Field of the Invention

The present invention relates to an apelin-containing culture medium and a method using the said medium for the derivation of mesenchymal stem cells (MSCs) from human pluripotent stem cells. Furthermore, the present invention relates to derivation of mesenchymal stem cell which can be used in research and clinical applications with the said culture medium and method.

Background of the Invention

Stem cells are main cells which can differentiate into the various cells that constitute the tissues of an organism, and they are often referred to as undifferentiated cells before transforming into other cells. Mesenchymal stem cells are used to restore and treat many tissues and organs since they can divide, self-renew, and differentiate into cells of mesodermal origin, such as bone, cartilage, fat, heart, muscle, etc. Mesenchymal stem cells are a type of adult stem cells. Although the use of mesenchymal stem cells is safer, easier and more advantageous relative to other stem cells, a substantial number of these cells must be obtained and produced to achieve therapeutic effect. In order to obtain sufficient amounts, either a large amount from the source should be isolated or prolonged in vitro cell division is required. However, senescence of mesenchymal stem cells due to their limited lifespan leads to the loss of functions, such as proliferation, differentiation, and migration, which are essential for their therapeutic efficacy. Therefore, there is a need for methods which will enable derivation of large amounts of healthy mesenchymal stem cells under in vitro conditions.

Mesenchymal stem cells are isolated and cultured from different tissues of an adult individual and can be used within the scope of research and application. Derivation of these cells from pluripotent stem cells and productionlarge amounts of cells in culture in a healthy way are important in terms of preventing the proliferation of heterogeneous cell populations and generation of a personalized cellular therapy source.

Pluripotent stem cells are classified as embryonic stem cells and induced pluripotent stem cells, and can differentiate into many cell types thanks to their pluripotent properties. Several attempts have been made to use pluripotent human stem cells, which can differentiate into all cells, as a stem cell therapy product. To date, several methods and protocols have been developed for the derivation of mesenchymal stem cells from pluripotent stem cells. Mesenchymal stem cells which are differentiated by using bFGF are very similar to the original mesenchymal stem cells in terms of morphology and expression of markers. However, their potentials of differentiation, especially differentiation to adipogenic lineage, may be limited.

The derivation, characterization, culture and differentiation of pluripotent stem cells are crucial for stem cell studies. Development of protocols which can be applied clinically will constitute the basis of stem cell therapy-based applications. Inefficient differentiation of pluripotent stem cells in culture hinders derivation of clinically relevant terminally differentiated cells. Since the main source of mesenchymal stem cells is somatic lateral plate mesoderm, pluripotent cells were first differentiated into mesoderm and then into mesenchymal stem cells by using the necessary morphogens and considering mesoderm signaling pathways. The mesenchymal stem cells obtained in this way and the original mesenchymal stem cells were not only identical in terms of morphology and surface markers, but also showed multipotency and low immune reaction.

One of the issues to be considered and overcome in order to be able to use mesenchymal stem cells derived from pluripotent stem cells instead of original mesenchymal stem cells in clinical trials is to test whether there is differentiation in oncological genes when induced pluripotent stem cells are differentiated into mesenchymal stem cells. In addition, since the purity and quality of mesenchymal stem cells are important, cells should be selected and sorted such that the mesenchymal stem cell markers will be positive and pluripotent markers will be negative, and trials should be performed on animal models before being transferred to the clinic to eliminate the risk of oncogenesis. Moreover, other unstable factors should be eliminated, and all steps must be carried out in a xeno- free culture medium.

In the literature, studies have been conducted regarding the use of human pluripotent stem cells for solving the problems in the method of culturing mesenchymal stem cell and for the derivation of mesenchymal stem cells. Differentiation from human pluripotent stem cells into mesenchymal stem cells generally requires an induction procedure. Also, there are challenges such as the mesenchymal stem cells produced by these methods maintaining their basic state and the low production efficiency. For these reasons, there are limitations to the use of mesenchymal stem cells as ideal cell therapy products in the fields of regenerative medicine and cell therapy. Inability to obtain enough mesenchymal stem cells, the use of invasive methods to reach stem cells, heterogeneity observed due to factors such as health status and age among different tissues and individuals, rapid aging during proliferation in culture and tissue incompatibility observed in allogeneic therapies are the problems observed in mesenchymal stem cell culture and therapies.

