METHODS OF CULTURING MESENCHYMAL STROMAL CELLS

FIELD OF THE INVENTION The invention relates to mesenchymal stromal cells (MSCs) and methods for culturing the same.

BACKGROUND OF THE INVENTION

Mesenchymal stromal cells (MSCs) are defined as adherent cells that do not express hematopoietic markers and can undergo chondrogenic, adipogenic and osteogenic differentiation. They are typically isolated from the bone marrow, adipose tissue, placental tissue and umbilical cord tissue and blood, but they can be isolated from almost every tissue in the body ^{1, 2} . To date, MSCs have been shown to be immunomodulatory, enable engraftment of hematopoietic cells, and secrete trophic factors that support angiogenesis, cell survival, and anti-fibrosis ¹ . MSCs are considered good therapeutic candidates for treatment of degenerative and inflammatory diseases due to this multifunctional ability. Thus, there is enormous interest in translating MSC research into successful clinical products. Currently, there are four approved commercial products and over 600 registered global clinical trials worldwide. Although, MSCs have demonstrated pre-dinical efficacy in a wide variety of disease indications, their efficacy in clinical studies has been equivocal ³ . Most pre-clinical studies, and virtually all phase II trials, have relied on early passage MSCs produced on a limited scale, whereas late stage trials, of necessity, have required larger expansion protocols.

Typically, early phase MSC trials have relied on 2 dimensional (D) planar technologies using tissue culture flasks to produce between 100-200 million cells for 10-20 patients ⁴ . Later-phase trials have used a combination of planar technologies and more scalable technologies including microcarrier-based systems in a stirred tank ⁵ or hollow-fiber bioreactors ⁶ , compact multi-layers (HYPERStack) ⁷ or multi-layer bioreactors (Integrity Xpansion unit) ⁷ . However, large scale production in either planar or scalable systems requiring frequent passaging, with or without cryopreservation, appears to have resulted in sub-optimal performance of MSCs ^{8, fl} , in part explaining the poor performance of MSCs in phase ill clinical trials in several diseases, including ulcerative colitis and ischemic stroke ¹⁰ (Athersys), cardiac repair ¹¹ (3 studies by Milteny Biotec) and acute kidney injury ¹² (Allocure). Thus, there is a need to generate ready-to-use, high quality and improved- potency MSCs to capitalize on the research investment, and therapeutic potential of these cells. 3D cell organization provides enhanced cell-cell interactions and closely mimics the natural microenvironment of a tissue, compared with traditional 2D monolayer cultures and have been shown to improve anti-inflammatory, immunodmodulatory, and anti-tumor properties of MSCs ( Petrenko et al. Stem Cell Research & Therapy (2017) 8:94). However, these effects are lost when MSCs are grown in serum-free conditions ( Petrenko et al.Stem Cell Research & Therapy (2017) 8:94) Currently, most studies on the evaluation of MSC properties in 3D spheroid cultures employ fetal bovine serum (FBS) as a classical culture media supplement. However, the utilization of animal-derived .products during the manufacturing of cellular therapeutics for human use is not recommended. Only a few studies have so far been published on investigation into the effect of FBS substitutes of human origin or chemically defined medium on the MSC spheroid generation and functional properties of spheroid-derived cells. Recently, Yloslato et al. (Cytotherapy. 2014;16:1486-500) showed that cell activation in a 3D environment depends critically on the culture medium. Spheroids can also be formed in chemically defined xeno-free medium, but the composition of the medium is critical for assembly of the cells into compact spheres and the patterns of genes expressed by MSCs. Therefore, the question about the optimization of xeno-free 3D culture conditions for the preparation of MSCs products with enhanced therapeutic properties is highly relevant. Besides adaptation of the cell culture environment and media composition, the widespread application of 3D MSCs culturing requires development of efficient large-scale 3D fabrication methods (Petrenko et al.Stem Cell Research & Therapy (2017) 8:94). Various 3D generation techniques have already been established. Among these, the most widespread are the hanging drop approach, application of low-adhesive substrates, membrane-based aggregation and the forced aggregation method. Use of these methods for large-scale production of cellular 3D MSCs is based on the limited area of standard culture dishes, is labor-intensive processes of establishment, and the harvesting of generated 3D cultures, is economically ineffective, membrane-based method leads to high variations in size , morphology, and function of the produced 3D MSCs.

Application of such high-throughput approaches represents unique opportunities in drug discovery applications and toxicology studies; however, clinical use of 3D MSC will probably require significantly higher cell (or 3D MSCs) numbers, and thus the developed technology in the patent application provides highly reproducible, simple and cost- effective technique for large-scale production of these novel 3D MSCs.

SUMMARY OF THE INVENTION

In an aspect, there is provided a method for culturing mesenchymal stromal cells (MSCs) comprising: a) providing a population comprising mesenchymal stromal cells; and b) exposing the population under conditions that allow the MSCs to self-aggregate and in the presence of IL-6, or an IL-6 analogue, to produce culture engineered MSCs (ce-MSCs), wherein the conditions are serum free.

In an aspect, there is provided a method for culturing mesenchymal stromal cells comprising: a) providing a population comprising mesenchymal stromal cells; and b) exposing the population in a low attachment container in the presence of IL-6, or an IL-6 analogue, to produce culture engineered MSCs (ce-MSCs). In an aspect, there is provided a method for culturing enhancing mesenchymal stromal cells comprising: a) providing a population comprising mesenchymal stromal cells; and b) exposing the population in an impeller based bioreactor in the presence of IL-6, or an IL-6 analogue, to produce culture engineered MSCs (ce-MSCs.

In an aspect, there is provided MSCs preparing using the method described herein. In an aspect, there is provided a method of treating inflammation comprising administering to a patient in need thereof, the ce-MSCs described herein. Inflammation in a patient may be associated with, for example, acute respiratory distress syndrome.

In an aspect, there is provided a method of treating a degenerative disease, such as osteoarthritis, comprising administering to a patient in need thereof, the ce-MSCs described herein.

In an aspect, there is provided a method of treating cancer comprising administering to a patient in need thereof, the ce-MSCs described herein.

In an aspect, there is provided a use of the ce-MSCs described herein in the preparation of a medicament for the treatment of an inflammatory, degenerative or cancerous diseases.

In an aspect, there is provided a use of the ce-MSCs described herein for the treatment of an inflammatory, degenerative or cancerous diseases.

In an aspect, there is provided the ce-MSCs described herein for use in the treatment of an inflammatory, degenerative or cancerous diseases.

BRIEF DESCRIPTION OF FIGURES

These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:

Figure \\ BM-ceMSCs show decreased expression of important surface markers for cell-cell interaction - CD 106, H LA-ABC, and CD54 surface markers are analyzed by flow cytometry. 3 MSCs donors are tested; representative sample is shown. Histogram plot with fluorescent intensity (A, B, C) of each marker is shown, black border fill - isotype control, gray border fill - BM-ceMSCs, black border and gray fill - naive MSCs. (D) Mean fluorescent intensity (MFI) in BM-ceMSCs for each marker are similar to the isotype control. Each point represents one donor. · - CD106,■ - CD54, o - HLA-ABC,— - is calculated mean MFI value of 3 donors.

Figure 2 -BM-ceMSCs immunophenotype and trilineage differentiation potential remains unchanged - Characterization of BM-ceMSCs and n-MSCs for cell surface markers is done by flow cytometry (CD90+, CD105+, CD73+, CD14-, CD19-, CD34-, CD45-, and HLA-DR-) and trilineage differentiation potential (Fig 9) by phase contrast microscopy showing unchanged MSC identity (n=3 MSC donors; Table data and image representative of 1 MSC donor; image magnification is 10x).

Figure 3: BM-ceMSCs show increased TSG-6 expression - BM-ceMSCs show significant increase in anti-inflammatory marker, TNF-a stimulated gene 6 (TSG-6) expressions. 3 MSCs donors are tested.■ - MSC donor 1 , T - MSC donor 2, · - MSC donor 3. y-axis show values relative to β2 microglobulin housekeeping gene for

each different conditions on x-axis.

Figure 4: Significant subsiding of paw edema in mice treated with BM-ceMSCs - (A) Paw thickness is measured at different time points for all the treatment groups (n-11 /group, n=5 per time point) post cell injection (· - LPS control,■/□ - Donor 1/Donor 2 BM-ceMSCs, Donor 1/Donor 2 n-MSCs, o - PBS control; * = p<0.005). (B) Dunnett test is used to compare each treatment (BM-ceMSCs, naive MSCs) with a single control (LPS treated). p=0.025 for BM-ceMSCs vs. control and p=0.23 for naive MSCs vs. control. (C) Shows inflammatory infiltration rating from 1-10 (1- low infiltration; 10- high infiltration) by 4 independent blinded observations of the HE histology slides. Black bar - naive MSCs, light gray bar - LPS control, black pattern bar - BM-ceMSCs, light gray pattern bar - PBS control.

