogramming efficiency.

Figure 4f TGF-β pathway activation during reprogramming measured as SMAD2/3 activity of a reporter line. Medium in microfiuidics is exposed to significantly higher conditioning of cell-released endogenous signals. Error bar, meanis.d. (n=3).

Figure 4g TGF-β dynamics during reprogramming in microfiuidics under non-treated (- ), and exogenousiy supplemented TGF-βΙ conditions for the whole duration of reprogramming (n=2).

Figure 4h Feeder-free reprogramming efficiency in microfiuidics under conditions described in (g), and in the presence of TGF-β inhibitor SB431542. SB2-2, 2 μΜ inhibitor concentration for the whole duration of reprogramming; SB2-10, 2 μ inhibitor concentration for the first 6 d, followed by 10-μΜ during the second half of the process

(/7=10).

Figure 5a Freshly-derived hiPSC in microfiuidics are competent to produce patient- specific differentiated functional ceil types. Extracellular soluble components of the signaling pathways play a major role during cell reprogramming and specification into the three germ layers.

Figure 5b μ-hiPSC colonies, freshly generated in microfiuidic channels and specifically differentiated towards the three germ layers, selectively express early germ-layer commitment markers, as detected by qPCR analysis. Error bars, meanis.d. (n=10).

Figure 5c Cardiomyocytes from late-stage specification in microfiuidics of freshly generated μ-hiPSC display cTNT expression and sarcomeric organization (see also Supplementary Fig. 5, Supplementary Video 5). Scale bars, 50 μητι. Figure 5d Maturation of primary μ-hiPSC into the hepatic lineage. Colonies differentiated into large areas of mature cells, displaying functional markers (CK18, albumin, PAS). Scale bars, 250 μιτη, 50 μ\η (albumin). Figure 5e Schematic summary describing the integrated process of reprogramming and differentiation in microfluidics without intermediate passaging.

Figure 5f Vision of high-throughput derivation of human tissues for biological assays on a population scale.

Figure S1 a Extrinsic Signaling during Reprogramming. In human pluripotent stem cells, exogenously- (medium) and endogenously-promoted (cell-secreted) FGF and TGF-β pathways sustain the master genetic network of NANOG, POU5F1 and SOX2. Figure S1 b, The extra-cellular soluble components of the signaling pathways were identified at the intersection of genes related to the extra-cellular region and genes involved in signaling pathways. The temporal expression of these components during reprogramming was analyzed from data reported by Takahashi et a!. ¹⁸ . Multiple of these genes that are progressively up- (red) or down-regulated (green) during reprogramming take part in signaling pathways implicated in pluripotency maintenance and self-renewal.

Figure S1 c, In conventional well culture systems, endogenous factors get diluted in the medium bulk by diffusion; on the contrary, reducing medium volume, the concentration of endogenous factors is maintained high in close proximity to the cells.

Figure S2a, Hierarchical clustering based on microarray expression date (GSE50206) of genes belonging to the intersection of GO: Extracellular region with TGF-β, WNT, MAPK, HIF and AKT-PI3K pathways. Temporal evolution of gene expression during reprogramming shows convergence to pluripotent profiles. Color bar: relative median- centered gene expression presented as a green-black-red-colored heatmap (log2).

Figure S2b, K-means clustering of the same genes as in (a) into three main transcriptional patterns (invariant, down-regulation and up-reguiation) during reprogramming (49 days). Ceils at the end of reprogramming show transcriptional profiles consistent with stable hiPSC and hESC.

Figure S3a Technological development of microfiuidic cell cuituring for reprogramming. Schematic of the two classes of substrates tested: adsorbed extra-cellular matrix (ECM) only by physical interaction, and cross-linked EC by covaient chemical bonding.

Figure S3b, Eight different surface treatments were tested within microfiuidic channels: A: adsorbed 0,6% Gel on glass, B: adsorbed 0, 1 % Gel, C: glass silanization by APTES, D: methacrylated glass via TMSPMA, E: 2% Gel A, covalenfly bound to methacrylated glass, F: adsorbed Fn, G: 0, 1 % Gel, covalently bound to APTES treated-giass via giutaraidehyde, H : 0, 1 % GelMA, covalently bound to methacrylated glass. Error bars, mean ± s.d. (n 3). Figure S3c, Representative images of BJ cultures on differently functionalized surfaces. Time points (day): A, D4; B, D9; C, D12; D and E, D14; F, D18; G and H, D24, Nomenclature A-H: same as in (b). Scale bars, 500 μιτι.

Figure S3d, Reprogramming microfiuidic chip design with complete manual management is performed by pipetting solutions into a reservoir. By picking up exhausted medium from the outlet, fresh medium introduced in culture channel. Each chip is built on common microscope glass slides and can be placed in a standard Petri dish with a phosphate buffer bath to ensure a sterile humidified environment. No other equipment is needed. Figure S3e, Exploded and assembled representation of the microfluidic platform with an integrated automated medium distribution system; colors highlight culture channels and medium by-passes (pink; thick and thin, respectively), medium distribution network (black), and pneumatic control circuit (gray). Below, the section of a single channel is shown schematically to display automated operation mechanism (layers not on scale): the medium distribution system works by pneumatic control with pulsed periodic medium changes within the culture channel during the day.

Figure S4a, Transfections were performed either for 4 h or overnight; fluorescence was detected 24 h after beginning of transfection, different transfection reagents were used: Stemfect (SF), StemMACS (S ), and RNAiMAX (RiM); ceils transfected in microfiuidics show higher efficiency in terms of percentage of nGFP+ cells and much lower mmRNA consumption; error bars, mean±s.d. (n=4). Figure S4b, mmRNAs were delivered every 24 h for three times by SF or SM on BJ cells with a seeding density of 120 cells/mm ² , either for 4 h or overnight. The two different mmRNA amounts indicated are expressed per 100 cells. Scale bar, 250 μιτι. After 72 h nGFP* cells were nearly 90% using SF. Figure S4c, Quantification of Figure S4b. Employing SM reagent, a smaller amount of transfected ceils was visible, although markedly expressing nGFP. Error bars, mean ± s.d. (n=3).

Figure S5a Reprogramming factors delivery efficiency. Schematic representation of the physical processes accounted for in the stochastic mathematic model: cells are represented as discrete areas on the culture surface, and RNA-containing vesicles move in the medium above by Brownian motion within a lattice. Pjump, probability of a movement between neighboring positions on the lattice or towards cell surface; Pads, probability of vesicle adsorption into a cell. Dimensions are not on scale. Figure S5b, Experimental (large dots) and mathematical model-derived (small dots) transfection efficiency for different incubation periods (RiM). Error bars, mean ± s.d. (n>4). Figure S5c, in siiico simulation of mmRNA content distribution in the cell population, as a function of mmRNA amount and transfection duration.

Figure S5d, Protein expression in early reprogramming phase. BJ cells were daily transfected with 5.5 pg/100 cells of 7-mmRNA mix, including nGFP mmRNA, during reprogramming. images were taken 24 h and 48 h after the first transfection. Scale bar, 100 μητ

Figure S5e, Quantification of nuclei fluorescence intensify determined by analysis of images obtained as in (d). To account for substrate differences (polystyrene vs. glass), fluorescence was normalized by background intensity. Cells transfected in microfluldics (μΡ) exhibit a significantly higher fluorescence intensity of nGFP compared to those in well. Error bars, mean ± s.d. (/?>60G ceils).

Figure S5f, Cell lysates from the same samples, both wells and single microfiuidic channels, were analyzed by Western blot for POUF5F1 (OCT4) and KLF4. Ceils transfected in microfluidics express a higher protein level also of these reprogramming factors.

Figure S5g, Fibroblast morphological mesenchymal-epithelial transition and transfection progression in microfluidics during reprogramming verified by nGFP expression. Scale bars, 500 and 150 D m (macro) refer to each column of Figures.

Figure S6 Extraction methods of patient-specific freshly generated hiPSC from microfluidics. Manual picking: a single colony (arrow) was mechanically dissected and picked with a glass tool after coring the PDMS surface with a biopsy punch (both the hole and the core flipped upside-down are shown). Colony fragments were seeded on MEF in a well and conventionally expanded. Shear stress-based extraction: hiPSC (left, arrows) were detached and collected at the outlet of the microfluidic channel by applying DPBS perfusion at high rate. Gathered cells were then seeded on MEF. Opening of the microfluidic channel and subsequent manual picking of single colonies results in a straightforward method to maintain hiPSC cionality upon isolation and ceil line generation. While shear stress can dissociate each original hiPSC colony in multiple smaller clusters, seeding into a well in sparse condition permits to randomly choose and isolate hiPSC arbitrary clones few days later. Alternatively, it is possible to perfuse colonies twice for 5 min each with 0.5 mM ethyienediaminetetraacetic acid, a standard solution for expansion of piuripotent stem cells in feeder-free conditions; Loosen hiPSC are then detached and collected perfusing with hiPSC medium. Scale bars, 750 ίη (channels), 250 μϊτ¾ (on MEF). Figure S7a In situ and off-chip characterization of hiPSC derived from BJ and HFF fibroblasts. Immunofluorescence of early reprogramming phase (day 6) in a microfluidics. E-CAD antibody was used to detect BJ cells undergoing mesenchymal- epithelial transition. After 5 daily transfections most of the ceils express epithelial cadherin. Scale bars, 250 μπι.

Figure S7b, Characterization of BJ-derived hiPSC extracted from microfluidic channels. Pluripotency markers by immunofluorescence and alkaline phosphatase expression. Scale bars, 100 μνη. Figure S7c, pluripotency genes expression by RT-PCR.

Figure S7d, upon differentiation stimuli, hiPSCs are able to generate embryoid bodies randomly expressing markers of three germ layers.

Figure S7e, normal karyotype Figure S7f, Characterization of HFF-derived hiPSC. In situ immunofluorescence demonstrates obtained colonies are NANOG ⁺ , OCT-4 ⁺ and SSEA-4 ⁺ ; analyses were performed 3 days after the last reprogramming transfection. Karyotype analysis does not show abnormalities in HFF-derived hiPSC after 12 passages (e, μΡ-hiPSC #357). Scale bars, 200 μηι.

Figure S7g, Upon subcutaneous injection in Rag2 ^~/" YC ^"/" mice, hiPSC derived in microfluidics underwent spontaneous differentiation generating teratomas with structures typical of the three germ layers.