Derivation of mesenchymal stem cells from pluripotent stem cells will eliminate these problems. The protocols used for the derivation of mesenchymal stem cells from pluripotent stem cells requires optimization. Performing the process of deriving mesenchymal stem cells from pluripotent stem cells in culture in a short time and by using inexpensive techniques are the problems that need to be solved to obtain cells that can be applied in therapy. For these reasons, establishing protocols for derivation of mesenchymal stem cells from pluripotent stem cells, and keeping the obtained cells in culture by preserving their stem cell properties are important and necessary for treatment protocols.

Apelin (also known as APLN) is a peptide that is encoded by the APLN gene in humans. United States patent document no. US9982234B2, known in the state of the art, discloses that when stem cells are cultured in an apelin-containing medium composition, the self-renewal ability, proliferation without changing the characteristics of the stem cells and telomerase activity of the stem cells are increased. However, there are no patents or articles on the use of apelin in the differentiation of mesenchymal stem cells from pluripotent stem cells.

In addition, the role of Apelin in differentiation into several cells and its protective effect on cells have been disclosed in articles. Yet, these studies are not related to the derivation of mesenchymal stem cells (MSCs) from pluripotent stem cells and maintaining their potential in culture. Summary of the Invention

The present invention relates to an apelin-containing culture medium and a method using the said medium for the differentiation of human pluripotent stem cells into mesenchymal stem cells (MSCs), which overcomes all of the above- mentioned problems and provides additional advantages to the relevant technical field.

The present invention relates to a method which can be used for the differentiation of mesenchymal stem cells from pluripotent stem cells, and the method is characterized in that the culture medium contains apelin peptide. The invention relates to the use of an apelin-containing culture medium at specific time intervals in method steps.

The said method disclosed in the present invention ensures that mesenchymal stem cells are derived by activating the apelin receptor signaling pathway at certain time intervals and their proliferation and characteristics are maintained.

The present invention also relates to an apelin-containing culture medium formulation which can be used for the differentiation of mesenchymal stem cells from pluripotent stem cells.

In this context, apelin peptide treatment enables mesenchymal stem cell (MSC) differentiation efficiency and maintenance of mesenchymal properties of the obtained cells. This patent relates to the derivation of mesenchymal stem cells (MSCs) from pluripotent stem cells and the maintenance of their potential in culture, in this manner.

One of the advantages achieved with the present invention is to develop a method and culture medium formulation, which allows unlimited derivation of mesenchymal stem cells, which are obtained in limited amounts from adult individuals under normal conditions, from pluripotent stem cells.

An objective of the present invention is to develop a method and culture medium formulation which allows repeated derivation of mesenchymal stem cells, which are obtained from adult individuals under normal conditions, from pluripotent stem cells to prevent their rapid aging in culture.

Another objective of the invention is to develop a method and culture medium formulation in which cultured mesenchymal stem cells can be passaged for a long period of time, without undergoing senescence in culture.

The main objective of the present invention relates to an effective differentiation method for deriving safe cells from pluripotent stem cells that can be used clinically The present invention may be used in mesenchymal stem cell (MSC)- derived cell therapy, therapeutic cloning and reprogramming applications, mesenchymal stem cell (MSC)-derived secretome, growth factor, cytokine, chemokine applications, mesenchymal stem cell (MSC) derivation applications for research purposes, and autologous and allogeneic banking applications for cell banking purposes. Thanks to the present invention, it will be possible to obtain bankable mesenchymal stem cells from pluripotent stem cells. The developed method and the content of the culture medium formulation are designed to be applied in a time-dependent manner by being made in the form of a kit for triggering the differentiation.

Brief Description of the Figures

“A Method and Culture Medium Formulation which can he Used for the Derivation of Mesenchymal Stem Cells from Pluripotent Stem Cells”, which was performed for achieving the objective of the present invention, is illustrated in the accompanying drawings, in which:

Figure 1. Culture media formulations used in the process of differentiation of pluripotent stem cells into mesenchymal stem cells and the time periods in the method steps.

