WO2016193836A1

WO2016193836A1 - Method of preparing mesenchymal stromal cells for specific indication and composition thereof

Info

Publication number: WO2016193836A1
Application number: PCT/IB2016/052636
Authority: WO
Inventors: Udaykumar KOLKUNDKAR; Suresh Kannan; Sudha BALASUBRAMANIAN; Parvathy VENUGOPAL; Priyanka SWAMYNATHAN; Anish Sen Majumdar
Original assignee: Stempeutics Research Private Limited
Priority date: 2015-06-01
Filing date: 2016-05-09
Publication date: 2016-12-08

Abstract

The present disclosure relates to a method of preparing stromal cell based compositions. The said compositions comprise pooled cells which are arrived by pooling of cells from individual donors. Said pooling of cells is based on the analysis of characteristics such as but not limiting to growth kinetics, gender, immunosuppression, gene profiling and secretome profiling. Thus, the present method can be used to prepare pooled stromal cells for specific clinical indications/applications as per the requirement.

Description

METHOD OF PREPARING MESENCHYMAL STROMAL CELLS FOR SPECIFIC INDICATION AND COMPOSITION THEREOF

TECHNICAL FIELD

The present disclosure relates to stem cell technology. In particular, a method of preparing stem cell based compositions is provided. The said compositions comprise pooled cells which are arrived by selective pooling of individual cells from characterized Donor cell banks (DCB). Specific/selective pooling of DCBs is based on the analysis of characters such as but not limiting to growth kinetics, gender, immunosuppressive studies, gene profiling and secretome profiling. Such composition is used to target specific indication or disease condition.

BACKGROUND OF THE DISCLOSURE

Mesenchymal stem cells (MSCs) are found in almost all post natal tissues such as bone marrow, adipose tissue, dental pulp, umbilical cord, amniotic fluid, limbal tissue etc. and are capable of differentiating in-vitro and in-vivo along multiple pathways. Their regeneration capability, multilineage differentiation potential and immune-suppressive capacity make them optimal tools for cell-based tissue engineering and regenerative medicine. Although bone marrow (BM) is the traditional and the most well characterized source of human MSCs, it has certain limitations such as, a highly invasive procedure is used for procuring BM from the donors and there is a decline in proliferation efficiency and differentiation potential of BM-MSCs with aging. Hence, alternative sources of MSCs need to be pursued.

The origin of MSCs may determine their fate and functional characteristics. Being from a fetal-derived cord, that is discarded after birth, wharton's jelly (WJ)-derived MSCs have higher proliferation and ex vivo expansion capabilities than adult MSCs. Although, MSCs from different origins share important biological properties, till date it is not clear which type or source of MSC should be selected for the different therapeutic approaches. The umbilical cord can provide an inexhaustible source of stem cells for therapy without involving any invasive procedures or ethical concerns. MSCs have been isolated from different compartments of the umbilical cord, and Wharton's jelly (WJ) is the embryonic mucous connective tissue lying between the amniotic epithelium and the umbilical vessels and is a rich source of MSCs. However, there are no methods available for developing efficient cell compositions targeted towards specific disorders. Therefore, there is an immense need for development of methods to prepare cell based compositions which are highly potent/ efficient and indication specific. The present disclosure achieves the said feat by providing such efficient compositions and corresponding methods. STATEMENT OF THE DISCLOSURE

Accordingly, the present disclosure relates to a method of preparing pooled mesenchymal stromal cell based composition, said method comprising acts of: a) culturing mesenchymal stromal cells of individual donors to a first predetermined passage to obtain individual donor cell banks (DCBs), b) characterizing each donor cell bank (DCB) at the first predetermined passage, c) culturing the mesenchymal stromal cells of DCBs up to a second predetermined passage and pooling the mesenchymal stromal cells from plurality of donor cell banks (DCBs) based on the characterization of step (b) to obtain working cell bank (WCB), and d) culturing the mesenchymal stromal cells of the working cell bank (WCB) up to a third predetermined passage to obtain the pooled mesenchymal stromal cell based composition; a mesenchymal stromal cell based composition comprising mesenchymal stromal cells and cryopreservation medium wherein said composition is prepared by the method as described above; and a method of managing a disease, said method comprising act of administering the composition comprising mesenchymal stromal cells and cryopreservation medium to a subject in need thereof. BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

Figure 1: shows a flow diagram for establishing and characterizing donor cell bank and further preparing indication specific working cell bank.