Figure S8a Feeder-dependent reprogramming in microfluidics. Highly efficient reprogramming within the microfiuidic setup. A binary image of NANOG ⁺ (green) and TRA-1-60 ⁺ (red) colonies, detected by fluorescence microscopy 48 h after the last reprogramming mmRNA transfection, was overlapped with the bright-field image of the corresponding 10 microfiuidic channels. The same channels of Fig. 3b are shown.

Figure S8b, image of 10 channels within a microfiuidic chip at day 18 of reprogramming with feeders. Primary derived hiPSC are visible as white translucent spots inside each channel. Scale bar, 3 mm. A magnification of channel 4 shows 4 colonies. Channel width is 1.5 mm.

Figure S8c, Reprogramming efficiency as a function of mmRNA dosage, maintained constant during the process. 1 x refers to the optimal transfection dosages in well and microfiuidic systems, 24 and 4 pg/100 cells, respectively.

Figure S9a Feeder-free reprogramming of BJ cells performed with SF and SM. BJs were seeded at 10 cell/mm ² and transfected for 18 days (SF) or 1 1 days (SM). Time course of nGFP expression during the first 6 days of reprogramming. Scale bar, 250 m. Figure S9b, TRA-1-80* colonies are schemaiicaily marked inside each channel at their actual position. Compared to SF, feeder-free reprogramming with S generated a significantly higher number of colonies with a more homogeneous distribution along each microfluidic channel.

Figure S10a, Nested ANOVA analysis considering ail reprogrammed samples (biological and technical independent replicates) reprogrammed in optimized feeder- free conditions with incremental mmRNA dosage during the first days. Variability between different cell batches or microfluidic chips is not statistically significant and results in similar p-vaiues.

Figure S10b, Reprogramming in feeder-free condition in microfluidics generates a considerably higher amount of hiPSC colonies per unit area compared to a standard well plate, both when performed at optimal initial cell density (5 cell/mm ² ), and with a 10-fold higher initial cell density (50 ceil/mm ² ). In the latter case, daily mmRNA amount has been doubled during the first week to accommodate exponential growth of a higher amount of cells. Despite this, the amount of hiPSC colonies generated per unit area is drastically reduced. Error bars, mean ± s.d. n=6.

Figure S10c, Morphological cell changes during reprogramming progression performed in microfluidics as described in (b). Conventional seeding density in microfluidic optimized reprogramming is 4.88 ± .60 cell/mm ² , /τ=32, consistent with the expected value of 5 ceil/mm ² .

Figure S11 a, RE cells at day 13 of the reprogramming protocol, after daily mmRNA transfections, within a microfluidic channel (phase contrast and nGFP fluorescence). Figure S11 b, Extracted hiPSC at passage 1 (p1 ) directly cultured in feeder-free conditions and expanded at passage 3 (p3).

Figure S11 c, Immunofluorescence characterization of RE-derived hiPSC confirms the expression of endogenous NANOG, OCT-4 (POU5F1), SSEA-4, and TRA-1-60. Scale bars 200 μηι, except (b, left) 25 m.

Figure S12 Comparison of hiPSC colony expression profiles. Microarray datasets from hiPSC colonies obtained in this work either in microfluidics or in well, are compared to datasets in GEO and Synapse databases (GSE50206, GSE42445, synl 447097, syn1449098). Principal component analysis shows mmRNA colonies obtained in this work (pO, p3) have expression profiles similar to pluripotent stem cell samples from literature. Moreover, differences among hiPSC colonies obtained in this work in well plates or in microfluidics (μΡ) are within the biological variability of pluripotent stem cells obtained by other groups.

Figure S13 Downscaie of mmRNA transfection in systems characterized by different medium height. mmRNA concentration used in well is maintained constant and mmRNA quantity rescaie accordingly to medium height. The 200- m-tall microfluidic channel represent the standard used in this paper and promotes high concentration of endogenous factors. By decreasing the medium height, no substantial reprogramming efficiency reduction is observed, while transfection (day 8) efficiency decreases with the reduction of mmRNA quantity introduced in the system. Reprogramming performed in 1 ,000^m-tall channels with the same mmRNA quantity (0.5 ng mm ^"2 ) used in 200 μηι height microfluidic optimised condition (Fig. 3a; efficiency of 121 ± 38) has a ~1 Q0- fold reduction in the reprogramming efficiency index.

Figure S14a Characterization of cells undergoing reprogramming in microfluidics. Three conditions were tested: non-treated control (-), inhibition of TGF-βΙ signaling (SB, 2 μΜ) and exogenous TGF- βΙ . Images were taken at day 7 after the first transfection. Scale bar, 50 μιη. Quaniification of the percentage of E-CAD* ceils is shown below. Expression of the epithelial marker E-CAD is high in each conditions, although TGF- β1 -treated cells do not give rise to any hiPSC colony (Figure 4h). Figure S14b, Reprogramming at day 12. While exogenous TGF-βΙ induces fibroblasts elongation counteracting mesenchymal to epithelial transition, treatment with SB (either 2 or 10 μΜ) promotes the definition of an epithelial-like cell layer without substantial ceil aggregation and rise of small hiPSC dusters (-). Figure S15a, Comparison of requirements for reprogramming in microfiuidics and well (left) and downscaling strategy of the conventional process to obtain functional ceil types from hiPSC in single-seeding procedure (right).

Figure S15b, Freshly-derived hiPSC were differentiated for 5 days in EB medium allowing the derivation of different cell phenotypes derived from the same colony. RT- PCR analysis (right) reveals the presence of markers of the three germ-layers.

Figure S1 Sc, Freshly-derived hiPSC were also differentiated towards a functional cardiac phenotype for 14 days. At the end of the differentiation protocol, extensive areas within the culture channel show tissue rearrangement (left). Scale bar, 750 μηι. RT-PCR (right), from single-channel total RNA extraction, evidenced the presence of advanced cardiac development and functional maturation. RT, reverse transcriptase; c+, samples used as a positive control. Figure S16 Schematic representation of the closed microfluidic network for the reprogramming process. The system is composed by environmental controller (EC), temperature controlled reservoirs (TCR), microfluidic units (uF), fiuidic connections (FC), gas pressure lines (GPL), pneumatic pressure lines (PPL). Environmental controller, EC, controls the temperature and the gas partial pressures, including N2, 02, and C02, for the uF1 , uF2, uF3 and TCR3. uF1 is microfluidic multi-inlet unit that delivers the transfection solution to the uF3 (ceil culture microfiuidic platform); continuous lines are flow channels connecting the unit to external, dashed lines are channels to pneumatically control the valve system. uF1 has a system of micro-valve for selectively loading of transfection solutions from the TCR1. uF1 has also a system to purge the air trapped in the tubing connection between TCR1 and uF1. uF2 is microfiuidic multi-inlet unit that delivers the media to the uF3 (ceil culture microfiuidic platform); continuous lines are flow channels connecting the unit to external, dashed lines are channels to pneumatically control the valve system, uF2 has a system of micro-valve for selectively loading the media from the TCR2. uF2 has also a system to purge the air trapped in the tubing connection between TCR2 and uF2.

uF3 is a muitiiayered microfiuidic composed of a 3-layer chip: control layer (CL), flow layer (FL) and culture layer (CCL) as described previously.

TRC1 is a temperature controlled reservoir that holds the aliquots of the transfection solutions (n=16 was used). TRC1 maintains the temperature at -20°C and when it required, the single aliquots are selectively heated at 37 °C by using a local electric resistance. The transfection solution are previously produced by fast cooling.

TRC2 is a temperature controlled reservoir that holds the aliquots of media (n=4 was used). TRC2 maintains the temperature at +4°C. The media are previously produced. TCR3 is a temperature controlled reservoir that holds the reservoirs of the waste coming the uF3 (n=2 was used).

Fiuidic connections (FC) are made by capillary tubing (Teflon is normally used material). Fluid flow are preferentially controlled by pressure drop along microfiuidic circuit. Gas pressure lines (GPL), pressurize the reservoirs hold in TCR1 , TCR2. Ail GPL are equipped with sterile filter. Pneumatic pressure lines (PPL), PPL1 , PPL2 and PPL3 controls the micro-valves of the uF1 , uF2 and uF3, respectively. PPL are controlled by external pneumatic transducer and remote computer.

Ail system is connected under sterile conditions and, once closed, is completely sealed from the external environment. Figure S17 It shows the images of transfected cells (nuclear GFP, left images) and their nuclei (Hoechst, right images) using fresh transfection solution (upper panel), -80 frozen transfection solution (middle panel) and - 88 transfection solution (lower panel). Quantitative analysis show that the efficiencies of transfection at the particular conditions of the experiments are: 32+5% for fresh transfection (upper panel); 15+2% for transfection solution frozen at -80°C (middle panel) and 18+2% for transfection solution frozen at -186X (lower panel).

DETAILED DESCRIPTION

A first object of the invention is a method of reprogramming adult cells to iPSC the method comprising

-seeding adult cells in a microfluidic cell culture chamber

-submitting the seeded cells to reprogramming by delivering into said cells suitable reprogramming factors and/or inducing into said cells the synthesis of suitable reprogramming factors, with suitable vectors

-culturing said cells until iPSC are obtained

wherein the height of the culture medium comprising said suitable reprogramming factors and/or vectors in the microfluidic cell culture chamber with respect to the bottom of the chamber is in the range from 500 to 50 pm.

According to the invention, any known protocol for cellular reprogramming may be followed in carrying out the reprogramming method of the invention.

As clearly demonstrated in the experimental section below and in the Figures, the method of the invention is highly effective and enhances the efficiency of literature reprogramming protocols up to 50 folds (cfr Figures 4 and 5). The increase in the efficiency is due, as shown in Figures 4 and 5, to the reducing of the medium volume/cell surface ratio of 5-10 folds compared to a well plate (figure 1c).

At this small microliter scale, the extrinsic factors released by the cells are rapidly concentrated. Autocrine and paracrine signals easily accumulate in the small medium volume and, consequently, they appear to stabilize signaling pathways fundamental for acquiring pluripotency.

According to the invention, the height of the culture medium comprising all the material necessary for the cell reprogramming, such as suitable vectors that introduce or induce the synthesis of suitable reprogramming factors into the cell, in the microfluidic cell culture chamber with respect to the bottom of the chamber will be in the range from 500 to 50 μηι, by way of example from 300 to 100 μηι. In a further embodiment, the height of the medium in the microfluidic cell culture chamber with respect to the bottom of the chamber will be from 250 to 150 μηι and in a yet further embodiment the height of the medium in the microfluidic cell culture chamber with respect to the bottom of the chamber will be of about 200 μηι.