Figure 2. Morphology of the cells, which were differentiated from pluripotent stem cells into mesenchymal stem cells, on days 10 and 24. Scale bar: 50pm.

Figure 3. Surface marker analysis of the cells, which were differentiated from pluripotent stem cells into mesenchymal stem cells, on day 10 (3A) and day 24 (3B) and the ratios of positive and negative surface markers.

Figure 4. Analysis of mesenchymal markers by gene expression in groups differentiated into mesenchymal stem cells.

Figure 5. Analysis of cell viability by MTS assay in the obtained mesenchymal stem cells. *P< 0.05

Figure 6. Analysis of cell proliferation by Trypan Blue in the obtained mesenchymal stem cells.

Figure 7. Division time (doubling time) of the obtained mesenchymal stem cells. Figure 8. Staining of control and Apelin MS cells after differentiation into bone, cartilage and fat. Von Kossa stains calcium minerals and is a marker of bone differentiation. Alcian Blue stains glycosaminoglycans and is a marker of cartilage differentiation. Oil Red stains lipid granules and is a marker of fat differentiation.

Abbreviations:

N2B27 : name of the serum -free medium used, bFGF: basic fibroblast growth factor,

CHIR: Wnt activator (CHIR99021),

DMEM: reduced glucose medium (Dulbecco’s Modified Eagle Medium),

Gl-6: Groups 1-6.

DPSC: Dental pulp stem cell

Apelin-13: Apelin peptide

Detailed Description of the Invention

In the studies of the present invention, the role of the Aplnr signaling pathway on derivation of mesenchymal stem cells from pluripotent stem cells through mesoderm was determined. In this context, the role of Aplnr signaling pathway on mesoderm cell migration and differentiation into mesenchymal stem cells has been studied and determined by using pluripotent stem cells.

Within the scope of the present invention, the effective differentiation of pluripotent stem cells into mesoderm and mesenchymal stem cells comprises the steps of obtaining three-dimensional embryoid bodies and then transferring them into monolayer culture medium, thereby obtaining mesenchymal stem cells. This allows effective cell differentiation to occur as a method for mimicking true embryonic development. The present invention comprises mesenchymal stem cells produced by this method, cell therapy products containing mesenchymal stem cells, and a standardized and optimized culturing method and an apelin-containing culture medium which can be used in clinical applications to produce mesenchymal stem cells from human pluripotent stem cells.

As used herein, the term "stem cells" refers to main cells which can be renewed unlimitedly to form the cells of tissues and organs. Stem cells are totipotent, multipotent, pluripotent, oligopotent or unipotent cells.

As used herein, the term "pluripotent stem cells" refers to pluripotent stem cells which can differentiate into all three germ layers that constitute a living body, and examples of these include embryonic stem cells and induced pluripotent stem (iPS) cells.

As used herein, the term “differentiation” refers to a process by which cells become specialized in terms of structure or function during cell division, proliferation, and growth, whether in embryonic development or in the adult individual.

As used herein, the term “mesenchymal stem cell” refers to adult stem cells present in different tissues of adult individual and to mesenchymal stem cells differentiated from the mesoderm germ layer by using pluripotent stem cells.

As used herein, the term “embryoid body” refers to an aggregate formed by inducing the differentiation of pluripotent stem cells. They are 3 -dimensional structures. The present invention relates to the use of a culture medium for the differentiation of human pluripotent stem cells into mesenchymal stem cells (MSCs), wherein the culture medium contains apelin peptide. The addition of apelin peptide to the culture medium can be adjusted between 0.1 micromolar to 1 micromolar depending on the cell type to be used. Preferably, the concentration of apelin peptide in the culture medium is 0.5 micromolar (pM).

The said culture media to which apelin peptide is added after preparation is any one of N2B27 and DMEM culture media.

N2B27 culture medium preferably comprises at least one of L-Glutamine, bFGF (basic fibroblast growth factor) and CHIR (CHIR99021; Wnt pathway activator) or mixtures thereof. In a preferred embodiment of the invention, N2B27 culture medium comprises L-Glutamine, bFGF, and CHIR and their present concentrations (in volume/total volume) are 1% L-Glutamine, 8-12 ng/ml bFGF (preferably 8ng/ml, lOng/ml or 12ng/ml), and 3uM CHIR.