Figure 5: Differential in-vivo bio-distribution of BM-ceMSCs - BM-ceMSCs show significant differential tissue distribution vs naive MSCs. Number of human DNA copies/Sng/μL total DNA in lung tissue for BM-ceMSCs is lower than naive MSCs. qPCR done in triplicates for 2 donor MSCs in paw edema model. Black solid bars - naive MSCs, black pattern bar - BM-ceMSCs. (*- p<0,05; # - below minimum detection limit) Figure 6 Significant reduction of peritoneal inflammation in zymosan-induced peritonises model by BM-ceMSCs vs. naiVe MSCs: (A) inflammatory MHCII+ monocyte/macrophages (indicative of pro-inflammatory monocytes/macrophages) levels at 24 hours post zymosan (0-1 mg/mL) treatment; n=4 animals / group; BM-MSCs - zymosan + naive BM-MSCs; ceBM-MSCs - zymosan + ceBM-MSCs; error bar - 95% CI (B) pro-inflammatory cytokine levels in zymosan-induced peritonises model by BM- ceMSCs: At 4 hours post-zymosan (0.1mg/mL) treatment; n=4 animals/group; * - p<0.05 vs. nMSCs group; # - p<0.05 vs. zymosan only group; Delta change in treatment groups (Zymosan only, nMSCs, ceMSCs) is relative to the no treatment group. Figure 7: BM-ceMSCs are more immunoevasive than naive MSCs - BM-ceMSCs from 3 donors are analyzed for their immunoevasiveness when co-cultured with IL-2 stimulated natural killer (NK92) cell line using ^s1 Cr Cytolytic assays (40:1 NK92: MSC ratio). Solid black bar - naTve MSCs, black pattern bar - BM-ceMSCs. Percentage lysis is calculated using the formula: (E - S)x100/(M - S), where E is the ⁵¹ Cr release ( ⁵¹ Cr present in supernatant) from an experimental sample, S the spontaneous release in the presence of complete medium and M the maximum release upon cell lysis with 10% Triton X-100. Data is presented as the mean percentage lysis of triplicate samples with 95% confidence interval (CI).

Figure 8 - CD4+ Th cell inhibition is conserved between BM-ceMSCs and naUVe MSCs. There is no statistically significant difference between licensed BM-ceMSCs and na ^' ive MSCs in terms of inhibition of Th cell proliferation. Naive MSCs are allowed to recover for 3-5 days post-thaw, and are tested under licensed (TNF-a, IFNy, TNF-a+IFNv) conditions. Closed circles - Positive (CD4+ Th cell with activation beads); open circles - (CD4+ Th cells without activation beads); closed triangles - BM-ceMSCs + TNF; open triangles - naive MSCs + TNF; closed diamonds - BM-ceMSCs + IFN; open diamonds - naive MSCs + IFN; closed squares - BM-ceMSCs + TNF & IFN; open squares - naive MSCs + TNF & IFN; data shown for 3 MSCs donors in triplicate testing (9 data points on graph); 1 CD4+ Th cell donor; * - p<0,05; error bar - 95% CI

Figure 9: Immunomodulatory effects of MSCs with and without early vs. late osteoarthritic (OA) synovial fluid (SF) - SF irrespective of the OA stage inhibits CD4+ Th cell proliferation (1.4 fold change for OA SF-activated vs. 2 fold change for activated vs. non-activated controls; p=0.002). BM-ceMSCs did not show any positive or negative effects on activated Th cells proliferation (1.65-fold change for OA SF-BM-ceMSCs vs. 1.4-fold change for OA SF-Activated; p>0.05). Naive MSCs however show stimulatory effects on activated Th cells proliferation (2.3 fold change for OA SF-na ^' iVe MSCs vs. 1.4 fold change for OA SF-Activated, p=0.020). There is thus additional inhibition of activated Th cell proliferation by BM-ceMSCs (1.65 fold change) vs. naive MSCs (2.3 fold change) p=0.004 in the presence of SF from OA patients. · - no activation (CD4+ Th cell without activation beads), ■ - activation (CD4+ Th cell with activation beads), OA SF no

activation, OA SF activation,♦ - OA SF BM-ceMSCs, o - OA SF na ^' ive MSCs. y-axis show fold expansion of CD4+ Th cells post seeding from day 0 to day 4. Assay is done at 1 :20 (MSCs:Th cell) ratio. Naive and ceMSCs controls were tested separately in different experiment (Fig 8), wherein both na ^' ive and ceMSCs inhibited CD4+ T cell proliferation without any stimulatory effects. Figure 10: Immediately post-thaw, BM-ceMSCs are more immunomodulatory than their naive MSC counterparts - 3 MSCs donors are tested for their CD4+ Th cell immunomodulatory properties at 1:20 (MSCs:Th cell) ratio. Data shows≥ 3 fold decreases in CD4+ Th cell proliferation with BM-ceMSCs vs. naTve MSCs / activated Th cell control. Solid black bar - positive (CD4+ Th cell with activation beads), Solid white bar - negative (CD4+ Th cells without activation beads), gray solid bars - naTve MSCs, black pattern bars - BM-ceMSCs. Cells are tested immediately post-thaw, thawing is done at 37"C in water bath, cryo media is removed by 1x washing with PBS. (*=p<0.05). y-axis show fold expansion of CD4+ Th cells post seeding from day 0 to day 4.

Figure 11 : ceMSCs show improved polarization of monocytes/macrophages to M2 subtypes vs. naive MSCs. (A,C) BM-derived MSCs; (B,D) Umbilical Cord Tissue (UCT)- derived-MSCs. (A, B) MSCs (BM-ceMSCs vs. nMSCs, n =3 independent donors) were co-cultured at 3:1 monocyte/macrophage:MSC ratio. M2 subtype is defined as CD163 high and CD206 high positive cells; M1 subtypes are defined as CD86 high and HLA-DR high. Black bar - BM-ceMSCs; gray bar - nMSCs; Μφ - na ^' ive monocytes/macrophage. Error bars - 95% CI. (C, D) ceMSC-polarized monocytes/macrophages secrete higher levels of IL-10; N=3 MSC donors are tested in sextuplicate. MSCs (BM-ceMSCs & riMSCs) were co-cultured at 3:1 monocyte/macrophageiMSC ratio. ΜΘ - nalVe monocyte/macrophage; Open circles (C) & gray bar (D) - ΜΘ + nMSCs; open squares (C) & black bars (D) - ΜΘ + BM-ceMSCs. Error bars - 95% CI

Figure 12: ceWISCs inhibit monocyte/macrophage polarization to inflammatory subtypes more effectively than na ^' ive MSCs. (A) BM-derived MSCs; (B) UCT-derived MSCs; (C) Adipose tissue (AT)-derived-MSCs; n=3 MSCs donor for BM and UCT; n ~2 MSCs donors for AT. Open circles - MO - naive monocyte/macrophages; black circles - M1 - inflammatory type monocyte/macrophages; open/black circles M2 - antiinflammatory type monocyte/macrophages. MSCs (BM-ceMSCs & nMSCs) were co- cultured at 3:1 monocyte/macrophage:MSC ratio under pro-inflammatory conditions; y- axis shows Reactive oxygen species ROS levels expressed as relative luminance units measured using Lucigenin (Sigma) luminescence assay (Yamazaki T, et al., Tropical Medicine and Health. 201 1 ;39(2):41-45. Error bars - 95% CI

Figure 13: ceMSCs functional properties remain unperturbed with changes in basal manufacturing medium (A) BM-derived MSCs; (B) UCT-derived MSCs; (C) Adipose tissue (AT)-derived~MSCs; n=3 MSCs donor for BM and UC; n =2 MSCs donors for AT. Open circles - MO - na ^' ive monocyte/macrophages; black circles - M1 - inflammatory type monocyte/macrophages; open/black circles M2 - anti-inflammatory type monocyte/macrophages. MSCs (BM-ceMSCs & nMSCs) were co-cultured at 3:1 monocyte/macrophage.'MSC ratio under pro-inflammatory conditions; y-axis shows ROS levels expressed as relative luminance units. Error bars - 95% CI

Figure 14: BM-ceMSCs show increased chemotaxis to TNFα stimuli: BM-ceMSCs show significant migration toward TNF-a (6ng/mL). Data shown is of 3 MSC donors. The centre of mass (COM) of n=30 cells is represented by the spatial average of all cell positions. The difference in the COM between initial values (0 minute) and at the end (72hours) of the experiment is termed the displacement of COM. Images are taken at 0 minute, 15 minute, 30 minute, 60 minute, 2h, 4h, 6h, 24h, 48h, and 72h post TNF-a exposure. *=p<0.05, **=p<0.01 , solid black bars = na ^' ive MSCs, black pattern bars = BM- ceMSCs. Figure 15: Differential gene expression profile of BM-ceMSCs vs. naive MSCs: BM- ceMSCs show increased gene expression of antHnflammatory, immunomodulatory, and anti-tumor genes vs. nMSCs. N= 3 MSCs donors are tested in duplicate. Blue dots - na ^' fve MSCs; red bars - BM-ceMSCs; data shown as

Figure 16: Differential gene expression profile of UCT-ceMSCs vs. na ^' fve MSCs: ceUCT-MSCs show increase gene expression of anti-inflammatory, immunomodulatory, and anti-tumor genes vs. nMSCs. N= 3 MSCs donors are tested in duplicate. Blue dots - nMSCs; red bars - BM-ceMSCs; data shown as

Figure 17: Differential gene expression profile of AT-ceMSCs vs. naive MSCs: ceAT- MSCs show increase gene expression of anti-inflammatory, immunomodulatory, and antitumor genes vs. nMSCs. N=2 MSCs donors are tested in duplicate. Blue dots - nMSCs; red bars - BM-ceMSCs; data shown as

Figure 18: BM-ceMSCs show differential miRNA expression - BM-ceMSCs show increase in miR-16 (known anti-angiogenic marker) levels (10 fold) relative to na ^' fve MSCs. No induction or inhibition is seen for miRs (21 , 24, 26a and 27a) known to be involved during differentiation of MSCs. y-axis show miR fold induction (solid black bars) in BM-ceMSCs relative to naive MSCs for each different miR on x-axis.