Figure 2: shows the morphology of individual and pooled cells (N, R, S and NRS; O, P, Q and OPQ and Q, R, S and QRS) at Passage 5. Magnification 10X.

Figure 3: shows the proliferation rate/ growth kinetics and CFU-F assay of passage 2 to passage 5 cells. Fig 3(A): shows the proliferation rate of cells from individual donors and pooled cells (N, R, S and NRS; O, P, Q and OPQ and Q, R, S and QRS) from passage 2 to passage 5; their total cell number; cumulative population doubling and population doubling time. Figure 3(B): shows characterization based on the CFU-F assay of pooled cells (NRS, OPQ and QRS) from passage 2 to passage 5 indicating that pooled and upscaled cells in Mesencult medium retained the clonal formation efficacy till passage 5.

Figure 4: shows the surface marker characterization of cells from individual donors (having phenotypes N, R, S;) versus pooled cells (NRS); OPQ;QRS) at passages 5. , indicating that pooling does not alter surface marker expression.

Figure 5: shows the trilineage differentiation potential of pooled cells of NRS, OPQ and QRS at passage -5 indicating that pooling does not affect trilineage differentiation.

Figure 6: shows Karyotype analysis of pooled cells by Giemsa -Banding (G-Banding) and Flourescent in situ hybridization (FISH) for X & Y specific probes.

Figure 6(A): shows the Karyotype analysis (G-Banding) of pooled cells (NRS; OPQ and QRS) at passage 6, indicating that at the end of passage 6, the cells from all the three pools (NRS, OPQ and QRS) exhibit normal karyotype. Mosaic karyoview is observed for the QRS mixed pool in the ratio of (XY 75 : XX 25).

Figure 6(B): shows the FISH results of QRS mixed pool. Evaluation of percentage of two clones using X/Y centromeric probe by FISH technique on interphase nuclei for better delineation of the two clones. Hybridization shows the presence of one signal for X chromosome (Green) and one signal for Y chromosome (Red) in 75% [XY] and two signals for X (Green) in the remaining 25% of cells analyzed [XX]. The FISH results corroborate with the karyotype data of mixed pool QRS.

Figure 7: shows the immunosuppression of cells from individual donors N, R, S at passages P4 and P6.

Figure 8: shows the gene expression profiles of individual and pooled cells.

8(A): shows the gene expression profile of individual (N, R, S) and pooled cells (NRS) at P6 for expression of Pro angiogenic cytokine, (CXCL5) anti- inflammatory gene (TGF b) and pro inflammatory markers (TNF -alpha & IL lb) and tumor suppressor marker P53 at passage 6.

Figure 8 (B): shows the gene expression profile of individual and pooled cells (N, R, S and NRS) at passage 6.

Figure 8 (C): shows the gene expression profile of individual and pooled cells (O, P, Q and OPQ) at passage 6. Figure 8 (D): shows the gene expression profile of individual and pooled cells (Q, R, S and QRS) at passage 6.

Figure 8(E): shows a summary of gene expression profile of pooled cells (NRS, OPQ and QRS) [summary of figure 8B, 8C and 8D].

Figure 8(F): shows a comparison of gene expression profile at passage 6 for three pooled donors (NRS, OPQ and QRS).

Figure 8(G): shows the comparison of gene expression profile of individual and pooled cells (N, R, S and NRS; O, P, Q and OPQ and Q, R, S and QRS) at passage 6.

Figure 9: shows the secretome analysis of individual and pooled cells (N, R, S and NRS; O, P, Q and OPQ and Q, R, S and QRS) conditioned media collected at passage 6.