DMEM culture medium comprises glucose, and its present glucose concentration is preferably Ig/L.

In the present invention, apelin peptide is added to at least one of these culture media, and “culture medium” which contains apelin is formed. As used herein, the expression “culture medium” includes culture media free of apelin.

The present invention also relates to a method for culturing stem cells by using a culture medium containing apelin peptide for the differentiation of human pluripotent stem cells into mesenchymal stem cells (MSCs). Thanks to apelin, the potential to obtain mesenchymal stem cells is increased with the use of the cells at different stages during the differentiation after exiting from pluripotent state. In this way, the time interval suitable for the addition of apelin was determined. It was observed that the mesenchymal stem cell properties of the obtained cells increased, and they preserved these properties.

During this process, apelin plays a role in promoting differentiation toward mesoderm and toward mesenchyme therefrom.

The present invention comprises the formation of embryoid bodies from human pluripotent stem cells and the formation of mesenchymal stem cells from these bodies. Embryoid bodies should be induced to allow their differentiation into mesenchymal stem cells. With the protocol in Figure 1, induction of mesenchymal stem cells is performed independent of apelin.

The present invention relates to a method for culturing the stem cells by using a culture medium containing apelin peptide for the differentiation of human pluripotent stem cells into mesenchymal stem cells (MSCs), and comprises the following steps: a) forming embryoid bodies from human pluripotent stem cells in suspended culture medium; b) attaching the formed embryoid bodies to a surface in the culture medium; c) directing the induction that is initiated in serum-free N2B27 medium to the differentiation of mesenchymal stem cells in serum containing DMEM medium to enable the differentiation of embryoid bodies into mesenchymal stem cells; d) adding apelin to the culture medium while maintaining the presence of mesenchymal stem cells. In step (a) of the method of the present invention, embryoid bodies are formed from human pluripotent stem cells. At this stage, human pluripotent stem cells are cultured under conditions suitable for forming embryoid bodies. In order to form the said embryoid bodies, human pluripotent stem cells are cultured in N2B27 culture medium, which is prepared with the addition of 1% L-Glutamine,I2 ng/ml bFGF and 3uM CHIR, and in suspended culture in an incubator with a temperature of 37° C and %5 CO2 atmosphere for 3 days (Figure 1).

In step (b) of the method of the present invention, the embryoid bodies formed in step (a) are attached to a surface. This step allows the transfer of embryoid bodies from 3 -dimensional culture to 2-dimensional culture. The formed embryoid bodies are transferred to the gelatin-coated surface in N2B27 culture medium with the addition of 1% L-Glutamine and lOng/ml bFGF on day 3 (while proceeding to step (c)) and kept in the incubator until day 5. Thereby, the cells adhere to the surface after 3 -day suspended culture and perform differentiation (Figure 1).

In step (c) of the method of the present invention, on day 5, culturing is continued in N2B27 culture medium with the addition of 1% L-Glutamine and 8ng/ml bFGF. After incubation until day 8, DMEM medium containing Ig/L glucose is administered.

In this step (c), DMEM (comprising Ig/L glucose) culture medium was used in the steps of cell differentiation (until day 24) and culturing. In this way, transition of the cells to mesenchymal stem cell phenotype was aimed. The protocol steps have such transitions, and it is a 24-day protocol (Figure 1).

In step (c) of the method of the present invention, apelin is added to the culture medium while maintaining the presence of mesenchymal stem cells. 0.5 pM apelin peptide is added to the culture medium. Peptide addition was carried out in 6 different experimental groups at different time intervals depending on the groups (Figure 1).

The said method disclosed in the present invention is a 24-day protocol, which involves the addition of apelin to the culture medium and is performed starting from day 3 (starting from step (c)). It was not performed for the group Gl, which is the control group. It was performed for the group G2 between days 3 and 5, for group G3 between days 5 and 8, for group G4 between days 8 and 10, for group G5 between days 10 and 24, for group G6 between days 3 and 24. Apelin treatment is a single treatment for each group in a way to cover its own time interval. For the group G6, the protocol was applied once in the specified day intervals (to cover that day interval) and as a booster every other day between the tenth and twenty-fourth day.