Figure 19: There appears to be no proliferative advantage of BM-ceMSCs vs. naive MSCs: BM-ceMSCs and nMSCs are seeded at different densities under nMSCs conditions. After 6 days of culture total DNA content is measured by spectrophotometry. Gray bar - nMSCs; open bars - BM-ceMSCs. N=3 BM-MSCs tested in triplicate. Error bar - 95% CI.

Figure 20: Change in cancer cell numbers in co-culture with BM-ceMSCs, but not na ^' ive MSCs (A) Cancer cell numbers in coculture with BM-ceMSCs: Data is expressed in terms of the average fold change relative to the initial seeding density of 20 000 cells (denoted by dotted line). (B) nMSC and ceMSC proliferation in coculture with cancer cells. Data is expressed in terms of the average fold change of the initial seeding density of 2000 cells (denoted by dotted line); open bar - MSCs alone; dash bar - K562 + MSCs. Error bars - 95% CI. N=3 bone marrow MSCs donors are tested in quadruplicates.

Figure 21 : BM-ceMSCs inhibit proliferation of tumor cell lines more effectively than naive MSCs: (A) Cell cycle arresting effects of BM-ceMSCs on HT-1080 (Skin), SKOV3 (Overy), and MCF-7 (breast) tumor cell lines. Tumor cell were co-cultured with BM-derived MSCs (nMSCs and BM-ceMSCs) at the indicated tumor cell : MSCs ratios. N=3 bone marrow MSCs donors, (B) dot plot graph showing cell cycle arresting effects of BM- ceMSCs on K562 tumor cell iine. Square - K562 alone; circles - K562 + nMSCs; triangles - K562 + BM-ceMSCs; Tumor cells : MSCs ratio - 10:1. Error bars - 95% CI

DETAILED DESCRIPTION

In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details. MSCs have shown tremendous therapeutic potential in pre-clinical studies, although late- phase clinical trials have recently faced some failures. It is important to improve MSCs quality, potency and scalability for human use, in an economic and compliant manner.

There is described herein, a novel cost-effective, minimal manipulation culture engineering method that enhances MSCs anti-inflammatory properties by physical (self- assembly aggregation) and cytokine-based stimulation. These culture engineered MSCs (ce-MSCs) were tested for improved anti-inflammatory, improved chemotactic, improved immunomodulatory properties in vitro, and in vivo acute inflammatory models; additionally ce-MSCs show an activated state of gene expression which is preserved even after cryo preservation and immediately, post-thaw. Ce-MSCs were shown to have improved anti-inflammatory and immunomodulatory effects in vivo in acute inflammation models. ceMSCs were tested for their improved in vitro r migratory capability and shown to have differential biodistribution in vivo relative to none culture engineered MSCs. Ce-MSCS have been derived from bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), and have all shown an activated state of gene expression, and suppressed polarization of monocytes/macrophages to M1 sub-types. , Specifically, cultured engineered MSCs (ceMSCs) show higher (>3 fold) expression of TSG-6 protein vs. naive MSCs (nMSCs), and greater polarization of monocytes (ceMSCs -≥20 fold vs, nMSCs - ≥9 fold vs. control). In vivo administration in an acute paw edema model shows reduced paw edema and immune cell infiltration (ceMSC - 3.5 score; nMSCs - 10 score; Control - 8 score; n=13, p<0.005). ceMSCs reduced levels of inflammatory MHC-II positive monocoytes and pro-inflammatory cytokine in lavage fluid vs. nMSCs (p<0.005) in zymosan induced peritonitis model (n=12). Activation, pre-freeze, is maintained during the cryopreserved state, as evidenced by increased TSG-6 protein expression and decrease in T helper cell proliferation (≤1 fold) in freshly thawed ceMSCs vs. freshly thawed nMSCs. The technology can also be used post thaw, which restores potency of cryopreserved MSCs to levels observed with fresh, non-frozen ceMSCs Cultured engineering of three tissue (Bone marrow, umbilical cord tissue, and adipose tissue) derived MSCs shows inhibition of monocyte/macrophage polarization to inflammatory subtypes more effectively than naive MSCs. Functional properties remain unperturbed with changes in basal manufacturing medium for ceMSCs derived from three tissue source (Bone marrow, umbilical cord tissue, and adipose tissue). ceMSCs vs. nMSCs are fundamentally in an activated state, based on gene expression profiling data. ceMSCs show increased polarization of monocyte/macrophages to M2 subtypes (19-fold increase) relative to na ^' fve MSC-mediated polarization of monocyte/macrophages (8 fold increase). Importantly, using our culture engineering method increases cell cycle arrest of a tumor cell line (HT- 1080, K562, SKOV3, MCF7) in G0/1 cell cycle phase (4 fold) and decrease in G2/M cell cycle phase (3-fold) when co-cultured with ceMSCs relative to n-MSCs. The ceMSCs demonstrated superior, off-the-shelf, ready-to-use properties that are important for their therapeutic utility. Our method therefore provides a reasonable solution to improve MSC potency without dramatically altering costs or regulatory approval pathway for autologous and/or allogeneic MSCs. Our approach also provides an alternative to costly culture rescue methods in improving posi-thaw efficacy of MSCs. In an aspect, there is provided a method for culturing mesenchymal stromal cells (MSCs) comprising; a) providing a population comprising mesenchymal stromal cells; and b) exposing the population under conditions that allow the MSCs to self-aggregate and in the presence of IL-6, or an IL-6 analogue, to produce culture engineered MSCs (ce-MSCs), wherein the conditions are serum free.

As used herein, mesenchymal stromal cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells). Characterization of cells as MSCs is well understood in the art and is described, for example in Dominici et al„ 2006 Ovotherapy.

IL-6 analogues include, without limitation, hyper-IL6 (Fischer M, et al„ (1997) Nat. Biotechnol. 15:142-145), IL-6 receptor activators such as lipopolysaccharides (Najar, Mehdi et al. Immune Network 17.2 (2017): 89-102) and IL-6 receptor antibodies.

In an embodiment, the IL-6 is present at 0.1 - 15 ng/ml.

In some embodiments, the exposing is for less than 7 days, for about 2-3 days, or for about 4-5 days or for≤24 hours. In some embodiments, the population is at a concentration of 750-2000 cells/cm ² , 2000- 3000 cells/cm ² , or 3000-5000 cells/cm ² .

In some embodiments, the conditions allow the MSCs to loosely self-aggregate. In some embodiments, the conditions that allow the MSCs to self-aggregate is enhancing in a low- attachment container. In an embodiment, the low-attachment container comprises a Corning® Ultra-Low Attachment surface or Nunc® low cell binding surface, in an embodiment, the low-attachment container has at least a 70%, 80%, 90%, 95%, 99%, 99.5%, 99.8%, or 99.9% reduction in cell attachment compared to a Corning ® standard surface treated tissue culture plate, when seeded with 2.6 x 10 ⁵ cells/well of a 6 well plate- Loose aggregation removes the need for enzymatic dissociation and/or vigorous mechanical dissociation to generate single cells. Single cells can be readily generated using simple mechanical agitation or mixing or shaking or gentle pipetting. This provides highly reproducible, simple and cost-effective technique for large-scale production of 3D MSCs. Our loose aggregation approach may advantageously a) remove the limitations of approaches currently being used such as hanging drop approach, application of low- adhesive substrates, membrane-based aggregation and other forced aggregation-based methods. Limitations of the use of these methods for large-scale production of cellular 3D MSCs is based on the limited area of standard culture dishes, involves labor-intensive processes of establishment and harvesting of 3D cultures, is economically ineffective at clinical and commercial scale, membrane-based method leads to high variations in size , morphology, and function of the produced 3D MSCs. Further these other methods may have limited clinical utility for systemic infusions as there is a risk of pulmonary emboli with infusion of non-dissociated or partially dissociated forced aggregates that are tightly held. In some embodiments, the conditions that allow the MSCs to self-aggregate is an impeller based bioreactor, such as the PBS™ bioreactor system, Quantum® Cell Expansion System, Mobius® 3 L Bioreactors or equivalent systems, preferably under serum free formulation media conditions and which is independent of serum free basal media formulations used. In some embodiments, the population comprising MSCs is a population of substantially all MSCs.

In some embodiments, the population comprising MSCs is a population comprising na ^' /ve MSCs.

In some embodiments, the method further comprises selecting adhered ce-MSCs. In some embodiments, the method further comprises cryopreserving the ce-MSCs.

In some embodiments, the method further comprises cryopreserving the na ^' iVe MSCs. The cryopreserved na ^' fve MSCs may be thawed before activating under culture engineering conditions as described above. In some embodiments, the enhancement may be done for ≤5 days,≤3 days,≤2 days, or≤24 hours. In an aspect, there is provided a method for culturing mesenchymal stromal cells comprising: a) providing a population comprising mesenchymal stromal cells; and b) enhancing the population in a low attachment container in the presence of IL-6, or an lL-6 analogue, to produce culture engineered MSCs (ce-MSCs). In an aspect, there is provided a method for culturing mesenchymal stromal cells comprising: a) providing a population comprising mesenchymal stromal cells; and b) enhancing the population in an empeller based bioreactor in the presence of IL-6, or an IL-6 analogue, to produce culture engineered MSCs (ce-MSCs). In some embodiments, the MSC are obtained from different tissue sources including but not limited to bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, placenta, amniotic fluid, dental pulp, synovium tissue.