In standard protocols, the height of the medium in a well with different area is of about 1000-2000 μηι. Standard microfluidic cell culture chamber, as the chambers suitable for carrying out the method of the present invention have a bottom (seeding) surface of about 10-30 mm ² and height of 50-500 μηι. Hence, the volume of the medium in the present method is strongly reduced with respect to the state of the art.

Surprisingly, this reduction does not affect the efficiency of the method, when the same conditions (and therefore concentration/compositions) are used. In particular, as evident from the Figures, when the final concentration of the reprogramming working solution is maintained in the comparative test, in a standard 1000 μηι medium height over a seeding surface of about 10 mm ² , the same efficiency is observed with 200 μηι medium height over a seeding surface of about 10 mm ² . If, the final concentration of the reprogramming working solution is optimized, e.g. by increasing it about 3-2 or about 2.5 folds, the efficiency of reprogramming increases dramatically (see, for example, Figures 4 and S13).

As Figure 5 demonstrates, the effectiveness of the method is strongly linked to the fact that the reprogramming signals easily accumulate in the small medium volume of the method disclosed herein, therefore, the highly efficient reprogramming, according to the present invention, can be carried out with any reprogramming protocol known in the art and, therefore with any known suitable vector known in the art.

The method of the invention is highly effective and allows an efficient reprogramming also from a very small amount of cells. Typically, according to the invention, the seeded cells can be at least 1 cell/mm ² , or at least 5 cells/mm ² or at least 8 cells/mm ² , or at least 10 cells/mm ² , in other words, the cells can be seeded at 1-1000 cells/mm ² , in particular at 1-100 cells/mm ² , and, as exemplified in the example section at 10-50 cells/mm ² .

In standard protocols a markedly higher number of cells is necessary in order to obtain some cell reprogramming. The high efficiency of the method of the present invention allows reprogramming also starting from a very small number of cells.

Although episomal or non-integrating vectors are preferred for obvious safety reasons, the method of the inventor can in principle be carried out with any suitable vector known in the art, such as viral vectors (by way of example lentiviral vector, a retroviral vector, an adenoviral vector or a sendai virus vector), episomal vectors, mRNAs, modified mRNAs, microRNAs, small molecules.

The skilled person will be able to select the desired reprogramming protocol (suitable set of reprogramming factors and suitable vectors) among the protocols in the art and to apply said protocol to the method of the invention without use of inventive skills. According to the present invention any adult cell will be suitable for reprogramming with the method herein disclosed. The differentiated or adult cells that may be reprogrammed may be fetal, neonatal or adult somatic cells, including both cells from healthy patients either affected or predisposed to diseases, for example cancer, include both human and animal cells and, in particular, of established cell lines, or immortalized primary cells.

In a further aspect of the invention, the cells are differentiated primary lines, such as fibroblasts derived from human biopsies, foreskin, skin and skeletal muscle; renal epithelial cells (e.g. derived from urine samples); amniocytes; bone marrow mesenchymal stem cells and nucleated cells derived from peripheral or cord blood. According to an aspect of the invention, the adult cells can be deriving from any superior animal, in a preferred embodiment from a mammal and in a more preferred embodiment from a human.

It is to be noted that, due to the extremely high efficiency of the method of the invention, the method is in particular suitable for the reprogramming of senescent cells or of cells have senescent features due to disease. According to the present disclosure, the reprogramming solution, i.e. the cell culture medium comprising said suitable reprogramming factors and/or vectors is changed with an equal amount of fresh reprogramming solution, i.e. the cell culture medium comprising said suitable reprogramming factors and/or vectors every 6-18 hours. In a particular embodiment, the change may be carried out every 8-12 hours or, more in particular every 8 hours, or every 12 hours.

The inventors have also surprisingly verified that the efficiency of the method of the invention is sufficiently high to allow the use of a pre-stored cryo-preserved ready-to- use reprogramming working solution comprising said suitable reprogramming factors and/or vectors and a suitable cell culture medium. In standard protocols, the reprogramming solution has often to be freshly prepared each time from working solutions of each component due to the fact that several components may degrade rapidly. Although the use of a cryo-preserved working solution decreases the efficiency of the cellular reprogramming of about 50% as reported in the experimental section below, the method of the invention is so efficient that the use of a previously prepared reprogramming working solution, cryo-preserved in working aliquots, when changing the reprogramming working solution, results, nevertheless, in an extremely high efficiency of reprogramming. According to the present invention, the reprogramming ready-to-use working solution may be cryo-preserved once prepared in ready-to-use dosed vials at a temperature of from -60°C to -200°C or of from -70°C to -190°C, or of from -75°C to -188°C, or of from -78°C to -187°C, or of about -80°C or of about -186°C. According to one embodiment of the invention, the method of reprogramming adult cells to iPSC as disclosed may further comprise one or more additional steps for the differentiation of the obtained iPSC. The experimental section and the related Figures show that the iPSC obtained with the reprogramming method of the invention can undergo functional differentiation, which requires the activation or repression of specific signaling pathways at defined temporal windows. It follows that reprogramming and differentiation can take place within the same microfluidic device in a single step or one step. The "onestep" differentiation and reprogramming can occur preferably when purity of iPSC is at least of 80%. By way of example, contracting cardiomyocyte-like cells showing remarkable troponin- T expression and sarcomeric organization were obtained in microfluidics by sequential exogenous promotion and inhibition of WNT signaling (Figure 6c, Supplementary Figure 13). Polygonal-shaped CK18+ hepatocyte-like cells, occasionally poly- nucleated, were also obtained promoting Activin and WNT pathways of freshly generated μ-hiPSC (Figure 6d). Hepatocyte-like cell functional maturation of extensive morphologically differentiated areas was confirmed by albumin secretion and glycogen storage.

Therefore, the disclosure reports the development of a seamless 30-day process of reprogramming and differentiation within the same microfluidic device, for the production of functional hepatocyte- and cardiomyocyte-like cells from human fibroblasts (figure 6e). In other words, according to any embodiments of the present invention, the process of reprogramming and differentiation within the same microfluidic device or within the same microfluidic channel, that is a one-step reprogramming and differentiation process, allows to produce mature functional hepatocyte-like cells.

In a particular aspect, the method according to any of the embodiments described above, can be carried out in a microfluidic system, preferably a closed microfluidic system.

In fact, microfluidics is well suited to comply with GMP requirements, because the system is closed and remotely controlled, maximizing robustness and strongly decreasing the probability of contaminations.

In particular, the microfluidic system includes at least a stock station configured to include one or more reprogramming solutions; at least a culturing device including one or more microfluidic culture cell chambers configured to allow cell adhesion and a delivery device to deliver solutions from the stock station to the culturing device. The system is configured to keep the stock station or the one or more reprogramming solutions of the stock station at a cryo temperature condition to obtain a cryo-preserved ready-to-use reprogramming solution.

In one embodiment of the invention, the stock station includes a plurality of aliquot holders (for example vials) each configured to include a dose or aliquot (or fraction) of a bigger pre-prepared cryo-preserved ready-to-use reprogramming solution. The use of the plurality of stock holders allows a selective use of each solutions/fractions. The stock holders can be kept at the cryo-temperature condition, temporarily or for a predetermined period of time. Then each holder or vial can be heated to get a working temperature, such as 37°C, according to a predetermined timing. For example, the stock station includes one or more heaters configured to heat each the cryo-preserved ready-to-use reprogramming solution. Preferably the stock station includes a container configured to house a plurality of said holders and a cooler configured to keep the one or more holders at the cryo temperature condition.

In one embodiment of the invention, each holder is provided with a heater, the heater being configured to heat the corresponding holder. Preferably, one or single heater is associated with one or single corresponding holder.

It follows that a reprogramming procedure can take place as follows.

A reprogramming solution can be prepared and incubated. More in particular, the solution is prepared once and divided in fractions to be kept as reservoirs. In other words, a plurality of separated reprogramming ready-to-use solutions are hold as stock solutions (thus avoiding daily preparation) and transferred in each holder. The holders can be intended as an aliquot holder, or dose holder. Each holder can be fast-cooled using systems at different temperatures: ranging from -20°C to -250°C. The solutions can be frozen 20 hours before reprogramming. Then the holder are kept in the frozen condition in a rack or container.

The holders can be sized to hold a solution suitable for carrying out a culture in the culturing device for a pre-determined period of time, such as for example 24 hours. Then, heating of one of aliquot holders can be programmed every 24 hours to delivery only the dose or aliquot of one holder to the culturing device. Timing for heating the aliquot holder and delivering the dose to the culturing device can be selected according to many factors, such as for example the type of cultures, the reprogramming solution, size of the chambers and reprogrammed cells to be obtained.

According to a preferred embodiment the system includes an environmental controller, temperature controlled reservoirs, microfluidic units, fluidic connections, gas pressure lines, pneumatic pressure lines. Environmental controller can control the temperature and the gas partial pressures, including for example N2, 02, and C02. Preferably a microfluidic multi-inlet unit is present. The multi-inlet unit can deliver the reprogramming solution to the microfluidic cell culture chambers or platform; continuous lines are flow channels connecting the unit to external.

The microfluidic cell culture chambers or platform can include one or more micro-valve for selectively loading of transfection solutions from the temperature controlled reservoirs. The microfluidic cell culture chamber can include a system to purge the air trapped in the tubing connection between cell culture microfluidic chambers and multi- inlet unit. An additional microfluidic multi-inlet unit can be present. The additional microfluidic multi-inlet unit delivers the media to the cell culture microfluidic platform. The additional microfluidic multi-inlet unit can have a system of micro-valve for selectively loading the media from the temperature controlled reservoirs.

More in particular, with reference to Figure S16, the system can be composed by environmental controller (EC), temperature-controlled reservoirs (TCR), microfluidic units (uF), fluidic connections (FC), gas pressure lines (GPL), pneumatic pressure lines (PPL). Environmental controller, EC, can control the temperature and the gas partial pressures, including N2, 02, and C02, for the uF1 , uF2, uF3 and TCR3. uF1 can be microfluidic multi-inlet unit that delivers the transfection solution to the uF3 (cell culture microfluidic platform); continuous lines can be flow channels connecting the unit to external, dashed lines are channels to pneumatically control the valve system. uF1 can have a system of micro-valve for selectively loading of transfection solutions from the TCR1. uF1 can include also a system to purge the air trapped in the tubing connection between TCR1 and uF1. uF2 can include microfluidic multi-inlet unit that delivers the media to the uF3 (cell culture microfluidic platform); continuous lines are flow channels connecting the unit to external, dashed lines are channels to pneumatically control the valve system. uF2 include a system of micro-valve for selectively loading the media from the TCR2. uF2 has also a system to purge the air trapped in the tubing connection between TCR2 and uF2.

uF3 ca be a multilayered microfluidic composed of a 3-layer chip: control layer (CL), flow layer (FL) and culture layer (CCL) as described previously.