Figure 10: shows the STR analysis of individual and pooled cells (N, R, S and NRS; O, P, Q and OPQ and Q, R, S and QRS) at passage 6.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to a method of preparing pooled mesenchymal stromal cell based composition, said method comprising acts of:

a) culturing mesenchymal stromal cells of individual donors to a first predetermined passage to obtain individual donor cell banks (DCBs);

b) characterizing each donor cell bank (DCB) at the first predetermined passage; c) culturing the mesenchymal stromal cells of DCBs up to a second predetermined passage and pooling the mesenchymal stromal cells from plurality of donor cell banks (DCBs) based on the characterization of step (b) to obtain working cell bank (WCB); and

d) culturing the mesenchymal stromal cells of the working cell bank (WCB) up to a third predetermined passage to obtain the pooled mesenchymal stromal cell based composition.

In an embodiment of the present disclosure, the mesenchymal stromal cells are Wharton jelly derived mesenchymal stromal cells.

In another embodiment of the present disclosure, the mesenchymal stromal cells are cultured in a xeno-free culture medium comprising Basal medium, 20% Proliferation Supplement, L- Glutamine, penicillin and streptomycin. In yet another embodiment of the present disclosure, the first predetermined passage is passage 2. In still another embodiment of the present disclosure, the characterization in step (b) is carried out by performing analyses selected from a group comprising growth kinetics, gender analysis, gene expression, secretome analysis and immunosuppression analysis, or any combination thereof. In still another embodiment of the present disclosure, the pooling in step (c) is performed by combining the mesenchymal stromal cells of characterized donor cell banks (DCBs) showing similar properties.

In still another embodiment of the present disclosure, the second predetermined passage is passage 4.

In still another embodiment of the present disclosure, the pooled mesenchymal stromal cell of the composition obtained in step (d) is characterized by performing analyses selected from a group comprising gender analysis, karyotype analysis, growth kinetics, gene expression analysis, secretome analysis, immunosuppressive analysis, trilineage potential analysis, surface marker characterization, tumorigenicity, STR analysis and clonal expression, or any combination thereof.

In still another embodiment of the present disclosure, the third predetermined passage is passage 6.

In still another embodiment of the present disclosure, the pooled mesenchymal stromal cell based composition is specific to an indication selected from group comprising but not limiting to Rheumatoid arthritis, Diabetes mellitus, graft-versus-host disease, diabetic foot ulcer, acute myocardial infarction, liver cirrhosis, stroke, multiple sclerosis and critical limb ischemia, or any combination thereof. The present disclosure relates to a mesenchymal stromal cell based composition comprising mesenchymal stromal cells and cryopreservation medium wherein said composition is prepared by the method comprising steps of:

b) characterizing each donor cell bank (DCB) at the first predetermined passage;

c) culturing the mesenchymal stromal cells of DCBs up to a second predetermined passage and pooling the mesenchymal stromal cells from plurality of donor cell banks (DCBs) based on the characterization of step (b) to obtain working cell bank (WCB); and d) culturing the mesenchymal stromal cells of the working cell bank (WCB) up to a third predetermined passage to obtain the pooled mesenchymal stromal cell based composition.

In an embodiment of the present disclosure, the cryopreservation medium is animal protein- free defined cryopreservation medium or a cryopreservation medium comprising ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent.

In yet another embodiment of the present disclosure, the composition is cryopreserved, and wherein said cryopreserved composition comprises mesenchymal stromal cells at a concentration ranging from about 10 million cells per ml to 25 million cells per ml of the medium.

The present disclosure relates to a method of managing a disease, said method comprising act of administering the composition comprising mesenchymal stromal cells and cryopreservation medium to a subject in need thereof.