In an aspect, there is provided MSCs preparing using the method described herein.

In an aspect, there is provided a method of treating inflammation comprising administering to a patient in need thereof, the ce-MSCs described herein. Inflammation in a patient may be associated with, for example, acute respiratory distress syndrome.

In an aspect, there is provided a method of treating a degenerative disease, such as osteoarthritis, comprising administering to a patient in need thereof, the ce-MSCs described herein. In an aspect, there is provided a method of treating cancer comprising administering to a patient in need thereof, the ce-MSCs described herein.

Many cancers might be treatable by the MSCs described herein. Cancers include adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/ens tumors, breast cancer, castleman disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (gist), gestational trophoblastic disease, hodgkin disease, kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia (acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, chronic myelomonocytic), liver cancer, lung cancer (non-small cell, small cell, lung carcinoid tumor), lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer (basal and squamous cell, melanoma, merkel cell), small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor. In some embodiments of the treatment method, the ce-MSCs have not been previously cryopreserved.

In some embodiments of the treatment method, the ce-MSCs have been previously cryopreserved. Preferably, the ce-MSCs are administered 5 days or less, 4 days or less, 3 days or less, 2 days or less, 1 day or less, or less than 1 day after thawing. In an aspect, there is provided a use of the ce-MSCs described herein in the preparation of a medicament for the treatment of an inflammatory, degenerative or cancerous diseases.

In an aspect, there is provided a use of the ce-MSCs described herein for the treatment of an inflammatory, degenerative or cancerous diseases. in an aspect, there is provided the ce-MSCs described herein for use in the treatment of an inflammatory, degenerative or cancerous diseases.

In certain embodiments, the ceMSCs demonstrate 40 - 80 fold decrease in expression of important cell-cell interaction surface marker CD106, vs. na ^' ive MSCs (Fig 1A, D).

In certain embodiments, the ceMSCs demonstrate 30 - 50 fold decrease in expression of important cell-cell interaction surface marker HLA-ABC, vs. na ^' ive MSCs (Fig 1 B, D).

In certain embodiments, the ceMSCs demonstrate 30 - 50 fold decrease in expression of important cell-cell interaction surface marker CD54, vs. naive MSCs (Fig 1C, D).

In certain embodiments, the ceMSCs do not show changes in identity based on immunophenotypic markers CD90 (99.9%), CD105 (98%), CD73 (99.9%), CD14 (0.2%), HLA-DR (0.1 %), CD34 (0.1%), thus meeting the minimal definition of MSCs (Dominici et al., 2006 Cyotherapy) (Fig 2). In certain embodiments, the ceMSCs do not show changes in tri-lineage differentiation potential (chondrocytes, osteocytes, or adipocytes) thus meeting the minimal definition of MSCs (Dominici et al., 2006 Cyotherapy) (Fig 2).

In certain embodiments, the ceMSCs demonstrate 30 - 50 fold decrease in expression of important cell-cell interaction surface marker CD54, vs. naive MSCs.

In certain embodiments, the ceMSCs demonstrate 40 - 80 fold decrease in expression of important cell-cell interaction surface marker CD106, vs. naive MSCs.

In certain embodiments, the ceMSCs demonstrate 30 - 50 fold decrease in expression of important cell-ceil interaction surface marker H LA-ABC, vs. naive MSCs. In certain embodiments, the ceMSCs show simultaneous increase in anti-inflammatory, immunomodulatory, and cell cycle arrest of tumorigenic cells.

In certain embodiments, the ceMSCs show increased anti-inflammatory properties such that the marker TNF-a stimulated gene-6 (TSG-6) gene with and without TNF-a stimulation is increased by≥2 fold (Fig 3). In certain embodiments, the ceMSCs show increased anti-inflammatory properties such that they result in significant subsiding of paw edema in a non-immunocompromised animal over an acute time period of 72 hours, 48 hours, 24 hours, or 6 hours (Fig 4 A, B, C).

In certain embodiments, the ceMSCs show significant differential in vivo tissue distribution such that they show significant reduction of≥4 folds in lung entrapment vs. naive MSCs at 24 hours (Fig 5).

(n certain embodiments, the ceMSCs show increased anti-inflammatory properties such that they result in significant subsiding of peritoneal inflammation in a non- immunocompromised animal over an acute time period of 4 hours, 24 hours, and 48 hours (Fig 6 A, B) In certain embodiments, the ceMSCs show significant subsiding of inflammatory cell infiltration at 24 hour time point in the peritoneal cavity in a non-immunocompromised animal over an acute inflammatory stimuli (Fig 6A)

In certain embodiments, the ceMSCs show significant subsiding of pro-inflammatory cytokine levels at 4 hour time point in the peritoneal cavity in a non-immunocompromised animal over an acute inflammatory stimuli (Fig 6B)

In certain embodiments, the ceMSCs show significant differential in vivo tissue distribution such that they show significant reduction of >4 folds in lung entrapment vs. naive MSCs at 24 hours. In certain embodiments, the ceMSCs show increased immunoevasive properties such that they show reduced cytotoxicity of ≥3 fold, ,≥40 fold,≥35 fold, or >25 fold, when co- incubated with a Natural Killer (NK92) cell line at 40:1, 20:1 , 10:1 , or 5:1 (NK92:MSCs) ratio respectively (Fig 7).

In certain embodiments, the ceMSCs demonstrate immunomodulation properties such that they show significant inhibition of≥9 fold,≥4 fold, or≥3 fold of CD4+ T helper cell proliferation at 1:10, 1:20, or 1:40 (CD4+ T helper cells: MSCs) ratios respectively with or without TNFα (6 ng/mL), IFNy (500 units/mL), and TNF-a+IFN-γ and with 10% fetal bovine serum media (Fig 8).

In certain embodiments, the ceMSCs demonstrate immunomodulation properties such that they show significant inhibition of≥3 fold of CD4+ T helper cells proliferation with and without early vs. late osteoarthritic synovial fluid (80% diluted with serum free media) relative to naTve MSCs which show stimulatory properties (Fig 9). Na ^' ive MSCs showed stimulatory properties with synovial fluids and significantly increased proliferation of CD4+ T helper cells relative to ceMSCs In certain embodiments, the ceMSCs demonstrate immunomoduiation properties such that they show significant inhibition of CD4+ T helper cells proliferation immediately post- thaw relative to naive MSCs (Fig 10) In certain embodiments, the ceMSCs derived from different tissue source (bone marrow, umbilical cord tissue) demonstrate polarization of monocytes/macrophages . to antiinflammatory macrophage (M2) subtypes such that they show significant increase of≥50 fold of mean fluorescence intensity (MFI) of CD163 surface marker on non-manipulated monocytes/macrophages (Fig 11 A, B).

In certain embodiments, the ceMSCs derived from different tissue source (bone marrow, umbilical cord tissue) demonstrate polarization of monocytes/macrophages to antiinflammatory macrophage (M2) subtypes such that they show significant increase of >30 fold of MFI of CD206 surface marker on naive monocytes/macrophages (Fig 11 A, B). In certain embodiments, the ceMSCs derived from different tissue source (bone marrow, umbilical cord tissue) demonstrate polarization of monocyte/macrophages to antiinflammatory macrophage (M2) subtypes such that they show significant increase of IL-10 (pg/mL) levels (Fig 11 C, D)

In certain embodiments, the ceMSCs derived from different tissue source (bone ^' marrow, umbilical cord tissue, and adipose tissue) demonstrate polarization of monocyte/macrophages to anti-inflammatory macrophage (M2) subtypes such that they show significant decrease in reactive oxygen species (ROS) levels under inflammatory conditions. ROS levels are expressed as relative luminance units measured using Lucigenin (Sigma) luminescence assay (Fig 12 A, B, C) (Yamazaki T, ef al., Tropical Medicine and Health. 2011;39(2):41-45).

In certain embodiments, the ceMSCs derived from different tissue source (bone marrow, umbilical cord tissue, and adipose tissue) that are manufactured using different serum free basal media formulation demonstrate polarization of monocyte/macrophages to antiinflammatory macrophage (M2) subtypes such that they show significant decrease in reactive oxygen species (ROS) levels under inflammatory conditions and is not dependent on serum free basal media formulation. ROS levels are expressed as relative luminance units measured using Lucigenin (Sigma) luminescence assay (Fig 13 A, B, C) (Yamazaki T, et al., Tropical Medicine and Health. 2011;39(2):41-45. In certain embodiments, the ceMSCs show increased anti-inflammatory properties such that they show significant in-vitro chemo-taxis migration of ≥3 fold with TNF-a as chemoattractant vs. na ^' iVe MSCs (Fig 14).

In certain embodiments, the ceMSCs demonstrate cell cycle arresting properties on tumor cell lines such that they show significant increase in hsa-miR-16 which is a known cell cycle arresting marker on tumor cell lines by≥10 fold vs. naive MSCs (Fig 15).

In certain embodiments, the ceMSCs derived from different tissue source (bone marrow, umbilical cord tissue, and adipose tissue) demonstrate differential gene expression profile of 30 genes including: hADAMTS4 , hADAMTSS, CCR7, CXCL13, CXCL8, CYCLIND1 , hHGF, hIDO, hlL10, hlL12, IL-1 B, iNOS, MMP13, MMP3, PD-L1 , PD-L2, PRG-4, Ptgs2/COX2, SOCS1 , SOCS3, SOX9, TGFB, TIMP1 , TLR2, TLR3, TLR4, TSG-6, NFKBIA, TWIST1 , VEGFA (Figs 16, 17, 18).