TRC1 can be a temperature controlled reservoir that holds the aliquots of the transfection solutions (n=16 was used). TRC1 maintains the temperature at -20°C and when it required, the single aliquots are selectively heated at 37 °C by using a local electric resistance, or other heaters. The transfection solutions are previously produced by fast cooling.

Each single heater can be selectively associated with a single aliquot to allow selective delivery of the ready-to-use reprogramming solution to the cell culture microfluidic chambers or platform.

TRC2 can be a temperature controlled reservoir that holds the aliquots of media (n=4 was used). TRC2 maintains the temperature at +4°C. The media are previously prepared.

TCR3 can be a temperature controlled reservoir that holds the reservoirs of the waste coming the uF3 (n=2 was used).

Fluidic connections (FC) can be made by capillary tubing (Teflon is the normally used material). Fluid flow is preferentially controlled by pressure drop along microfluidic circuit. Gas pressure lines (GPL) pressurize the reservoirs hold in TCR1 , TCR2. All GPL are equipped with sterile filter. Pneumatic pressure lines (PPL), PPL1 , PPL2 and PPL3 control the micro-valves of the uF1 , uF2 and uF3, respectively. PPL are controlled by external pneumatic transducer and remote computer.

The whole system is assembled under sterile conditions and, once closed, is completely sealed from the external environment.

According to one embodiment of the invention, the closed microfluidic network applied for the cell reprogramming satisfies the following requirements: the reprogramming process takes at least 10-16 day; it requires daily delivery of transfection or reprogramming solutions; it also requires at least 2 media changes per day. The system preferably operates under sterile conditions.

The operating steps for performing reprogramming process within the closed microfluidic network can include the following steps:

A) microfluidic network preparation ) Sterilization of different units of the network and all components used for building the network;

) Connecting the uF3 to pneumatic pressure line (PPL), under sterile conditions, for making the uF3 fully functional;

) Automatic loading the coating solutions in the cell culture microfluidic platform (uF3) for surface functionalization under sterile conditions;

) Automatic loading the cell suspension in the cell culture microfluidic platform (uF3) under sterile conditions;

) Loading of the reservoirs (for example n=16 are prepared) with the aliquots of the transfection solutions and fast cooling them with cooler down to temperature ranging between -80°C and -190°C;

) Transfer the reservoirs of the transection solutions within the TCR1 working, for example, at -20°C;

) Loading of the reservoirs (for example, n=4 are prepared) with the aliquots of the different media (n=3) and with a washing solution (n=1) used in the reprogramming process;

) Transfer the reservoirs of the media within the TCR2 working for example, at +4°C;

) Assembling of the microfluidic network making sealed connection among the different units of the network under sterile conditions;

0) Degassing the microfluidic uF1 , uF2, uF3 and all connections between them through automatic procedure using the micro-valves integrated in the system;1) Delivery of media through each cell culture microfluidic chamber;

icrofluidic network reprogramming, each step is repeated every day (from step 1 1ep 16 ):

2) Changing the media (using TCR2) in any of the cell culture microfluidic chambers (uF3) through uF2 (t=0h); washing solution (TCR2) is used to clean the dead microfluidic volume;

3) Heating up and thawing one single aliquot (TCR1) containing transfection solution by selectively turning the electric resistance on; 14) Delivering the transfection solution to uF1 and purge the air contained between TCR1 and uF1 ;

15) Delivering the transfection solution in any of the cell culture microfluidic chambers (uF3) through uF1 (t=8h); washing solution (TCR2) is used to clean the dead microfluidic volume;

16) Changing the media (using TCR2) in any of the cell culture microfluidic chambers (uF3) through uF2 (t=12h); washing solution (TCR2) is used to clean the dead microfluidic volume;

C) Maintaining and extracting of microfluidic generated iPSCs for example, after 12-16 day of reprogramming:

17) Changing the media (using TCR2) in any of the cell culture microfluidic chambers (uF3) through uF2 (t=0h); washing solution (TCR2) is used to clean the dead microfluidic volume;

18) Changing the media (using TCR2) in any of the cell culture microfluidic chambers (uF3) through uF2 (t=12h); washing solution (TCR2) is used to clean the dead microfluidic volume;

19) Repeating steps 17 and 18 for example, for 2-5 days;

20) Open the microfluidic cell culture chamber (uF3) by detaching/cutting the top microfluidic layer under sterile conditions;

21) Mechanically extracting the iPSC colony under sterile conditions;

22) The iPSC colonies are re-plated in other microfluidic platform or Petri dish or frozen for cryo-preservation.

EXAMPLES

Technological downscale of reprogramming process

By analyzing previously published microarray data on the temporal progression of reprogramming, the authors observed that defined sets of extracellular soluble components of the signaling pathways are temporally regulated during the process, either positively or negatively (Figure 1 b, Supplementary Figure 1 , Supplementary Spreadsheet 1). According to this observation, they hypothesized that the perturbation of endogenous signaling pathways through the modulation of their extrinsic components can strongly impact on the reprogramming process. To this aim, the inventors downscaled the volume of cell culture medium to few microliters, reducing medium volume/cell surface ratio of 10 folds compared to a well plate (Figure 1 c). At this small microliter scale, the extrinsic factors released by the cells are rapidly concentrated. Autocrine and paracrine signals easily accumulate in the small medium volume and, consequently, they are likely to stabilize signaling pathways fundamental for acquiring pluripotency.

A downscaled reprogramming process was hence designed within a microfluidic platform (Figure 2a, Supplementary Figure 2, Supplementary Video 1). The microfluidic culture chambers have a surface comparable to that of a standard 96-well plate, whereas a medium height of only 100-300 μηι, in particular of about 200 μηι ensures significant accumulation of endogenous factors. This experimental setup contains culture chambers with the same geometry as other microfluidic platforms previously used to demonstrate long-term culture of pluripotent stem cells, germ layer differentiation, and delivery of viral particles (Giobbe, G. G. et al. Functional differentiation of human pluripotent stem cells on a chip. Nat. Methods 12, 637-640 (2015); Takahashi, K. et al. Induction of pluripotency in human somatic cells via a transient state resembling primitive streak-like mesendoderm. Nat. Commun. 5, (2014); Giulitti, S., Magrofuoco, E., Prevedello, L. & Elvassore, N. Optimal periodic perfusion strategy for robust long-term microfluidic cell culture. Lab. Chip 13, 4430-4441 (2013)). These microfluidic platforms are extremely versatile since they can be also designed and used without particular skills and the aid of liquid handling equipment: microfluidic culture channels can be managed by simple manual pipetting as in a common multi- well (Supplementary Figure 2). To ensure overall process robustness and accurate culture conditions, the inventors also automated the microfluidic setup by large-scale microfluidic integration, with a system of pneumatic valves that are remotely controlled to distribute medium into the 32 culture chambers of a single device with precise timing (Figure 2a). This microfluidic system is particularly prone to be set up according to Good Manufacturing Practice (GMP) as it can be run in a completely remotely controlled closed-loop process.

The inventors opted for a fast transient expression of reprogramming factors using modified mRNA (mmRNA) in order to remove the exogenous contribution when necessary. Typical mmRNA lifespan in cells is 24 h, thus daily transfections are required for sustained expression over several days, and the procedure highly benefits of microfluidic automation. The inventors first provide experimental evidence that mmRNA transfections can be efficiently performed within the microfluidic system. Using mmRNA encoding a nucleus-targeted green fluorescence protein (nGFP), the inventors obtained 80% nGFP cells by a single transfection in microfluidics, with a 60-fold reduction of the overall amount of mmRNA per cell used, compared to an equally efficient transfection in well (Figure 2b, Supplementary Figure 3). Optimized mmRNA delivery conditions, in terms of transfection duration and mmRNA concentration, were obtained by means of a stochastic mathematical model (Figure 2c, Supplementary Figure 4). At optimal conditions of mmRNA delivery, two key players in the reprogramming process, OCT4 and KLF4, show the highest expression at the protein level in microfluidics (Figure 2d, Supplementary Figure 4).

High efficiency and purity of reprogramming at micro-scale

It was next examined whether human fibroblasts could be effectively reprogrammed to hiPSC by sequential delivery of mmRNAs within the microfluidic platform. In addition to the four so-called Yamanaka factors that induce cell reprogramming (OCT4, SOX2, KLF4, and C-MYC), the inventors daily transfected LIN28 and NANOG, as suggested by Thompson and co-workers, and nGFP, to verify the sustained delivery efficiency during the process (Figure 2e, Supplementary Figure 4). To maximize chances of success, the inventors initially performed reprogramming in the presence of mitotically inactive feeder cells. μ-hiPSC colonies were first recognized morphologically as early as 7 days after the first mmRNA transfection and more colonies formed during the following days of protocol (Figure 3a). Randomly chosen colonies were thoroughly characterized after clonal extraction by manual picking (Supplementary Figure 5) for expression of pluripotency markers, absence of karyotypic abnormalities, and ability to generate teratomas in vivo and early-stage progenitors of the three germ layers in vitro, both via embryo body formation and in monolayer (Figure 3b-e, Supplementary Figure 6). Immunofluorescence analysis was also performed in situ to verify the expression of mesenchymal-to-epithelial transition (MET) and pluripotency markers at different stages of the reprogramming process (Figure 3f-g, Supplementary Figure 6).

Reprogramming yield was quantified as an efficiency index, calculated by counting the number of hiPSC colonies expressing pluripotency markers (TRA-1-60 and NANOG) at the end of the protocol per 100 cells initially seeded (Figure 4a). After establishing an incremental mmRNA dosage strategy to balance exogenous delivery, cell proliferation, and cell death, the inventors unexpectedly obtained a very high reprogramming yield in microfluidics, up to 16 (6±4) under feeder conditions (Figure 4a and 4b, Supplementary Figure 7).