In an embodiment of the present disclosure, the disease is selected from a group comprising Rheumatoid arthritis, Diabetes mellitus, graft-versus-host disease, diabetic foot ulcer, acute myocardial infarction, liver cirrhosis, stroke, multiple sclerosis and critical limb ischemia, or any combination thereof. Mesenchymal Stem Cells are known for their use in treating various auto immune diseases. They have the characteristics of modulating immune response by secreting various cytokines and growth factors. But the properties of MSCs largely depend on the donor characteristics and the clinical capabilities of the MSCs vary across the donors. For example, MSCs from some donors might have higher proliferation rate, high immunosuppressive capacities than compared to other donors. So as to minimize the donor to donor variation, MSCs from different donors are pooled. In the present disclosure, pooled MSCs show reduced variability with regards to properties such as proliferation rate, gene expression, immunosuppressive capacities etc., which has been shown by comparing individual samples vs. pooled samples. WJ-derived MSCs share some properties unique to fetal derived MSCs like having faster proliferation and greater ex-vivo expansion capabilities than adult MSCs. MSC transplantation requires harvesting a large number of cells. MSCs are present in very low percentage in their niches and a vast number of cells are required for their subsequent use in therapeutic applications. Hence, it is important to define optimal culture conditions which would maximize the yield of MSCs from WJ of umbilical cord within a reasonable time frame. As used herein, the terms "mesenchymal stromal cell", "mesenchymal stem cell" and "MSC" are employed interchangeably within the instant disclosure.

As used herein, the terms "cell composition", "cell product", "Investigational Medicinal product (IMP)", "final MSC composition" are used interchangeably within the instant disclosure and means the cell product comprising pooled MSCs obtained by the method of the instant disclosure.

As used herein, "management" or "managing" refers to preventing a disease or disorder from occurring in a subject, decreasing the risk of death due to a disease or disorder, delaying the onset of a disease or disorder, inhibiting the progression of a disease or disorder, partial or complete cure of a disease or disorder and/or adverse effect attributable to the said disease or disorder, obtaining a desired pharmacologic and/or physiologic effect (the effect may be prophylactic in terms of completely or partially preventing a disorder or disease or condition, or a symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease or disorder and/or adverse effect attributable to the disease or disorder), relieving a disease or disorder (i.e. causing regression of the disease or disorder). Further, the present disclosure also envisages managing/treating said disorder by administering therapeutically effective dosage of the composition/MSCs prepared by the method of the invention. The present disclosure relates to a general method of establishing improved stem cell compositions targeting specific disease conditions wherein the method uses the concept of indication specific pooling of stem cells based on stem cell characteristics. The said method involves establishing well characterized disease specific cell banks referred to as working cells banks (WCBs). The disease specific working cell bank is used in developing the disease specific therapeutic cell products/compositions.

In an embodiment of the present disclosure, 'indication specific composition', 'indication specific pooled mesenchymal stromal cell based composition', 'indication specific MSC composition' or 'indication specific MSCs/mesenchymal stromal cells' refers to composition/MSCs which has been prepared for management/treatment of a specific disease(s) or disorder(s) or medical condition(s)/indication(s). In an exemplary embodiment, said disease or disorder or condition/indication is selected from a group comprising but not limiting to Rheumatoid arthritis, Diabetes Mellitus (DM), graft-versus-host disease (GVHD), Diabetic Foot Ulcer (DFU), Critical limb Ischemia (CLI), Acute myocardial infarction (AMI), Liver cirrhosis (LC), stroke and Multiple sclerosis (MS).

In an embodiment of the present disclosure, the method involves establishing a well characterized Donor cell Bank (DCB) or Master cell bank (MCB) of each stem cell donor and thereafter indication specific Working Cell Bank (WCB) is established by pooling DCBs. The WCB is optionally cryopreserved and later the WCBs are further used in developing indication specific cell product/composition for clinical/therapeutic application. Such an indication specific cell product system ensures reduced variability in the products manufactured from the single established WCB.

In a crucial aspect of the present disclosure, the steps involved in obtaining and characterizing DCBs and subsequent establishment of WCB is important which determines the quality and specificity of the final composition/product towards indications/diseases.