In certain embodiments, the ceMSCs demonstrate no proliferative advantage properties such that they show non-significant increase in cell number when cultured under naive MSCs conditions up to 6 days relative to naive MSCs (Fig 19)

In certain embodiments, the ceMSCs demonstrate anti-tumor properties such that they show decrease of tumor cell line (chronic myelogenous leukemia: K562, ATCC® CCL- 243™) total cell numbers when co-cultured with ceMSCs relative to naive MSCs co- cultures (Fig 20 A, B) In certain embodiments, the ceMSCs demonstrate anti-tumorigenic properties such that they show accumulation of a tumor cell lines (fibrosarcoma : HT-1080, ATCC® CCL- 121™; overy adenocarcinoma: SKOV3, ATCC® HTB-77™; breast adenocarcinoma: MCF7, ATCC® HTB-22™; chronic myelogenous leukemia: K562, ATCC® CCL-243™) in G0/1 cell cycle phase by≥4 fold, relative to na ^' ive MSCs (Fig 21 A, B). In certain embodiments, the ceMSCs demonstrate anti-tumorigenic properties such that they show decrease of a tumor cell lines (fibrosarcoma : HT-1080, ATCC® CCL-121™; overy adenocarcinoma: SKOV3, ATCC® HTB-77™; breast adenocarcinoma: MCF7, ATCC® HTB-22™; chronic myelogenous leukemia: K562, ATCC® CCL-243™) in G2/M cell cycle phase by >3-fold, relative to nafve MSCs (Fig 21 A, B). In certain embodiments, the ceMSCs demonstrate anti-tumorigenic properties such that they show accumulation of a tumor cell lines (fibrosarcoma : HT-1080, ATCC® CCL- 121™; overy adenocarcinoma: SKOV3, ATCC® HTB-77™; breast adenocarcinoma: MCF7, ATCC® HTB-22™; chronic myelogenous leukemia: K562, ATCC® CCL-243™) in GO/1 cell cycle phase by≥5 fold relative to stimulated na ^' ive MSCs (Fig 21 A, B).

In certain embodiments, the ceMSCs demonstrate anti-tumorigenic properties such that they show accumulation of a tumor cell lines (fibrosarcoma : HT-1080, ATCC® CCL- 121™; overy adenocarcinoma: SKOV3, ATCC® HTB-77™; breast adenocarcinoma: MCF7, ATCC® HTB-22™; chronic myelogenous leukemia: K562, ATCC® CCL-243™) in GO/1 cell cycle phase by >7 fold relative to naive MSCs (Fig 21 A, B).

In certain embodiments, the ceMSCs demonstrate anti-tumorigenic properties such that they show decrease of a tumor cell lines (fibrosarcoma : HT-1080, ATCC® CCL-121™; overy adenocarcinoma: SKOV3, ATCC® HTB-77™; breast adenocarcinoma: MCF7, ATCC® HTB-22™; chronic myelogenous leukemia: K562, ATCC® CCL-243™) in G2/M cell cycle phase by≥3-fold relative tonalVe MSCs (Fig 21 A, B).

The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention.

EXAMPLES

Methods and Materials

Bone Marrow Cell Preparation

The University Health Network Research Ethics Board (REB) approved the acquisition of bone marrow (BM) from healthy donors (REB# 14-7483-AE). All participants provided written informed consent to participate in this study. Naive MSCs

Briefly, BM mononuclear cells were isolated by density gradient centrifugation on Ficoll- Paque (GE Healthcare Life Sciences, Mississauga, Canada), suspended in serum-free medium (SFM) (Lonza TheraPEAK MSCBM-CDTM medium, Lonza, Richnmond, BC) with recommended supplements (Lonza MSCGM-CD™ SingleQuots™ kit, Lonza). T175 flasks were coated with 22.75 mL of 5 μg/mL/cm ² recombinant fibronectin (BD Biosciences) in PBS for 2 hours at room temperature. The cells were seeded at 3000 cells/cm ² densities and incubated at 37°C with 5% CO ₂ . Medium was changed 2x per week Flasks were washed with PBS and harvested using TrypLE™ Select (Life Technologies, Burlington, ON) at 80-90% confluence.

Culture Engineered MSCs

To enhance MSCs by culture engineering, the culture expanded n-MSCs (as describe before) were harvested and additionally enhanced at a concentration of 0.2 x 10 ^s cells/mL in SFM with cytokine supplement [0.1 - 15 ng/mL lnterlukin-6 (IL-6) (Sigma & PeproTech), 0.1 -0.5% Human serum albumin (Sigma), and water for injection (Hospira). Cells were incubated at 37°C in a humidified atmosphere with 5% CO ₂ in 100 mL media in stirred suspension spinner flasks (Sigma) or in 10 mL media in T75 ultra-low attachment flasks (Corning). For stirred suspension spinner flasks, 25-35 RPM was used. Cells were enhanced between 2-9 days and media volume was doubled once post-seeding. . Immunophenotyping

MSCs were characterized for their surface expression of PE-CD90 (5E10), PE-CD105 (266), PE-CD14 (M5E2) (BD Biosciences); PE-CD73 (AD2), PE-HLA-ABC (W6/32), PE- CD106 (STA), PE-CD54 (HA58), PE-CD19 (HIB19), PE-CD45 (HI30), PE-CD34 (581), and PE-HLA-DR (L243) (Biolegend, San Diego, CA); propidium iodide (PI) (Sigma). For immunolabelling, cells were incubated for 45 minutes at 2-8°C. Viable PI- MSCs were characterized using FC500 flow cytometer (Beckman Coulter, Mississauga, ON) and analyzed using FlowJo (T reestar). Tri!ineage differentiation assay ceMSCs and n-MSCs were tested for their trilineage differentiation potential using StemPro differentiation kits (Life technologies) for osteogeneic, adipogeneic and chondrogeneic differentiation as per manufacturer's instructions. Staining was performed using Oil Red 0 (Sigma) stain for adipogenesis, Alizarin Red S (Sigma) for osteogenesis, and Alcian Blue 8GX (Sigma) for chondrogenesis.

⁵¹ Cr Cytolytic assays

MSCs were harvested, washed once with PBS and resuspended at 10x10 ^e cells/ml in AIM V medium (Life technologies) with CilMa ₂ ⁵¹ Cr04 (1 μCi/μl). Cells were incubated for 2 hours at 37°C and 5% CO ₂ , washed 3x and resuspended in X-vivo medium (Lonza). 5x10 ³ radiolabeled MSCs were added to each 96-well U-bottomed plate well along with NK-92 cells at an effector (NK92) to target (MSCs) ratio of 40:1 , 20:1, 10:1 or 5:1 (100 μL/well). Plates were incubated at 37°C, 5% CO ₂ for 4 h, and centrifuged at 285 g for 5 min. 100 μL supernatant was collected from each well and amount of ⁵¹ Cr present in supematants was determined using a Packard Cobra II Gamma Counter (Perkin Elmer, Waltham, MA). Percentage lysis was calculated using the formula: (E - S)x100/(M - S), where E is the ^s1 Cr release ( ⁵¹ Cr present in supernatant) from an experimental sample, S the spontaneous release in the presence of complete medium and M the maximum release upon ceil lysis with 10% Triton X-100 (Sigma). Data is presented as the mean percentage lysis of triplicate samples with 95% confidence interval (CI).

Gene expression assay by real-time (RT-PCR) assay for MSCs derived from three different tissue sources (bone Marrow, umbilical cord tissue, and adipose tissue)

Total RNA was isolated from ceMSCs vs. n-MSCs using TRIzol® Reagent (Life Technologies) and converted into cDNA with High-Capacity cDNA RT Kit (Life Technologies). Real-time RT PCR was performed using FastStart Universal SYBR Green Master mix (Roche, Indianapolis, IN). Samples were analysed in triplicate. Thermal cycling was performed with 7900HT System (Life Technologies); 95 ^a C, 2 min followed by 40 cycles of 95°C, 15s and 60°C, 20s. Relative expression levels were calculated using the 2 ^"M Ct method ¹³ , with B2M as housekeeping gene. TNF-α in vitro chemotaxis assay

In vitro chemotaxis migration assay was conducted using μ-Slide Chemotaxis slides (Ibidi, Madison, Wl) with and without tumor necrosis factor-a (TNFα; 6 ng/mL). The centre of mass of all cells (n=30) was represented by the spatial average of all cell positions. The difference in the centre of mass between initial values, and at the end of experiment was termed the displacement of centre of mass, as previously described ^{14, 15} .

CD4+ T helper (Th) cell proliferation assay

Peripheral blood monocytes (PBMCs) were isolated by density gradient centrifugation on Ficoll-Paque from fresh blood samples. CD4+ T helper (Th) cells were isolated by CD4+ T cell negative selection (Milteny Biotec, San Diego, CA) and stained with carboxyfluorescein succinimidyl ester (CFSE, Life Technologies) following the manufacturer's recommendations. 96-well plates were seeded with 5, 10 or 20 x10 ³ MSCs and 1x10 ⁶ CFSE-Th cells, with or without TNFα (6 ng/mL) + IFNy (500 units/mL), in complete medium: RPMI1640 with L-glutamine (80%), DMEM (10%), 1 mM sodium pyruvate, 1X Penicillin-Streptomycin and fetal bovine serum (FBS) (10%). Dyna beads Human T-Activator CD3/CD28 (Life Technologies) were used to activate Th cells. After 4 days, Th cells in suspension were harvested and mixed with PI, and read using a FC500 flow cytometer (Beckman Coulter, Mississauga, Ontario). Fold change in cell number was determined by algorithmic regression accounting for both proliferation and viability ¹⁸ . The effect of early-stage (n=5 donors) and late-stage osteoarthritis (OA) synovial fluid (SF) (n=5 donors) on MSC-mediated inhibition of Th cell proliferation was tested by performing the same assay described above in 80% synovial fluid diluted in the Th cell complete medium without FBS.