To uniquely identify the correlation between reprogramming and endogenous factors, the inventors performed reprogramming in chemically defined xeno-free conditions (Supplementary Figure 8), especially avoiding cell feeder layers, which would uncontrollably perturb medium composition, the inventors also showed that the balance between initial cell density and the transfection-induced toxicity has to be optimized; for instance, even using higher mmRNA dose, a ten-fold increase in cell seeding density decreases reprogramming efficiency (Supplementary Figure 9). Under feeder-free optimal conditions, the inventors obtained a reprogramming yield of up to 150 (124±36) (Figure 4a-c, Supplementary Figure 8). Reprogramming yield of up to 150 can be obtained by means of the optimized height of the culture medium with respect to the bottom of the chamber is in the range from 500 to 50 μηι. Without feeders, cells reprogrammed with about 50-fold greater reprogramming yield in micro-scale compared to a multi-well plate (2.1 ±0.7) and to the highest yield reported in literature (chlaeger, T. M. et al. A comparison of non-integrating reprogramming methods. Nat. Biotechnol. 33, 58-63 (2015) and Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618-630 (2010)). The inventors explored how different sources of variability could affect reprogramming efficiency in microfluidics, using nested ANOVA for different random factors, cell batches (n=3), microfluidic devices (n>2), and independent culture channels (n>2), for a total of n=32 experimental data points (Figure 4a). Nested ANOVA shows no significant effect of cell batches and microfluidic devices on the reprogramming efficiency index and confirm system robustness (Supplementary Figure 9). Moreover, μ-hiPSC purity at the end of reprogramming, measured as NANOG ⁺ /TRA-1 -60 ⁺ cells over total number of cells in culture, is 85% (84.8±5.7%, Figure 4c). Thus, microfluidic reprogramming demonstrates to be very homogeneous, as showed by imaging cells undergoing reprogramming every 12 hours for 17 days.

Although only few hiPSC colonies are usually expanded for further applications downstream of the reprogramming process, having a system that boosts the efficiency is very important to be able to generate hiPSCs also from cells more refractive to be reprogrammed, such as slowly proliferating and early senescent primary cells. A particularly minimal invasive cell source derivation to obtain hiPSCs from a patient is urine. The reprogramming efficiency of urine-derived epithelial cells by a non- integrating method has been reported to be variable from patient to patient, but less than 0.02 colonies obtained from 100 cells seeded. Using mmRNA within the microfluidic system, the inventors successfully reprogrammed urine-derived epithelial cells from three different patients (Figure 4d, Supplementary Figure 10), confirming a 50-fold increase in reprogramming efficiency yield compared to a well plate. Next, the inventors reprogrammed cells from patients affected by genetic diseases, either freshly isolated or stocked in cell banks (Figure 4e). A particularly interesting application of our system is the reprogramming of skeletal muscle fibroblasts isolated from patients affected by Duchenne muscular dystrophy (DMD) and with limited lifespan before senescence onset. To our knowledge, this is the first time that hiPSCs are obtained from the target tissue of a specific genetic disease, and could have important implications in in vitro studies of various diseases, due to the possibility of tissue- specific somatic mutations occurring during tissue aging or disease progression.

Amplified endogenous signaling enhances reprogramming

The results above showed that the confined environment strongly affects the reprogramming process. Specifically, extrinsic endogenous cues due to cell autocrine and paracrine signals could be responsible for promoting pluripotency reacquisition. To clarify this aspect, the inventors performed a microarray analysis of the expression profiles of colonies freshly obtained in microfluidics (ρθ ^F) and in a multi-well plate (Figure 5a). The inventors designed the experiment in order to assess also if potential differences in gene expression of hiPSC clones derived in microfluidics and in well could be reverted after expansion in standard conditions. Freshly generated colonies in microfluidics evidenced a distinctive expression profile that, after a three-passage conventional expansion, converged to the same expression profile of hiPSC derived in multi-well (Figure 5b, Supplementary Spreadsheet 2). However, differences did not involve the expression of main pluripotency genes especially operating as nuclear factors (Figure 5c). The expression profile of colonies obtained in all four conditions was within the variability of pluripotent stem cells reported in literature (Supplementary Figure 11). Functional annotation analysis showed that the most significantly enriched gene ontology (GO) categories characterizing the genes differentially expressed in the microfluidic system respect to a well are related to the extracellular space and the receptor-mediated signaling pathways. More specifically, multiple extra-cellular components of the TGF-β pathway were up-regulated in colonies freshly generated in microfluidics (Figure 5d, Supplementary Spreadsheet 3). To verify the contribution of the extracellular accumulation of cell-secreted factors in the reprogramming, the inventors designed two experimental strategies for perturbing endogenous signal accumulation by tuning the frequency of media changes or the volume of the microfluidic culture chamber.

Firstly, the inventors increased the frequency of medium change, expecting to increase the washing out of the cell-secreted factors ¹⁷ . Consistently with this hypothesis, Figure 5e shows that the efficiency of reprogramming is significantly decreased by increasing the frequency of media change from f=3 d ^"1 to f=6 d ^"1 , even with higher delivery of the pro-pluripotency exogenous factors present in the reprogramming medium.

Secondly, the inventors compared reprogramming efficiency in microfluidic culture chambers with different heights ranging from 200 to 1000 μηι (Figure 5f). Different combinations of mmRNA dose were used for maintaining constant mmRNA concentration and mmRNA quantity used either in optimized microfluidic or in standard well (all experimental conditions are reported in Supplementary Table 2). Using mmRNA concentration (0.5 ng/μ-.) of standard well, the inventors observed successful reprogramming and comparable efficiency among different conditions (Figure 5f). Remarkably, the 10-fold reduction of total amount of mmRNA from standard well to 200 μηι height microfluidic, which also provoked significant decrease of the transfection efficiency (2 fold at day 7, Figure 5f), do not prevent to obtain hiPSC colony with an average efficiency index of 3. Moreover, when the mmRNA quantity (5 ng/mm ² ) used in 200 μηι height microfluidic culture chamber was used in the 1000 μηι height chamber, the reprogramming efficiency drastically decreased from an average of 124 to -1.5 hiPSC colonies per 100 initial cells seeded, even if both conditions show no significant difference in transfection efficiency (Figure 5f).

Overall, these results support the hypothesis that in our standard microfluidic system (200-μηι height and low frequency of medium change) there is an optimal balance between endogenous and exogenous factors for cell reprogramming progression. As highlighted by the microarray results (Figure 5d), the inventors then investigated the role of TGF-β pathway. Since exogenous TGF-β is not present in the medium, the inventors then hypothesized that relevant differences could take place in this pathway activation along reprogramming in the two culture systems due to differential accumulation of cell-secreted TGF^-family ligands. Conditioned medium by cells undergoing reprogramming evidenced exceptionally strong activation of the downstream effector, SMAD2/3, in luciferase reporter cells (Figure 5g). Therefore, a different temporal profile of TGF^-pathway activity is a potential candidate for explaining the enhanced reprogramming efficiency obtained in microfluidics.

To explore further the role of TGF-β pathway, the inventors promoted or inhibited its signaling (Figure 5h-i). When exogenous TGF-βΙ was added in culture during the whole reprogramming process, it prevented morphological MET even in the presence of exogenous factors-mediated E-CAD expression (Supplementary Figure 12), and abolished reprogramming (Figure 5i). However, inhibiting the pathway abolished cell reprogramming as well (Figure 5i). In this latter case, cells did not generate hiPSC colonies, but exhibited an epithelial morphology at the end of the reprogramming protocol (Supplementary Figure 12).

It can be concluded that in the microfluidic confined environment cells are able to self- regulate signaling towards maximal reprogramming efficiency and TGF-β pathway plays an important but not trivial role during reprogramming. The pathway is activated with a characteristic temporal profile given by a small up-regulation at the first stages, a down-regulation when MET occurs, and a relevant up-regulation after MET. Exogenous and constant up- or down-regulation of the pathway strongly inhibited the reprogramming process. Other cell-secreted TGF^-family ligands could also be involved in pathway activation.

Direct conversion of freshly-derived μ-hiPSC into functional cell types It was next questioned whether the freshly generated μ-hiPSCs (ρθ μΡ) are competent to differentiate to specific phenotypes without an intermediate stage of expansion. The high purity of freshly generated hiPSCs (Figure 4c) allows defined differentiation, limiting undesired extrinsic signals from non-pluripotent cells. The signaling pathways regulating the exit from pluripotency program and the induction of specific germ layer differentiation should be in place (Figure 6a). Indeed, our results show that specific markers of the three germ layers are selectively expressed after directed differentiation of freshly generated μ-hiPSCs (Figure 6b).

Moreover, the inventors verified if freshly generated μ-hiPSCs (pO ^F) could undergo functional differentiation, which requires the activation or repression of specific signaling pathways at defined temporal windows. Contracting cardiomyocyte-like cells showing remarkable troponin-T expression and sarcomeric organization were obtained in microfluidics by sequential exogenous promotion and inhibition of WNT signaling (Figure 6c, Supplementary Figure 13). Polygonal-shaped CK18 ⁺ hepatocyte-like cells, occasionally poly-nucleated, were also obtained promoting Activin and WNT pathways of freshly generated μ-hiPSC (Figure 6d). Hepatocyte-like cell functional maturation of extensive morphologically differentiated areas was confirmed by albumin secretion and glycogen storage. It follows that reprogramming and differentiation within the same microfluidic device or within the same microfluidic channel produce mature functional hepatocyte-like cells (one-step reprogramming and differentiation process). The one- step reprogramming and differentiation process preferably take place with 80% purity of iPSC. Therefore, the inventors report the development of a seamless 30-day process of reprogramming and differentiation within the same microfluidic device, for the production of functional hepatocyte- and cardiomyocyte-like cells from human fibroblasts (Figure 6e).

DISCUSSION The process the inventors described have several advantages, both technological and scientific. As for the first aspect, the microfluidic system requires only few microliters of medium per day for each independent culture channel, with an overall 100-fold reagent savings compared to a well in a 6-well plate. Cost reduction coupled with system automation makes feasible the implementation of the process to hundreds of parallel reprogramming experiments. By way of example 32 parallel experiments can be carried out in a single chip.

Technological advances obtained by downscaling the reprogramming process in microfluidics are strongly correlated with the envisioned applications. Biologically, this system offers the possibility to play on cell culture microenvironment with high precision. In the examples provided inventors used mmRNAs to induce gene expression, because they are effective (non-integrating and giving high transfection efficiency), but practically cumbersome due to the daily transfections required for sustained expression. However, the transient nature of mmRNA allows a dynamic tuning of intra-cellular gene expression, achieving maximal control over the experiment, which can be easily implemented in an automated system.