In an embodiment of the present disclosure, the method involves preparing MSC based compositions, wherein said MSCs are derived from source selected from a group comprising Wharton's jelly, bone marrow, adipose tissue and dental pulp, or any combination thereof. In an exemplary embodiment, the MSCs are derived from Wharton's jelly (WJ).

In another embodiment of the present disclosure, the selection of DCBs for pooling and preparing the WCB passes through specific stages and this involves extensive characterization of the DCB and identification of DCBs with similar characteristics. Depending on the similarity of the selected characteristics, DCBs are pooled to generate WCB. One of the important steps involved in the establishment of indication-specific WCBs and subsequent final composition is the initial characterization of DCB at specific time points of culturing. The characterization results are the main criteria for selection of DCBs for pooling. The indication specific WCBs which are established after pooling the characterized DCBs are further expanded/passaged till specific time-point. The cell products/compositions prepared at such time-point of expansion will be indication specific and are potent for the said indication/disease. The pooled MSCs forming the final composition have high expression levels of bioactive factors such as cytokines and growth factors which are beneficial for the relevant indications thus making the compositions more efficacious for a specific indication/disease condition.

In an exemplary feature of the present disclosure, the method of preparing WJ-MSC based compositions broadly comprises acts of:

(a) culturing WJ- MSCs of individual donors up to a specific time-point i.e. 'passage 2 (P2)' to prepare individual donor cell banks (DCBs);

(b) carrying out analysis/characterization of each DCB at passage 2;

(c) Based on the results of aforesaid analysis/characterization at passage 2, the cells of DCBs are selectively pooled and expanded to prepare indication-specific working cell banks (WCBs) at passage 4;

(d) expansion of WCBs up to specific time-point i.e. 'passage 6 (P6)' and preparation of the final composition. In an embodiment of the present disclosure, the MSCs are derived from human donors selected from male, female, or a combination thereof. In another embodiment, the number of donors is at least two. In yet another embodiment, the number of donors can be three, four, five, six, seven, eight, nine or ten, and so on, as suitable.

In an embodiment, the MSCs of final composition (at P6) is further analysed/characterized for clinical potency.

In another embodiment of the present disclosure, the MSCs of DCB and the final composition are analyzed/ characterized for characteristics selected from a group comprising proliferation rate/ growth kinetics (population doubling and population doubling time), gender (male, female or mixed), immunomodulatory analysis (immunogenicity and immunosuppression analysis), gene expression (expression related to chondrogenesis, immunomodulation /angiogenesis etc.), secretome analysis (secretion of cytokines and growth factors), tumorigenicity assay, trilineage potential, surface marker characterization, STR analysis, clonal expression and karyotype analysis, or any combination of characteristics thereof.

In an embodiment of the present disclosure, the MSCs of DCB is characterized at passage 2 for characteristics- growth kinetics, gender, gene expression, secretome analysis and immunosuppression. In another embodiment of the present disclosure, the MSCs of final composition is characterized for characteristics- growth kinetics, gene expression, secretome analysis, immunosuppression, trilineage potential, surface marker characteristics, clonal expansion, karyotype analysis and STR analysis, or any combination thereof. In an embodiment of the present disclosure, the WCB comprises 'pooled' WJ-MSCs wherein the WCB pool is prepared based on the analysis/characterization of each DCB as described above. Further, said WCB is selected from a female pool (i.e. the pooled WJ-MSCs are from female DCBs), male pool (i.e. the pooled WJ-MSCs are from male DCBs) or mixed pool (i.e. the pooled WJ-MSCs are a combination male and female DCBs).

The present disclosure further provides for stem cell based pooled compositions targeting specific indications/diseases. More particularly, the disclosure provides potent indication specific pooled WJ-MSC compositions. In another non-limiting embodiment of the present disclosure, the compositions are prepared by the present method for indication/disease selected from a group including but not limited to Rheumatoid arthritis, Diabetes Mellitus (DM), graft-versus-host disease (GVHD), Diabetic Foot Ulcer (DFU), Critical limb Ischemia (CLI), Acute myocardial infarction (AMI), Liver cirrhosis (LC), stroke and Multiple sclerosis (MS) or any combination thereof. In an exemplary embodiment, preparation of Rheumatoid arthritis Working Cell Bank (WCB-RA), Diabetes Mellitus (WCB-DM) and Critical limb Ischemia Working Cell Bank (WCB-CLI) have been disclosed in the present disclosure.