The effects of cryopreservation on the MSC-mediated inhibition of Th cell proliferation was also determined by thawing MSCs, and immediately assaying for proliferation-inhibition effects on Th cells as described above. In vivo paw edema model

C57BL/6J mice (weight 25±5g) were used (n=11 per group). To generate paw edemas, bacterial lipopolysaccharide (LPS) (E.Coli 026:B6; Sigma-Aldrich) dissolved in saline was subcutaneously injected into the plantar region of the left hind paw. Animals were restrained by hand-held method during LPS injection. 1 hours post-LPS injection (to allow inflammation to set in), 50 μL of human MSCs (0.5 x 10 ⁶ cells) were injected (26 gauge) intravenously through tail vein injection; PBS (50 μL) was injected as the control. Paw thickness was measured using digital calipers post LPS injection at baseline, 2, 4, 6, 24 48, and 72hours (h). Histology and hematoxylin and eosin staining

After 24 and 72h post LPS injection, animals were sacrificed and the paws were dissected, followed by decalcification using ethylenediaminetetraacetic acid (EDTA; Sigma). Briefly, tissues were collected in RNAIater® Stabilization Solution (Thermo Fisher) and decalcified by immersing paws in PBS/0.5 M EDTA solution (3x changes) for 3-5 days. Paws were kept in 30% sucrose solution at 4°C overnight, transferred into cryomolds filled with 100% optimum cutting temperature (OCT) solution, in a horizontal orientation. These cryomolds were snap frozen on dry ice with absolute alcohol and stored at -80°C. Frozen paw molds were sectioned, fixed, and stained for standard hematoxylin/eosin (HE) for cytological evaluation of polymorphonuclear cells/infiltration. The paw images were rated 1-10 (1=low; 10=high) by 4 independent blinded observers by visual inspection for presence of inflammatory cell infiltrates.

Mouse Model of Peritonitis

Male C57BL/6J mice (Jackson Laboratories), 6-8 wk of age and housed on, a 12 h light/dark cycle, were used to study the anti-inflammatory action of bone marrow derived na ^' ive and ceMSCs on zymosan-induced peritonitis, The inflammatory compound Zymosan A (Sigma) was prepared at a concentration of 1 mg/mL in PBS and autoclaved for 15 min to sterilize. To induce inflammation, 1 mL of the 0.1% zymosan solution was administered i.p, Fifteen minutes later, 0.5 x 10 ⁸ of na ^' ive and ceMSCs were administered i.p. through a 20-gauge needle 150-200 μL of HBSS (Gibco). Peritoneal lavage was collected at 4, 24 and 48 hours post MSCs injections. The lavage volume was recorded and the cells removed by centrifugation at 500 x g for 10 min. Cellular infiltration in peritoneum was determined by flow cytometer ¹⁷ . Levels of inflammatory molecules TNFα, IL12p40, CXCL1 , CCL2, IL-10, IL-6 were determined from the peritoneal lavage using custom build available flow cytometry multiplex kit (Biolegends). The statistical significance in mean values was examined using graph pad with multiple T-test analysis using Holm-Sidak multiple comparisons method.

Monocyte isolation and polarization

Human monocytes were acquired from peripheral blood of previously consented healthy donors (REB#l5-9224-CE) by Ficoll gradient centrifugation, and purified by magnetic bead separation of CD14+ monocytes. These isolated monocytes were co-cultured for 2 days with and without naive MSCs or ceMSCs (n=3 donors). After 2 days, the cells were detached, washed 3x with PBS, and analyzed using FC500 flow cytometer, Monocyte/macrophages were characterized using CD86, HLA-DR, CD163 and CD206, where M1 subtypes are pro-inflammatory with CD86 and HLA-DR high, and M2 subtypes are anti-inflammatory with predominantly CD 163 high and/or CD206 high; CD206 expression on M2 subtypes in our hands is more variable, but is included to be consistent with literature. The statistical significance in mean values was examined using graph pad with multiple T-test analysis using Holm-Sidak multiple comparisons method. Alu sequence detection by real-time PCR

Real time PCR for ALU sequences detection was done using Alu primers ¹⁶ . 15-60 ng of total genomic DNA was used for each 10-μί reaction. Thermal cycling was performed with 7900HT System (Applied Biosystems) using FastStart Universal SYBR Green Master mix (Roche). Cycling setup was: 95°C, 2 min followed by 35 cycles 95°C for 15 s, 68°C for 30 s and 72°C, 30s.

MicroRNA analysis

MicroRNA analysis was conducted using Quanta Biosciences kits (MIR qScript™ microRNA cDNA Synthesis Kit and PerfeCTa® SYBR® Green SuperMix, ROX™). hsa- mi ^' R-16; hsa-miR-21; hsa-miR-24; hsa-miR-26a; hsa-miR-27a were analysed as per manufacturer's instructions.

Propidium iodide staining of tumor ceils to assess cell cycle arrest

Tumor cells (fibrosarcoma : HT-1080, ATCC® CCL-121™; overy adenocarcinoma: SKOV3, ATCC® HTB-77™; breast adenocarcinoma: MCF7, ATCC® HTB-22™; chronic myelogenous leukemia: K562, ATCC® CCL-243™) were co-cultured with bone marrow derived naive and ceMSCs (n~3 donors) in a 24 well plate at 10:1 or 5:1 (tumor: MSC) ratio. Briefly, MSCs were harvested as previously described, counted and seeded in a 24 well plate with SFM. Tumor cells without MSCs were used as positive controls. Non- stimulated MSCs were used as experimental controls. After 3 days of co-culture, the cells (MSCs & tumor cells) were detached, counted, and fixed with 70% ethanol. For cell-cycle analyses, the cells were labeled with PI (200 μl PI from 50 μg/mL stock solution, incubated for 30 min, at 37*C in the dark), and re-suspended in PBS. Samples were run on a FC500 flow cytometer and the data was analyzed using FLOWING SOFTWARE 2.5.1. The results are expressed as the mean (±95% CI) of three donors (each tested in triplicate). The statistical significance in mean values was examined with Bonferroni's multiple comparisons test after one-way ANOVA test.

Cell number assessment of tumor cells co-cultured with MSCs

Tumor cells (chronic myelogenous leukemia: K562, ATCC® CCL-243™) were co-cultured with bone marrow derived na ^' fve and ceMSCs (n=3 donors) in a 24 well plate at 10:1 (tumor: MSC) ratio. Briefly, MSCs were harvested as previously described, counted and seeded in a 24 well plate with SFM. Tumor cells without MSCs were used as positive controls. Only MSCs were used to check effects of tumor cells on MSCs numbers. After 2 days of co-culture, the cells (MSCs & tumor cells) were detached, and counted by trypan blue using hemocytometre. The results are expressed as the mean (±95% CI) of three donors (each tested in triplicate). The statistical significance in mean values was examined with Bonferroni's multiple comparisons test after one-way ANOVA test. Cryopreservation n-MSCs and ceMSCs were cryopreserved using current good manufacturing practice (cGMP) grade, xeno-free cryomedium CryoStor® CS10 (BioLife Solutions, Bothell, WA) which is optimized for MSCs Statistical Analysis

We used GraphPad Prism software (Prism 7) for calculating statistical significances between different groups, t-tests, nonparametric Mann-Whitney tests, confidence intervals of difference of means, Wilcoxon tests with confidence interval of median, and repeated measures one-way ANOVA followed by Dunnett multiple comparison tests. For chemotaxis we used chemotaxis and migration Tool (Version 2.0 - independent standalone software, and Version 1.01 - Plugin for ImageJ).

Results and Discussion

Characterization of ceMSCs and n-MSCs MSCs from 3 healthy donors were characterized for cell surface markers (CD90+, CD105+, CD73+, CD14-, CD19-, CD34-, CD45-, and HLA-DR-) and their trilineage differentiation potential as per International Society for Cellular Therapy (ISCT) minimal criteria ¹⁹ . As shown (Fig 2), culture engineering methods did not alter the identity of MSCs. We also observed a transient (24h post seeding) decrease in expression of intercellular adhesion molecule (ICAM) or CD54, vascular cell adhesion molecule (VCAM) or CD106, and human leukocyte antigens (HLA-ABC) on ceMSCs vs. n-MSCs (Fig 1). ceMSCs are more responsive towards an inflammatory stimulus ceMSCs showed an increase in TSG-6 gene expression a measure of anti-inflammatory states of MSCs ²⁰ relative to n-MSCs (p<0.005) (Fig 3). As shown, although the basal level of TSG-Θ on unstimulated n-MSCs varied between each donor (n=3), the TSG-6 gene expression levels were still higher for ceMSCs for each donor both with and without TNF- α (6 ng/mL) stimulaiton. Results also indicate that ceMSCs are more responsive towards an inflammatory stimulus (6 ng/mL of TNF-a) vs. n-MSCs. Similar response towards TNF- α by ceMSCs vs. n-MSCs was also observed under in vitro chemotaxis assay (Fig 14) demonstrating improved migratory properties of ceMSCs over nMSCs. Analysis of ceMSCs anti-inflammatory properties in vivo, after i.v. infusion

As shown (Fig 4a, b), ceMSCs significantly subsided inflammation in a LPS- induced acute paw edema model (paw thickness for ceMSCs (3.29 mm), relative to n-MSCs (3.55 mm), and controls (4.42 mm) at 6 hours (n=11 mice; p<0.005) H&E staining of paws was done to determine number of infiltrating inflammatory cells near the injection site (24h post LPS injection) and was shown to be significantly lower for ceMSCs (p<0.0005) vs. n- MSCs (p>0.05) relative to the LPS control group. Figure 4c shows the inflammatory infiltration rating (at 24h) done by 4 independent blinded observations on histologic sections; average rating for ceMSCs was 3.5, n-MSCs was 10, and LPS control was 8 (1=low infiltration - 10= high infiltration). Analysis of ceMSCs in vivo bio-distribution after intravenous infusion ceMSCs showed significant differential bio- distribution vs. n-MSCs (Fig 5) after systemic tail vein injection. The number of human DNA copies^L in lung tissue for ceMSCs was significantly (p<0.05) lower than n-MSCs, indicating that ceMSCs were better able to escape lung entrapment compared to n-MSCs (qPCR done in triplicates for 2 different MSC donor in the acute injury paw edema model).