It is obvious from the data provided that the method is not limited to the use of mRNA or mmRNA as vectors for the expression of the suitable reprogramming factors.

It is also clear that, microfluidics is well suited to comply with GMP requirements, because the system allows to carry out the method of the invention in closed and remotely controlled environment, maximizing robustness and strongly decreasing the probability of contaminations.

A number of groups worked on increasing reprogramming efficiency. A recent work showed that reprogramming is a deterministic process, demonstrating that there are not specific restrictions to cell reacquisition of pluripotency. However, the current macro-scale reprogramming processes produce only few colonies inter-dispersed within a layer of non- or partially-reprogrammed cells, evidencing that current reprogramming protocols are still sub-optimal.

The microfluidic process the inventors developed shows that an almost homogeneous reprogramming process (-85% of hiPSCs in culture at the end of the process) is achievable with proper temporally regulated cues. Moreover, a detailed knowledge of these cues is not necessary, because cells undergoing reprogramming are able to self- regulate and promote the process, once endogenous signaling is sufficiently accentuated by downscaling of the culture system. Nonetheless, this micro-scale system could be successfully used also for dissection of reprogramming mechanisms, given that the majority of cells in culture synchronously reacquire a pluripotent phenotype.

Extremely highly efficient and homogeneous reprogramming coupled with mmRNA technology opens the possibility to differentiate patient-specific μ-hiPSCs without the need of cell expansion. The overall protocol of reprogramming and differentiation into functional cardiomyocyte- and hepatocyte-like cells lasts approximately a month. Besides lowering costs, this time reduction has important implications on cell quality, reducing the probability of genetic aberrations. In comparison with other non-integrating reprogramming methods, such as Sendai virus, mmRNA technology shows tunable balance between transfection efficiency and cytotoxicity, which are a fundamental pre- requisite for feasible microfluidic reprogramming.

In perspective, the proposed downscaled process makes feasible the derivation of hiPSC from a large cohort of patients, and a rapid and reliable production of patient- specific somatic cells (Figure 6f). Continues efforts to develop methods for improving cell functional maturation by differentiation will further accelerate the use of this system for high-throughput studies ^26-28 , in particular for the systematic investigation of the influence of the genetic background on multiple cues (disease development, drug cytotoxicity and efficacy, response to environmental factors). METHODS

Cell culture. Human foreskin BJ fibroblasts (Miltenyi Biotec) and skeletal muscle fibroblasts (SkMF, Telethon Network of Genetic Biobanks, Italy) from patients with Duchenne muscular dystrophy were cultured in Dulbecco's modified Eagle Medium (DM EM, Life technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies). HFF-1 fibroblasts (ATCC) were cultured in DMEM with 15% FBS. Renal epithelial cells (RE) were isolated and expanded from three healthy donors, male and female, in RE medium (Lonza) as described in Zhou, T. et al. Generation of human induced pluripotent stem cells from urine samples. Nat. Protoc. 7, 2080-2089 (2012). Inactivated human newborn foreskin fibroblasts NuFF-RQs (AMS Biotechnology) were seeded on 0.2% type-A gelatin (Sigma-Aldrich) at 260 cells/mm ² in DMEM with 10% FBS, in case of use as a feeder layer for reprogramming and for Pluriton medium (Stemgent) conditioning. RE cells from patients with anti-trypsin-deficiency (ATD) were freshly isolated and provided by Fondazione IRCCS Policlinico San Matteo.

hiPSC were mechanically passaged on mytomicin-treated mouse embryonic fibroblasts (MEF, Millipore) with daily changes of hiPSC medium (DMEM/F12, 20% knockout serum replacement, 1 % NEAA, 1 % glutamine, 1 % β-mercaptoethanol (all Life Technologies), 20 ng/ml b-FGF (Peprotech). Alternatively, freshly derived hiPSC were directly cultured in feeder-free medium StemMACS™ iPS-Brew XF (Miltenyi Biotec) or transferred from MEF without any adaptation. hiPSC were passaged every 3-4 days on vitronectin XF treated plates using gentle cell dissociation reagent (both Stemcell Technologies). All cell lines were cultured at 37 °C and 5% C0 ₂ atmosphere.

Microfluidic platform. Microfluidic platforms were fabricated according to standard soft-lithographic techniques and molded in poly-dimethylsiloxane (PDMS), as disclosed for example in U.S. Patent Application No.: 15/006,505. Briefly, Sylgard 184 (Dow Corning) was cured on a 200^m-thick patterned SU-2100 photoresist (MicroChem) in order to obtain a single PDMS mold with multiple independent channels. The PDMS mold was punched and sealed on a 75 x 25 mm microscope glass slide (Menzel- Glaser) by plasma treatment. Channels were rinsed with isopropanol and distilled water to check proper flow, before autoclaving. A syringe step-motor pump (Cavro, Tecan) was used to periodically control medium flow rate into the microfluidic channels at 120 μΙ_Λτπη for 5 s. 0.5 ID Tygon tubings (Cole-Parmer) and 21G stainless-steel needles were used to connect the microfluidic channels to the pump head.

Microfluidic platform with integrated medium distribution system. Multilayer soft lithography was used to fabricate this type of microfluidic platform, composed of a 3- layer PDMS chip. Transparent photomasks were printed at 8000 dpi. A 4-inch silicon wafer (Siegert) was used for molding. Control mold was made by SU8 2050 (Microchem), obtaining 45 μηι square channels. Flow mold had 45-μηι round channels made by SPR 220-7 (Dow Corning); to avoid unintentional cross-section valves, 90-μηι square channels have been made over the previous by a second SU8 2100 layer. Culture channel mold has 220 μηι square section made by SU8 2100. Every mold was previously coated for 1 hour at room temperature with chlorotrimethylsilane vapors (Sigma-Aldrich). Sylgard 184 (Dow Corning) was mixed at 5: 1 ratio (base:cure agent) for the flow layer (FL), 20: 1 ratio for the control layer (CL) and 10: 1 ratio for the cell culture layer (CCL). All layers were partially cured at 333 K before peeling, cutting and punching: 45 minutes for FL, 60 minutes for CL and 70 minutes for CCL. After alignment, another 2 hours at 353 K completed the curing. The PDMS chip was finally bonded by plasma activation on a large glass slip (75 x 50 mm, Ted Pella) covered with a thin (<0.5 mm) 20: 1 PDMS layer.

The microfluidic platform was fully assisted by an automated medium delivery and distribution system into the culture channels. A periodic 120 μΙ_/ηιίη perfusion for 5 s was controlled twice a day by Cavro pumps (Tecan) and a lab-made software interface written in Labview (National Instruments). The experimental setup is shown in Figure 2a and Supplementary Figure 2. Surface coating in microfluidic setup. After autoclaving and before cell seeding, microfluidic culture channels were treated for surface functionalization. Extracellular matrix proteins were either adsorbed or chemically bound to the silanized glass bottom of each channel to provide a durable coating for cell culture (Supplementary Figure 2). For the adsorbed substrates either fibronectin (Fn, 10 μg/mL, Sigma-Aldrich) or type-A pork gelatin (Gel, 0.1 % and 0.6%, Sigma-Aldrich) were incubated within the microfluidic channels for 1 h at 37 °C before rinsing with DPBS. For glass silanization, surface was treated either with a 5% water solution of (3-aminopropyl)-triethoxysilane (APTES, Sigma-Aldrich) for 20 minutes, or a 0.3% ethanol solution of 3-(trimethoxysilyl)-propyl methacrylate (TMSPMA, Acros Organics) for 5 minutes. Covalent bonding of adsorbed Gel on APTES was obtained treating with 0.5% v/v glutaraldehyde for 10 minutes. Covalent bonding of Gel on TMSPMA was obtained treating Gel with methacrylic anhydride (Sigma-Aldrich) in PBS buffer for 1 h at 60 °C to produce an acrylate- reactive variant (GelMA). A TMSPMA-GelMA bonding was performed for 15 minutes by adding 0.1 % ammonium persulphate and Ν,Ν,Ν',Ν'-tetramethylenethylenediamine (Sigma-Aldrich) prior to GelMA (0.1 % or 0.6%) injection within each channel. After surface biofunctionalization, channels were extensively rinsed with DPBS prior to cell seeding. In multilayered platforms, the distribution layer was functionalized by directly incorporating 0.3% TMSPMA in the PDMS before curing.

Reprogramming. For technological validation, an mmRNA encoding for nuclear GFP (StemMACS Nuclear eGFP, Miltenyi Biotec) was used alone, carried into the cells by different transfection reagents: RNAiMAX (RiM, Life Technology), Stemfect (SF, Stemgent), and StemMACS Transfection Reagent (SM, Miltenyi Biotec), according to the transfection protocols below. Comparisons of transfection efficiencies, between microfluidics and wells, and between different transfection reagents, were carried out with the same cell density. SF was used if not specified. SM was used for feeder-free protocols. hiPSCs were generated via mmRNA-mediated reprogramming, adapting the protocols reported in literature for feeder and feeder-free hiPSC derivation, from multiple cell sources: human foreskin BJ and HFF-1 fibroblasts, skeletal muscle fibroblasts (SkMF) and urine-derived renal epithelial cells. In particular, reprogramming was optimized with incremental dosage of mmRNAs during the first four daily transfections, with 25%, 50%, 75%, 100% mmRNA amount of subsequent transfections at -0.5 ng/mm ² . As reported by others ³² the incremental mmRNA dosage for high reprogramming performances is important because a proper balance between efficient transcription factors delivery, cell proliferation rate and transfection-induced cell mortality has to be achieved.

For reprogramming with a feeder layer, NuFF-RQs were seeded at day -2. At day -1 , the reprogramming target fibroblasts (either BJ or HFF) were seeded at different densities (5, 10, and 26 cell/mm ² ) in DMEM with 10% FBS. At day 0, 2 h prior to the first mmRNA transfection, medium was switched to defined TGF^-free Pluriton reprogramming medium (Stemgent) supplemented with 200 ng/mL B18R (eBioscience) to suppress single-strand RNA-induced immune response mediated by type I interferons.