In an embodiment of the present disclosure, the final composition is in the form of a cell suspension.

In another embodiment of the present disclosure, the pooled cell population of WCB is cryopreserved in known commercial or in-house developed cryopreservation solution. The in-house cryo-formulation comprises of ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent.

In yet another embodiment of the present disclosure, the ionic buffer is selected from a group comprising PlasmaLyte A, ringer lactate solution, sucrose, and combinations thereof; the cryoprotectant is selected from a group comprising dimethylsulfoxide (DMSO), glycerol, triglycerol, poly-L-lysine, and combinations thereof; the protein is selected from a group comprising human serum albumin, recombinant plant derived xenofree human serum albumin, and a combination thereof; the energy substrate is selected from a group comprising trehalose, hydroethyl starch, and a combination thereof; and the anti-aging agent is selected from a group comprising L-Glutamine, poly-L-lysine, ectoine, and combinations thereof.

In still another embodiment of the present disclosure, the pooled cell population of WCB is cryopreserved in commercial available cryopreservation/cryo-formulation solution selected from but not limited to animal protein-free, defined cryopreservation medium CryoStor™ CS2, CryoStor™ CS5, CryoStor™ CS10, from Biolife solution and other known cryopreservation solutions used in the art can be used without limitation. In still another embodiment of the present disclosure, the P6 expanded indication - specific pooled cell population of 10 million to 25 million is cryopreserved in 1ml of the either in- house or commercially available cryopreservation solution. It is to be noted here that isolating or obtaining mesenchymal stem cells from a donor does not involve operation or surgery or any invasive means/methods performed by doctors or medical practitioners. In other words, a person having average skill in the field of stem cell technology can obtain or isolate mesenchymal stem cells from discarded umbilical cords using means which does not involve or require invasive steps (such as surgery) or the intervention of a doctor or a medical practitioner. For instance, in the present disclosure, wharton's jelly derived mesenchymal stem cells (WJ-MSCs) are isolated from discarded umbilical cords (obtained after child birth) by the inventors without performing any step of surgery or any means practiced by doctors or medical practitioners. Alternatively, mesenchymal stem cells can be obtained from sources including but non-limiting to stem cell banks- a repository of stem cells, as gifts from collaborating laboratories or a scientific person in this field of technology, and so on, but does not include any surgical/invasive step for obtaining said stem cells. Further, all the aforementioned approaches or sources of isolating/obtaining stem cells are within the scope of the present disclosure. In an embodiment of the present disclosure, the WJ-MSC is obtained from a cell bank. The term "Bank" in the present disclosure means source for obtaining the WJ-MSC. In an embodiment, such source is pre-processed and/or cryopreserved WJ-MSC or umbilical cords, stored for instant or future use. In an embodiment, the source is a Mesenchymal Stem Cell Bank. In embodiments of the invention, any depository that stores umbilical cord or WJ-MSC for a pre-determined period of time is a bank. Further, any medium such as apparatus or device or vessel or container that stores WJ-MSC for a pre-determined period of time is considered to be a bank. In an embodiment, information about the bank is procured from Cord blood registry. In another embodiment of the present disclosure, the WJ MSCs are isolated from the umbilical cords by enzymatic method. In yet another embodiment of the present disclosure, the WJ MSCs are isolated from the umbilical cords by explant culture method. In a preferable embodiment, the isolation by the enzymatic method is carried out after dissection and removal of the umbilical cord arteries, vein and amniotic epithelium. The cords are cut into 2-3 cm segments after disinfecting the cords. MSCs isolation from WJ cord is achieved by enzymatic dissociation with collagenase- 1 and hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) at about 37°C for about 16 hours followed by trypsin digestion at about 37°C for about lOmins for generation of WJ cell pellets. Suspended cell pellets are thereafter seeded onto tissue cultured pre-coated flask with MesenCultTM-XF Attachment Substrate, in MesenCult™-XF. Complete medium (XF medium), consisting of MesenCult™- XF Basal medium supplemented with about 20% MesenCult™-XF Proliferation Supplement (all from STEMCELL Technologies TM Inc., Vancouver BC, Canada), L-Glutamine to a final concentration of about 2mM, about 100 U/ml penicillin and about 100 μg/ml streptomycin. Cultures of WJ tissue suspended cell pellets are maintained at about 37 °C with about 5% C0₂ and about 21% 0₂ in a humidified atmosphere. For optimal cell growth, medium is changed at about 50% confluency and harvested at about 80% confluence.