In the present invention, the addition of apelin to the culture medium is administered from day 3, i.e., from step (c), such that it is performed as 0.5 pM for a minimum of 2 days (day 5 in the method) and a maximum of 21 days (day 24 in the method) until day 24. In a preferred embodiment of the invention, 0.5 pM apelin treatment was performed every other day for the group between days 10 to 24.

The mesenchymal properties of the cells were compared by adding 0.5 pM apelin to these culture media at various day intervals (Figure 1), and group 2 (G2) with 0.5 pM Apelin added between days 3 to 5, and group (G6) with 0.5pM Apelin added between days 3 to 24 were selected as the groups showing the best mesenchymal stem cell properties. Fibroblast cell morphology and plastic adherence, which are the phenotypes of mesenchymal stem cells, were observed for Gl (control group), G2 and G6 on both day 10 and day 24 (Figure 2). Within the scope of protocol optimization, surface marker analyses were performed on days 10 and 24, and it was decided that the 24-day protocol was suitable for the derivation of mesenchymal stem cells (Figure 3). There is an early mesenchymal stem cell formation on day 10 and an adult mesenchymal stem cell formation on day 24.

At the end of 24 days, the mesenchymal markers CD73, CD90 and CD105 showed a significant increase in gene size in the G6 group treated continuously with Apelin compared to the G1 group that was not treated with Apelin (Figure 4). The viability of the cells was measured by MTS and proliferation was measured by counting with Trypan blue every day for 72 hours and a significant increase in both viability and proliferation was observed in the group treated with apelin (Figures 5 and 6). In addition, the division time (doubling time) of the cells was reduced after Apelin treatment (G6). Cells were seeded in equal amounts and counted twice for 48 hours, and the division time was calculated. This surprisingly showed that Apelin treatment also affected cell proliferation and division rate and had a positive effect (Figure 7).

Mesenchymal stem cells produced by the method of the present invention have characteristics which can be used for treatment.

In the preferred embodiment of the invention, mesenchymal stem cells produced by the method of the present invention can be differentiated into adipocytes, osteocytes, chondrocytes, epithelial, endothelial and myocytes, neurocytes and cardiomyocytes.

In addition, the present invention comprises the use of mesenchymal stem cells obtained by the method of the present invention in cell therapy products. Specifically, the said cell therapy products can be used for the formation of adipocytes, osteocytes, chondrocytes, myocytes, neurocytes and cardiomyocytes and their differentiation into various cells according to the environments.

In addition, the present invention may pave the way for the use of secretions of mesenchymal stem cells obtained by the method of the present invention such as exosomes, cytokines, growth factors and chemokines in cell therapy products.

The following examples are to better illustrate the subject of the invention and the subject of the invention is not limited to these examples.

EXAMPLES

Example 1 - Implementation of the protocol

The protocol is a 24-day protocol, and it is carried out for obtaining embryoid bodies in suspended culture the first three days, while the rest is carried out in monolayer culture. After the third day, embryoid bodies were transferred to the coated surface, adhered thereto, and cells separated from these bodies were differentiated into MSCs (Figure 1).

Example 2 - Characterization of MSCs:

MSC differentiation is characterized by the appearance of fibrillary cells similar to fibroblast cells in culture (Figure 2). Besides, the cells should have the ability to adhere to the plastic surface (Figure 2). Cells should contain MDC surface markers on their surface as positive (Table 1).

MSCs must successfully perform mesoderm-derived bone, cartilage, and fat differentiation in culture. In this context, the cells obtained in this study were subjected to bone, cartilage, and fat differentiation and Apelin treatment increased the differentiation (Figure 8).

Table 1: Surface marker analyses of Control, Groups 2 and 6 on the tenth and twenty-fourth day of the protocol used for the derivation of MSCs. The percent values show what percentage of cells are positive and negative for each marker. While CD 44, 90, 105 are markers of MSCs, CD 45 is a marker which is not observed in MSCs.