The transfection mix was prepared according to the manual of the StemMACS mRNA Reprogramming Kit (Miltenyi Biotec) pooling two solutions: the first obtained diluting 5X 100 ng/μί. mmRNA of OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28, and nGFP, with stoichiometry 3: 1 : 1 : 1 : 1 : 1 :1 , in transfection buffer solution, and the second diluting 10X transfection reagent in transfection buffer solution. The two solutions were mixed in 2: 1 volume ratio (RiM and SF) or 3: 1 (SM), and the final solution was incubated for 15 min (RiM and SF) or 20 min (SM). Where not specified, SF transfection reagent was used. Transfections using the final mmRNA solution were started at day 0 and daily repeated for at least 12 days. In well, the final mmRNA solution was added dropwise gently rocking the plate, 4 hours prior to daily medium changes with B18R-supplemented Pluriton medium. In microfluidics, the final mmRNA solution was added to different percentages of B18R-supplemented Pluriton medium, pipetted inside a reservoir and automatically perfused inside each channel. Fresh B18R-supplemented Pluriton was added to the reservoir and automatically perfused after a transfection period of 4 h and 12 h thereafter. Experiments aimed at increasing medium change frequency have same mmRNA transfection incubation (4 h) and medium changes every 4 hours.

To compensate for progressive NuFF death during reprog ramming, NuFF-conditioned B18R-supplemented Pluriton medium was used from day 6. Pluriton medium was conditioned daily with 4 ng/mL b-FGF on a separate NuFF culture.

At the end of the transfection series, hiPSC were cultured for two days in Pluriton medium without B18R. Few experiments were stopped at this point for performing immunofluorescence analysis and determining a reprogramming efficiency index, defined as the ratio of the number of double stained NANOG ⁺ /TRA-1-60 ⁺ colonies per 100 target cells seeded. Otherwise, hiPSC colonies were picked and passaged as previously described. Microfluidic hiPSC colonies were collected either by coring the rubber of the microfluidic chip with a biopsy punch or by a preferential detachment using a high flow rate corresponding to a shear-stress of 25 Pa.

Feeder-free reprogramming was performed solely seeding BJ, HFF, SkMF, or RE cells at day -1 and using B18R-supplemented Pluriton medium for the whole duration of reprogramming. RE cells were also kept in RE medium during the first 5 days of reprogramming transfections.

Double-stained colonies for NANOG and TRA-1-60 obtained in microfluidics were catalogued as hiPSC with progressive numbering and labeled thereafter.

Immunofluorescence and colorimetric assays. Immunofluorescence analysis was performed either in conventional wells or microfluidic channels with the same protocols. Cells were fixed in 4% (w/v) paraformaldehyde (Sigma-Aldrich) for 10 min and stained with primary antibodies in 5% goat serum with 0.1 % (v/v) Triton-X-100 (Sigma-Aldrich). Membrane markers were stained without cell permeabilization. Primary antibodies: OCT4 and SSEA-4 (Santa Cruz), NANOG (Reprocell), TRA-1-60 and TRA-1-81 (Millipore), SOX2 (Novus Biologicals); AFP and BRACHYURY-T (Sigma-Aldrich), β-ΙΙΙ- TUBULIN (Abeam), CK18 (GeneTex), ALBUMIN (R&D). Alexa488 or Alexa594 conjugated rabbit or mouse secondary antibodies were used (Life technologies). Nuclei were stained with Hoechst 33342 (Life Technologies). Images were acquired with a DMI6000B fluorescence microscope with motorized stage (Leica Microsystems). The alkaline phosphatase (AP) assay was performed either by a live AP-kit in KnockOut DMEM (both Life Technologies) with a 45-minute incubation, or after cell fixation by AP-staining kit II (Stemgent) with a 10-minute incubation of the staining solution. Glycogen storage analysis was performed by periodic acid-Schiff (PAS) staining (Sigma-Aldrich) following manufacturer's specifications.

Image analysis. Image analysis was performed as described in Luni, C, Michielin, F., Barzon, L, Calabro, V. & Elvassore, N. Stochastic model-assisted development of efficient low-dose viral transduction in microfluidics. Biophys. J. 104, 934-942 (2013). Briefly, pairs of images of Hoechst 33342 stained nuclei and nGFP ⁺ cells were analyzed using the software MATLAB R2012b (The MathWorks). HOECHST images were binary transformed for nuclei localization, after contrast adjustment and morphological filtering. In the detected nuclei positions, mean nGFP fluorescence intensity was evaluated. A background correction was performed to account for differences in cell substrates and in image acquisition conditions. A threshold of fluorescence intensity was chosen to discriminate between nGFP positive and negative cells. A minimum of 3 images per channel/well (>100 cells per image) in a least 5 channels/wells were acquired at 10X magnification for quantification of nGFP ⁺ cells. At least 5 images per channel/well and 5 channels/well were acquired at 20X for quantification of single nucleus fluorescence intensity (>600 cells). Stochastic model. A mathematical model was developed to describe the transfection process of nGFP-encoding mmRNA at different concentrations (from 0.48 to 20 pg/100 cell in microfluidics) and transfection durations (1 , 2, 4 and 12 h). The model is in the form of a stochastic simulation algorithm to capture the discrete nature of mmRNA- containing vesicles and cells (Supplementary Figure 4).

Cells are described as a two-dimensional partially adsorbing boundary layer at the bottom of the culture volume. In each simulation, they are randomly positioned within a regular square grid, covering a 1-mm ² surface on the cartesian plane xz . The grid spacing, Ax and Δζ , was fixed at 57.7 μηι, corresponding to the side of a square having the same area as the average cell, determined experimentally. In the experiments the inventors observed that BJ cells, seeded at a density of 250 cell/mm ² , reached a density of 300 cell/mm ² after 24 h, when transfection occurred, and of 600 cell/mm ² after another 24 h, when nGFP images were taken. Thus, the inventors simulated the transfection process at 300 cell/mm ² and assumed that proliferation rate was not affected by cell transfection.

The stochastic model is discrete both in time and space. A vesicle in position

(x(t), y(t), z(t) in a time interval At will jump to the neighbor position on the lattice with probability P _Jump = DAt/ 5 ² (Erban, R. & Chapman, S. J. Reactive boundary conditions for stochastic simulations of reaction-diffusion processes. Phys. Biol. 4, 16- 28 (2007)), where δ , equal to 10 μηι, is the lattice spacing and the capture boundary layer (Moledina, F. et al. Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells. Proc. Natl. Acad. Sci. 109, 3264-3269 (2012)). At , the time interval simulated, is arbitrary as long as the inequality 2DAt«5 ² ho\ds, where D is the diffusion coefficient of vesicles in medium. D was estimated to be 10 ^"12 m ² /s by Stokes-Einstein equation: D

βπμΙΙ where k _B is Boltzmann constant, T absolute temperature (310.15 K), μ medium viscosity (691.6 10 ^"6 Pa s (Kestin, J., Sokolov, M. & Wakeham, W. A. Viscosity of liquid water in the range -8 °C to 150 °C. J. Phys. Chem. Ref. Data 7, 941 (1978)), approximated by water property), and R the vesicle radius assuming a spherical shape (approximately 300 nm (Russo, L, Berardi, V., Tardani, F., La Mesa, C. & Risuleo, G. Delivery of RNA and its intracellular translation into protein mediated by SDS-CTAB vesicles: potential use in nano-biotechnology. BioMed Res. Int. 2013, (2013)).

Named y the direction perpendicular to the plane of cell culture, the boundary condition at y = 0 (the cell plane) was defined as partially adsorbing: a vesicle is adsorbed with probability P _ads = c _mA k5 and reflected otherwise, where c _mA represents the vesicle concentration in the bulk, and k is a positive constant that describes the reactivity of the boundary and was fitted using transfection experimental data ( = 1.4· 10 ³ μηι ² ^ for RiM, and = 8 · 10 ³ μηι ² ^ for SF). Thus, the overall probability a vesicle enters a cell at the boundary is given by two contributions, the probability of reaching the boundary and the probability of adsorption:

P t.ransf jump ads

provided a vesicle is at distance δ from the boundary at the previous time point simulated.

The characteristic time of diffusion, τ , along the microfluidic culture channel height ( H = 200 μηι) was calculated by Einstein's relation: H ²

2D and it is approximately 5.5 h. Thus, the inventors simulated the process within the capture boundary layer, assuming the medium bulk plays as an infinite source of vesicles for transfections lasting up to 4 hours. While, for a transfection time, t _transf , of 12 hours, the inventors included a multiplicative corrective factor, ε , in (2), defined by the following expression:

H

The model was solved using MATLAB.

RT-PCR and qPCR analyses. Microfluidic channels were first perfused with D-PBS, then with iScript (Bio-Rad) for total RNA extraction. The solution was left in the channels for 2 min before collection. Total RNA was isolated with the RNeasymini kit (Qiagen), treated with DNase (Life Technologies), and quantified using NanoDrop spectrophotometer. RNA (0.1 μg) was reverse transcribed into cDNA (Life Technologies). The list of primers used is available in Table S4. PCR was performed with Platinum Taq polymerase (Life Technologies). Electrophoresis was perfomed in a 2% (w/v) agarose gel with SYBR Green (Life Technologies). qRT-PCR was performed with TaqMan gene expression assay probes (Life Technologies) according to manufacturer's instructions. Reactions were performed on ABI Prism 7000 machine and results were analyzed with ABI Prism 7000 SDS software. GAPDH expression was used to normalize Ct values of gene expression, and data were shown as relative fold change to control cells, using the delta-delta Ct method.

Western blotting analysis. Whole cell lysate was obtained by solubilization of cells in 10 of 5% deoxycholic acid (DOC, Sigma-Aldrich) and Complete protease inhibitor (Roche). PAGE was performed with 4-12% NuPAGE polyacrylamide gels and MOPS buffer (Life Technologies). Proteins were blotted on PVDF membranes (Life Technologies) and detected with Carestream films (Kodak). A 1 : 1000 dilution of primary antibodies and HRP-conjugated secondary antibodies (mouse, Bio-Rad; rabbit, Life Technologies) were used. Whole cell lysate from HES2 embryonic stem cells was used as OCT4 reference.

TGF-β assay. Cell-conditioned media were collected every 24 h during the reprogramming process. In the microfluidic system this corresponds to mixing the medium collected 3 times/day. To activate latent TGF-β, conditioned media were heat- treated for 5 min at 368 K prior to use. CAGA12 SMAD2/3 reporter HaCaT cell line was kindly provided by Stefano Piccolo's Lab (Department of Molecular Medicine, University of Padova), cultured in DMEM supplemented with 10% FBS. For luciferase assay, cells were plated in 24-well plates at 90% confluence and incubated overnight in DMEM without serum. Cells were then treated with conditioned media for 10 h, and supplemented with 1 μΜ TGF-β receptor inhibitor SB431542 (SB, Peprotech) where indicated. Luciferase expression was detected as described in Inui, M. et al. USP15 is a deubiquitylating enzyme for receptor-activated SMADs. Nat. Cell Biol. 13, 1368-1375 (201 1). Data were normalized on total protein content, determined through Bradford assay.