Analysis of ceMSCs anti-inflammatory properties in vivo, after i.p. infusion in mouse model of peritonitis

As shown (Fig 6 a,b), BM-derived ceMSCs (BM-MSCs) significantly reduce peritoneal inflammation in zymosan-induced peritonises model vs. naive MSCs. Inflammatory MHCII+ MOs (indicative of pro-inflammatory monocytes/macrophages) levels (Fig 6a) at 24 hours post zymosan (0.1 mg/mL) treatment in BM-ceMSCs (<15%) were lower vs. na ^' ive MSCs (>18%) and zymosan only controls (>19%). Pro-inflammatory cytokine levels (Fig 6b) in zymosan-induced peritonises model by BM-ceMSCs at 4 hours were lower vs. na ^' ive MSCs and zymosan only controls ceMSCs are more immunoevasive

MSCs from 3 healthy donors were analyzed for their immunoevasiveness when co- cultured with a natural killer (NK92) cell line using ⁵¹ Cr Cytolytic assays ²¹ . lL-2 stimulated NK92 cell line mediated killing of MSCs was 23.69% ± 7.13% (mean+95%CI, n=3) for ceMSCs vs. 53.02% ± 1 1.25% for n-MSCs at 40:1 (NK92: MSC) ratio (Fig 7). No killing by NK92 cells was observed at 20:1 , 10:1 , and 5:1 (NK92: MSC) ratios for ceMSCs vs n- MSCs (Figure 7).

Immediate post-thaw, Bone marrow ceMSCs are more immunomodulatory n-MSCs were unable to inhibit Th cell proliferation immediately post-thaw while ceMSCs retained this inhibitory capacity, (n=3 MSC donors) at 1 :20 (MSC: Th cell) ratio, regardless of MSC donor source; this assay was done with unlicensed MSCs. Fold expansion (4 days post seeding) of CD4+ Th cell population in the presence of ceMSCs was >3.5 fold lower than n-MSCs, and >A fold lower than activated Th cell control as measured by flow cytometry analysis (Fig 10). There were no significant differences between the inhibitions of activated Th cell proliferation in the presence of either ceMSCs or n-MSCs, which were allowed to recover 3-5 days post thawing (Fig 8) as shown by others ⁹ .

Interaction of bone marrow ceMSCs and naive MSCs with early vs. late osteoarthritic (OA) synovial fluid (SF)

3 healthy donor bone marrow MSCs were pooled and tested for their inhibition of CD4+ Th cells proliferation with and without osteoarthritic synovial fluid (OA SF). OA SF was added to stimulate a pro-inflammatory microenvironment as would be encountered in vivo, instead of licensing MSCs with IFN~v and/or TNF-a. OA SF from 5 OA patients each in early and late stages (Early stage: Kellgren-Lawrence grading scale (KL) i and II; Late stage: KL 111 and IV) was diluted to 80% with Th cell complete medium without FBS. As shown (Fig 9), with ceMSCs (OA SF-ceMSC), >2.5 fold inhibition of CD4+ Th cell proliferation was seen vs. n-MSCs (OA SF-na ^' ive MSCs) (p<0.004), regardless of OA stage. Th cells with SF (OA-SF activated) in general showed less proliferation vs. activated Th cells (activated), indicating inhibitory effects of OA SF on Th cells (p<0.002). Regardless of OA stage, ceMSCs did not additionally inhibit activated Th cells with OA SF, but ceMSCs were still better than n-MSCs, which resulted in enhancement rather than inhibition of Th cell proliferation. This observation requires further analysis on the use of n- MSCs to treat OA, as there may be pro-inflammatory effects.

Bone marrow derived ceMSCs are more immunomodulatory MSCs from 3 healthy donors were analyzed for their ability to polarize monocytes/macrophages (n=1 donor) into M2 subtypes. MSCs are known to polarize monocytes/macrophages into anti-inflammatory M2-subtypes ²² . As shown (Fig 11 A, B), ceMSC polarized na ^' iVe monocytes/macrophages into M2 subtypes to a greater extent (MFI values for CD163 - 24,675) compared to n-MSCs (MFI values for CD163 - 10,189). This also translated into increased ratio of M2 subtype MFI value (19 fold increase with respect to naive monocyte/macrophage control) with ceMSCs compared to n-MSCs (8 fold increase with respect to naive monocyte/macrophage control). ceMSC co-cultured monocyte/macrophages (n=1 donor), show increased IL-10 levels in the culture supernatant (>10 pg/mL) vs. naive MSCs co-cultured monocyte/macrophages (<2 pg/mL) (Fig 1 1 C, D). ceMSCs derived from different tissue source are more immunomodulatory ceMSCs derived from different tissue source show increase inhibition of monocyte/macrophage polarization to inflammatory subtypes vs. na ^' ive MSCs (Fig 12 A, B, C). The levels of reactive oxygen species (ROS) in co-culture of monocyte/macrophages with ceMSCs was <250 RLU (relative luminescence units) vs. in co-culture of monocyte/macrophages with naTve MSCs (>500 RLU). 3 donor Bone marrow MSCs, 3 donor umbilical cord MSCs, and 2 donor adipose tissue MSCs are used. ceMSCs derivied from different tissue source have unperturbed functional properties with changes in basal manufacturing medium ceMSCs derived from different tissue source show increase inhibition of monocyte/macrophage polarization to inflammatory subtypes vs. naive MSCs (Fig 13 A, B, C) irrespective of basal manufacturing media formulation. ROS levels in co-culture of monocyte/macrophages with ceMSCs was not significantly different between the two test media's and was lower than co-cu!ture of monocyte/macrophages with naive MSCs. 3 donor Bone marrow MSCs, 3 donor umbilical cord MSCs, and 2 donor adipose tissue MSCs are used. ceMSCs derived from different tissue source demonstrate differential gene expression profile 3 donor Bone marrow MSCs, 3 donor umbilical cord MSCs, and 2 donor adipose tissue MSCs were characterized for 30 gene expression (Fig 15, 16, 17) including: hADAMTS4 , hADAMTS5, CCR7, CXCL13, CXCL8, CYCLIND1 , hHGF, hlDO, hlUO, hlL12, 1L-1 B. iNOS, MMP13, MMP3, PD-L1 , PD-L2, PRG-4, Ptgs2/COX2, SOCS1 , SOCS3, SOX9, TGFB, TIMP1, TLR2, TLR3, TLR4, TSG-6, NFKBIA, TWIST1, VEGFA, B2M. ceMSCs showed increased in gene expression of genes related to anti-inflammatory, immunomodulatory, and anti-tumor genes, that demonstrates them to be fundamentally in a more activated state vs. nalVe MSCs.

Bone marrow ceMSCs demonstrate differential miRNA expression

3 healthy donor MSCs were characterized for miRNA (miR) expression including: has- miR-16; hsa-miR-21; hsa-miR-24; hsa-miR-26a; hsa-miR-27a. ceMSCs showed significantly increased miR-16 levels (10 fold) relative to n-MSCs (Fig 18). miR-16 is known to promote tumor cells G1 arrest, myogenesis, inhibits proliferation and angiogenic potential in MSCs ²³ , it indicates that ceMSCs are more cell cycle arresting than .n-MSCs. Less than 2-fold increase was observed in miRs (21 , 24, 26a and 27a) in ceMSCs vs. n- MSCs (Fig 18); these miRs are known to be involved in differentiations ²³ , indicating that MSC differentiation potential is not changed at the miRNA level in ceMSCs.

There appears to be no proliferative advantage of ceMSCs vs. naive MSCs. ceMSCs and nMSCs were seeded at different densities under nMSCs conditions. After 6 days of culture total DNA content is measured by spectrophotometry. No significant difference were seen between ceMSCs and naive MSCs (Fig 19).

Bone marrow ceMSCs inhibit increase in tumor cell number

3 healthy donor bone marrow MSCs were tested for their inhibition on K562 cell number growth (Fig 20A). K562 cells co-culture with BM-ceMSCs show decrease in cell number by <2 fold changes were seen relative to the initial seeding density 20,000 K562 cells vs. K562 cells co-culture with naTve MSCs (>3 fold changes were seen relative to the initial seeding density of 20,000 K562 cells). No change in cell number for BM-ceMSC and na ^' ive MSCs were seen (>10 fold changes were seen relative to the initial seeding density of 2,000 MSCs cells) when co-culture with K562 cells vs. MSCs only controls (Fig 20B).