While preferred embodiments have been shown and described, various modifications and substitutions may be made thereto without departing from the spirit and scope of the present disclosure. Accordingly, it is to be understood that apart from the preparation of WCBs or final compositions towards Rheumatoid arthritis, Diabetes Mellitus and Critical Limb Ischemia as provided in the below examples section, the method of the present disclosure can be employed to prepare WCBs/final compositions for treatment of all indications involving stem cell therapy and the same fall within the scope of the present disclosure.

In an embodiment of the present disclosure, the technology of the instant application is further elaborated with the help of following examples and figures. However, the examples should not be construed to limit the scope of the disclosure.

EXAMPLES

Example 1:

Isolation of WJ-MSCs and establishment of Donor Cell Banks (DCBs):

Fresh and healthy human umbilical cords are obtained with informed consent using the guidelines approved by the Institutional Committee for Stem Cell Research and Therapy (ICSCRT) and Institutional Ethics Committee (IEC), at the Manipal Hospital, Bangalore, India. Six umbilical cords named as N, O, P, Q, R, S are used for the present process. Further, this process can use any number of umbilical cords without limitation. The WJ-MSCs are isolated from each of these cords by non-surgical/non-invasive method as per the below protocol to establish 6 Donor Cell Banks (DCB). Thereafter, each individual DCB is characterized for population doubling time, gender, immunogenicity, immunosuppressive properties, gene expression and cytokines secretion (secretome analysis).

The procedure followed for the establishment of DCBs is as follows:

The Wharton's Jelly derived MSC from umbilical cords are isolated by explant culture method and/ or enzymatic digestion method (i.e. non-surgical methods). As an alternative, WJ-MSCs can also be obtained non-surgically) from Cell Banks or Depositories that store WJ-MSCs for predetermined period of time.

In the present example, explant culture method is employed for isolation of WJ MSCs from umbilical cord. Explants culture is based on the mesenchymal migratory capabilities and is also referred to as "plate and wait" method wherein the cord segments are simply plated in the culture medium and the MSCs are obtained by waiting for them to migrate out. The explant culture method is preferred as it is simple, reliable and less laborious, and yielding pure and viable cultures of human umbilical cord derived WJ-MSCs.

In the explant culture method followed in the present example, the individual cords measuring about 5-10 cms are used. The cords collected are subjected to multiple wash using normal saline (about 0.9% w/v Sodium Chloride) to clean the cord and remove all the blood clots. The cords are then cut into multiple bits and placed in normal saline with anti-anti and kept in the refrigerator for about half an hour. Before processing, the cords are given a quick rinse with about 70% ethanol for not more than about 30 seconds and followed by rinsing with normal saline couple of times.

The cords are then dissected longitudinally and the inner part of the cord is exposed. After removing the veins and arteries manually, the cords are cut into small pieces of size of about 0.4 to 0.7 cms. Approximately 40 to 50 such pieces are placed in 100 mm tissue culture dish containing serum free culture media. The explants are left undisturbed in 5% C0₂ incubator at about 37°C for about 3-4 days, after which fresh culture medium is added to the culture dishes. By this time, the MSCs start to migrate out and grow in the dishes. The MSCs isolated at passage 0 (P0) are further passaged to P2 to eliminate any other tissue and to obtain pure cell population. The cells at P2 are cryopreserved and stored. The WJ-MSCs from each individual cord are cryopreserved at P2 and are referred to as Donor Cell Banks (DCBs).