Teratomas. 3* 10 ⁶ cells were harvested, re-suspended in 20 of Matrigel and injected subcutaneously into Rag ^" ' ^" mice. Teratomas were evident 8 weeks after cell implantation. Teratomas were harvested and fixed in 4% PFA, followed by cryo- sectioning. Hematoxylin and eosin staining was performed according to standard protocols.

Karyotype. Q-banded karyotype was performed by the Cytogenetic and Molecular Genetics Laboratory at the University of Brescia (Italy).

Microarray and bioinformatics. A microarray computational analysis was performed on data of temporal progression of human fibroblast reprogramming (GSE50206) using MATLAB. Gene expression values were normalized by the 75th percentile shifts. Genes were selected belonging to the intersection of GO: Extracellular region with TGF- β, WNT, MAPK, HIF and AKT-PI3K pathways (according to KEGG database). Hierarchical clustering was performed with average linkage and Euclidean distance. Normalized data were clustered into 3 clusters using the k-means algorithm.

Microarray analyses performed in this study were executed 48 hours after the last reprogramming mmRNA transfection four freshly derived colonies of comparable size (-8000 cells) were selected both from the microfluidic system and from a parallel reprogramming experiment in well plates. After a 24-h conditioning in StemMACS iPS- Brew XF medium, each colony was split in two halves: the one half (passage pO) was used for total RNA extraction, the other one was further expanded in feeder-free conditions for 3 passages (p3) in well plates before total RNA extraction from another sectioned colony of comparable size (Figure 5a). Each colony was lysed using SuperAmp Lysis Buffer (Miltenyi Biotec) and stored appropriately according to the instructions of the SuperAmp Preparation Kit. The samples were sent on dry ice to Miltenyi Biotec, where amplification, cDNA quantification using ND-1000 Spectrophotometer (NanoDrop Technologies), evaluation of cDNA integrity using a 2100 Bioanalyzer (Agilent Technologies), and microarray analysis were performed. 250 ng of each of the cDNAs were labeled with Cyanine 3 and hybridized (17 hours, 318.15 K) to an Agilent Whole Human Genome Oligo Microarrays 8x60K v2. Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent's Microarray Scanner System (Agilent Technologies). Agilent Feature Extraction Software (FES) was used to read out the microarray image files. Extracted signals were analyzed using GeneSpring v12.6 software (Agilent Technologies). Gene expression values were normalized by the 75th percentile shifts, and baseline-corrected to the median of all samples. Differentially expressed genes between pairs of conditions were found using ANOVA with Tukey post-test (significance set at P<0.05), combined with a two-fold change expression threshold. Differentially expressed genes were checked for functional enrichment using DAVID bioinformatics database (http://david.abcc.ncifcrf.gov/). Principal Component Analysis (PCA) was performed with variance-based weights, and hierarchical clustering with Euclidean distance and nearest-neighbor linkage clustering method, using MATLAB. Microarray data are available at the National Center for Biotechnology Information Gene Expression Omnibus database under the series accession number GSE59534.

Comparison with microarray datasets deposited in public databases (GEO and Synapse Commons Repository) was performed after selection of relevant samples obtained with similar Whole Human Genome Agilent arrays (GSE50206, GSE42445, synl 447097, synl 449098). Single-batch data normalization was performed as above. Only probes common to all the datasets were taken into consideration, and genes whose detection was compromised in at least one sample were excluded from the analysis. After this filtering procedure, 18479 genes were further processed. Datasets merging was performed after batch-effect removal using an empirical Bayes method implemented in ComBat R-code ³⁹ . PCA was then performed in MATLAB as above. Aspecific differentiation. Embryoid bodies (EB). hiPSC colonies were treated with CTK (0.1 mg/mL collagenase IV, 0.25% trypsin, 0.01 M CaCI ₂ , and 0.2% KSR in dH20) for 30 s, mechanically scratched with a serological pipette and resuspended in EB medium (DMEM/F12, 20% knockout serum replacement, 1 % L-glutamine, 1 % NEAA, β-mercaptoethanol, all Life Technologies). EB were cultured in ultra-low adhesive plates (Corning) for 20 days and then transferred on custom-made PDMS micro-wells with a Matrigel (BD) coated glass bottom. Characterization was performed after 6 days. Aspecific differentiation in monolayer. EB medium (without β-mercaptoethanol) was used to aspecifically differentiate hiPSC colonies for 6 days. In microfluidics differentiation was performed perfusing the medium every 12 h. In wells medium was changed every 48 h. Early-germ layer differentiation. hiPSC were differentiated in the following germ- layer-specifing media. Ectoderm. DMEM/F12, 1 % NEAA, 1 mM L-glutamine, 0.1 mM beta-mercaptoethanol, 20% KnockOUT serum replacement (all Life technologies), 1 uM dorsomorphin. Mesoderm. Supplemented StemPro-34 (Life Technologies), 5 ng/mL b-FGF, 2 mM L-glutamine, 50 μg/mL ascorbic acid, 150 μg/mL transferrin, 0.3 ng/mL Activin-A, 10 ng/mL BMP-4, 46 μg/mL methyl-thio-glycerol. Endoderm. Days 1-2, RPMI, 2% B27 supplement, 100 ng/mL Activin-A, 50 ng/mL Wnt3a. Days 3-5, RPMI, 2% B27 supplement, 100 ng/mL Activin-A.

Cardiac differentiation. Small molecules were used to promote cardiac differentiation of hiPSC colonies. RPMI with B27 without insulin (cardiac basal medium, CBM, Life Technologies) with 10 μΜ CHIR99021 and was perfused in microfluidics every 12 h for the first 24 h. Thereafter the medium was changed every 24 h. CBM was used in the following 36 h and CBM with 4 μΜ IWP-4 in the next 24 h. CBM was then used until cardiac maturation at day 14 from the beginning of differentiation protocol. Medium change in wells was performed only at changes of medium composition as above.

Hepatic differentiation. hiPSC colonies obtained in microfluidics were maintained in StemMACS iPS-Brew XF medium to grow over the channel surface. RPMI-B27 was supplemented with 100 ng/ml Activin-A and 0.5 mM sodium butyrate for 3 days. Medium was changed to KO-DMEM, 20% Serum Replacement (both from Invitrogen), 1 mM L-glutamine, 1 % NEAA, 0.1 mM b-mercaptoethanol, 1 % DMSO (Sigma-Aldrich) for 6 days. Hepatic-like cells were maturated with L15 medium (Sigma-Aldrich) supplemented with 8.3% FBS, 8.3% tryptose phosphate broth, 10 μΜ hydrocortisone 21-hemisuccinate, 1 μΜ insulin (all from Sigma-Aldrich) and 2 mM L-glutamine containing 10 ng/ml hepatocyte growth factor and 20 ng/ml oncostatin M (both from R&D) for 6 days. Medium was changed every 12 h in the microfluidic system, and every 24 h in wells. Statistical analysis. For statistical analyses, single pairwise comparisons were analyzed using Student's t-test with P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***), indicating significance. Multiple comparisons were performed by one-way ANOVA with Tukey post-test, with P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***), indicating significance. Values are expressed by means and standard deviations (s.d.). The variability of reprogramming efficiency with different cell batches within different microfluidic devices and channels was verified by nested ANOVA, using Minitab 17 statistical software. Throughout the text, n indicates the number of replicates, referring to a combination of independent experiments performed at least in 2 different chips or in 2 different batches of cells.

Efficiency of the a pre-prepared cryo-preserved reprogramming solution

The reprogramming efficiency was tested with a pre-prepared cryo-preserved transfection solution (i.e. a reprogramming solution wherein transfection is the selected transformation protocol). Such a solution is useful for gaining time as well as for working in a closed and automatized microfluidic network. The reprogramming solutions for transfection were conserved as frozen solution at different temperatures. Conservation at +4°C is not possible because of the mmRNA instability. Usually the transfection solutions are prepared immediately before transfecting (freshly prepared). The transfection solutions is made by mixing mmRNA, the transfection reagent, the transfection buffer and the medium for cell culture as follow: the transfection mix was prepared according to the manual of the StemMACS mRNA Reprogramming Kit (Miltenyi Biotec) pooling two solutions: the first obtained diluting 5X 100 ng/μ-. mmRNA in transfection buffer solution, and the second diluting 10X transfection reagent in transfection buffer solution. The two solutions were mixed in 2: 1 volume ratio (RiM and SF) or 3: 1 (SM), and the final solution was incubated for 15 min (RiM and SF) or 20 min (SM). Where not specified, SF transfection reagent was used.

To avoid daily preparation of transfection solution, cryo-preservation of the transfection solution prepared once, in advance has been tested. The transfection solution was transferred into the aliquot holder. Each holder was fast- cooled using systems at different temperatures: -20°C, -80°C and -186°C. The transfection efficiency between freshly prepared and the different frozen transfection solutions was compared.

The results clearly showed that the efficiency of frozen solutions at both temperatures (-80°C and -186°C) is only reduced by 50% in comparison to the efficiency obtained with the fresh solution. Aliquots frozen at -20°C did not show any transfection activity. These results allow obtaining high-efficiency reprogramming also using frozen transfection aliquots. In particular, quantitative analysis shows that the efficiencies of transfection at the particular conditions of the experiments are: 32±5% for fresh transfection (upper panel); 15±2% for transfection solution frozen at -80°C (middle panel) and 16±2% for transfection solution frozen at -186°C (lower panel).

The experiments have been performed with the following methods. BJ cells (human fibroblasts) have been seeded within microfluidic channels at a cell density of 5 cell/mm ² with their culture medium (DMEM+10%FBS). The transfection solutions were frozen 20 hours before the transfection. After 24 hours, the cells have been transfected for 4 hours with 17.2 pg/cell of nGFP mmRNA. 20 hours after the transfection, the cells have been incubated with Hoechst for 5 minutes and fluorescence images of nGFP and Hoechst have been acquired. The transfection efficiency has been calculated by the ratio between nGFP positive cells over the total number of cells (represented by Hoechst positive cells).

Microarray data are deposited in the Gene Expression Omnibus (GEO) database with accession no. GSE59534.