Bone marrow ceMSCs inhibit tumor cell number by cell cycle arrest

3 healthy donor bone marrow MSCs were tested for their effects on tumor cells (fibrosarcoma: HT-1080, ATCC® CCL-121™ ¹ ; overy adenocarcinoma: SKOV3, ATCC® HTB-77™; breast adenocarcinoma: MCF7, ATCC® HTB-22™; chronic myelogenous leukemia: K562, ATCC® CCL-243™) cell cycle. Co-culture of bone marrow ceMSCs with tumor cell lines, arrested tumor cells at G0/1 or S cell cycle stage (Fig 21A,B). Increased cell cycle arrest of a tumor cell line (HT-1080, K562, SKOV3, MCF7) in G0/1 cell cycle phase (4 fold) and decrease in G2/M cell cycle phase (3-fold) were seen when co-cultured with ceMSCs vs. n-MSCs. Our culture engineering method consists of a series of physical manipulations that causes temporal changes to the extracellular matrix (ECM) interaction of MSCs with the growth surface, other MSCs, and cells. As a consequence, these culture engineered MSCs (ce-MSCs) have increased anti-inflammatory properties {in vivo and in vitro), differential migration, increased immune cell modulation post-thaw and in an inflammatory milieu, and increased expression of micro RNA (miR)-16, a known anti-angiogenesis factor.

Importantly, these enhanced properties are also present immediately after thawing ceMSCs, compared to n-MSCs, regardless of donor source (Fig 6). This immediate post- thaw benefit is highly relevant to clinical applications as it eliminates the need for MSCs to recover 3-5 days post-thaw ^s (Fig 6). The use of our approach, pre-cryopreservation as a last step following commercial expansion will reduce costs and logistic challenges. Companies such as MediPOST, which currently use a 5-day recovery period for their cryopreserved MSC product CARTISTEM® prior to administration ²⁴ would greatly benefit from our approach. Mesenchymal stromal cells (MSCs) through cell-cell contact and secretion of various cytokines and chemokines have been reported to both suppress e.g. via modification of Akt signaling ²⁵ and enhance tumor growth and tumor-driven angiogenesis ²⁶ . The tropism of MSCs for tumors e.g., hepatocellular carcinoma ²⁷ , brain tumors ²⁸ and sarcomas ²⁵ wide interest regarding their potential as a delivery vehicle for anti-cancer agents. Indeed, several reports have described the feasibility of using genetically-modified MSCs as anticancer delivery vehicles for anti-tumorigenic factors including tissue necrosis factor (TNF) ²⁸ , TNF related apoptosis inducing ligand (TRAIL) ²⁹ , and interferon (ΙΡΝ)-β ³⁰ . However, genetically-modified MSCs for clinical application in humans for cancer therapy may be hampered by concerns of both feasibility and safety ³¹ . These challenges may be addressed by using non-genetically enhanced MSCs that renders the MSCs both antiinflammatory and anti-tumor properties at the same time, ceMSCs show improved immunomodulation by suppressing T cell proliferation after cryopreservation / thawing, compared to nMSC. ceMSCs also showed greater polarization of monocytes/macrophages to anti-inflammatory M2 sub-types. Taken together ^' with the increased cell cycle arrest and decrease in tumor cell number of tested tumor cell lines (Fig 20, 21) and increased anti-angiogenic marker (miR-16) (Fig 8), we believe our culture engineering method increases anti-tumor potential of MSCs, and increasing their immunomodulatory effects on T cells, monocytes/macrophages and cancer cells. Additional in vitro and in vivo testing with other solid and hematological cancer cell lines and primary tumor cells will be needed to confirm these findings. ceMSCs are more immunoevasive than n-MSCs as evidenced by low NK92 cell-mediated cytolysis (Fig 5) of ceMSCs. This may be due to low levels of HLA-ABC, ICAM, and VCAM expression ²¹ (Fig 1 ) on ceMSCs relative to n-MSCs suggesting that ceMSCs may be better candidates than n-MSCs for allogeneic therapy. n-MSCs while acting in an immune privilege manner in an allogenic setting, particularly with Th cells of incompatible donors ^{32, 33} , are still able to elicit innate immune responses ^{34, 35} (Fig 7). These immune responses may be responsible for failures of some clinical trials using allogenic MSCs ³⁴ . ceMSCs could elicit improved therapeutic effects over n-MSCs in terms of reduced innate cell-mediated immune rejection, and increased anti-inflammatory properties. AIu sequence detecting human DNA showed differential biodistribution of ceMSCs vs. n- MSCs including reduced lung entrapment for ceMSCs. Around 80% of human MSCs are sequestered in the lung upon intravenous Injection, leading to emboli of MSCs in lung vessels, which are cleared rapidly within the first 24 hours, leading to complete elimination within 100 hours ^{36, 37} . This hampers the therapeutic effect of MSCs due to lack of homing to injured sites, and potentially limiting the paracrine effects. Limited biodistribution may contribute to misleading conclusions on dose and effectiveness of MSCs. ceMSCS offers a wider biodistribution of MSCs which may potentially improve paracrine effects over a longer duration. ceMSCs have a higher level of TSG-6 gene expression under both TNF-a stimulated and non-stimulated conditions vs. n-MSCs. There is little or no constitutive expression of TSG- 6 in adult tissues, but the protein is synthesized by fibroblasts and many other cell types in response to stimulation with several pro-inflammatory mediators. Human MSCs are particularly responsive to TNF-a and it is expressed at much higher levels than cultured fibroblasts. TSG-6 signalling reduces neutrophill infiltration and attenuates nuclear factor- kappa beta (NF-KB) signaling in resident macrophages ³⁸ . ceMSCs with higher TSG-6 expression are thus primed to be more potent anti-inflammatory modulators.

Intravenously infused MSCs improved cardiac function and decreased scarring in a mouse model of myocardial infarction occurs in part because the cells that are trapped in the lung as microemboli are activated to secrete the anti-inflammatory protein, TSG-6. However, these MSCs did not express TSG-6 until 12-24 h after forming microemboli in lungs at which time about half of the MSCs have been destroyed ³⁹ . Standard cultures of MSCs do not express TSG-6 ^' ' ⁰ but are activated to express the protein if incubated for 24h with tumor necrosis factor-a (TNFα). These observations suggest that aggregation of MSCs in culture may provide an effective procedure to pre-activate the cells ³⁹ and increase their anti-inflammatory properties, akin to what happens via microemboli formation in lung tissue when MSCs are systemically injected in vivo.

Our approach of culture engineering MSCs uses a self-assembly aggregation method, wherein MSCs are seeded onto an ultra-low attachment, high-volume flask, and licensed using IL-6. This method is compatible with clinical scale generation of MSCs. There has been some research on aggregating MSCs either as a procedure for enhancing chondrogeneic differentiation or to increase their therapeutic potential ^44"47 . 3D methods generally facilitate greater cell-cell contacts and interactions of cells with the extracellular matrix (ECM), allowing cells to adapt to their native morphology, which in turn influences signaling activity ^48"51 . To create a 3D environment, techniques have included the use of porous scaffolds, collagen gels, and forced multicellular aggregates ^52'54 , each of which have been successfully employed on MSCs ⁵⁵ . However current techniques are difficult to scale up limiting them to research use. Importantly, technologies like Aggrewell™ systems (STEMCELL technologies) generate strong aggregates that require enzymatic dissociation and are not readily scalable. Our method uses a self-assembly aggregation method, that generates loose agglomerates, and only requires mechanical rather than enzymatic dissociation. This translates into an easier regulatory pathway, falling under minimal manipulation, rather than a more involved drug/biologic regulatory pathway.

To our knowledge, this is first time that two independent minimal methods to activate and enhance MSCs (i.e aggregation and licensing by IL-6) are used in tandem. Although it is well established that IL-6 has positive effects on MSC growth, proliferation and activity ^56"58 , IL-6 has not been previously used as a licensing agent for activating and enhancing MSCs.

Other methods to activate and enhance MSCs using soluble factors include the use of TLR3/4 ligands, such as polyinosinic:polycytidylic acid (poly IC) and LPS to activate MSC sub-sets that are either pro-or anti-inflammatory ^{59, 60} , small molecule based approaches to enhance MSCs ⁶¹ , and genetic modification-based enhancements, such as knock-in of IL- 10 ⁶ⁱ or tumor necrosis factor-a/CD40 ligand ⁶³ This methods may require cell sorting to isolate MSCs sub-sets that are anti-inflammatory and present safety risks associated with genetic modification ⁸⁴ . Additionally, this methods can be used post-thaw only, not pre- cryopreservation, limiting its off-the-shelf utility of MSCs. The use of IL-6, at low concentrations as described and for a limited period of time (4 days), would add little to the cost of manufacturing MSC, less than $100/100 million MSCs. Since our approach results in minimal manipulation of MSCs, it would be expected to face lower regulatory hurdles for product approval, and results in easier marketability due to lower costs. Taken together, our culture engineering method significantly improves performance of MSCs anti-inflammatory properties, without changing cell identity. The scalable transient method uses minimal manipulation of MSC, can improve the therapeutic efficacy of MSCs pre-cryopreservation and/or, post-thawing, in a cost effective manner, and may be an important technology to enable the field to capitalize on the research and therapeutic potential of MSCs.. Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference.

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