From the above described methods, it is evident that the isolation of the WJ-MSCs used in the present disclosure does not involve invasive or surgical procedures as the explant culture method and the enzymatic digestion method do not require the intervention/performance of operation or surgery by a doctor or a medical practitioner. Alternatively, the WJ-MSCs used in the present method can also be sourced from depositories or cell banks.

Example 2:

Characterization of DCB for pooling to prepare indication specific working cell bank (WCB):

After the establishment of DCBs at P2, each of the DCBs is extensively characterized based on their proliferation kinetics/ growth kinetics, gender, immunogenicity, immunosuppressive properties, gene expression and secretome analysis. Depending on the disease/indication which needs to be treated, the DCBs showing similar properties are selected to be pooled together for establishing indication specific WCB at P4. Depending on requirements, WCB is expanded up to passage 6 to prepare final product- IMP.

For instance, to treat auto immune diseases like Rheumatoid arthritis (RA), the individual DCBs which have higher expression of anti-inflammatory gene (IL-10, TGFP) and low expression of pro-inflammatory genes (IL-Ιβ, TNFa ) as assayed at gene level and on secretomes level, are chosen along with population doubling and similar gender to be pooled.

WJ-MSCs from the prepared WCBs are expanded to P6 and are further characterized to analyse their proliferation kinetics/growth kinetics, trilineage potential, expression of MSC markers, immunogenicity, immunosuppressive properties, gene expression (especially anti and pro inflammatory markers), secretome analysis, and tumorigenicity assay. The characterization of the cells at passage 6 is an optional step which is performed to check the potency of the cells for clinical use. Figure 1 gives the illustration for the concept of pooling DCBs based on selected characteristics for preparing and establishing indication specific WCB for various indications and manufacturing indication specific cell product for clinical application. The illustration in figure 1 shows that the cells from 6 different cords are isolated to prepare DCB. The DCB cells are characterized at passage 2 for their population doubling (PD), gender, gene expression, secretome analysis etc. Based on the PD and gender, the first level screening for selection is performed. This is followed by gene expression analysis to select DCB for pooling to prepare an indication specific WCB at P4. Thereafter, the pooled WCB cells are expanded up to passage P6 (based on requirement) to prepare F P and P6 cells are extensively characterized to establish the potency for clinical application.

The analysis/characterization of DCBs is carried out as follows: (i) Growth kinetics (population doubling and population doubling time):

The population doubling time, number of population doublings in a passage and the cumulative population doubling over the passages are calculated. Thereafter, at least 2 DCBs having similar values for the above said parameters are pooled together. The 6 cords are cultured in serum free and xeno free culture media (MesenCult™-XF Basal medium, 20% MesenCult™-XF Proliferation Supplement, L-Glutamine, penicillin and streptomycin). Figure 3A shows the proliferation rate of pooled and individual samples. Figure 3A and table 1 shows the growth/ proliferation kinetics of the WJ-MSC from each DCB. Their mean population doubling time (PDT) and cumulative population doubling (CPD) is 28.8 ± 4.09 and 18.07 ± 1.21 respectively. These parameters are estimated for each DCB in order to compare with the parameters of the WCBs established after indication specific pooling of the DCBs.

Table 1 : Population kinetics of WJ-MSCs from each DCB

o 26.83 17.94

P 24.97 18.58

Q 25.36 18.86

R 29.76 18.11

S 29.14 19.89

(ii) Gender:

The gender details of the baby are collected during the birth. The cords of the same gender are pooled and expanded. Table 2 gives the details of the gender of the 6 DCB.

Table 2: Details of the gender of DCBs

(iii) Gene expression: RNA isolated from MSC of each individual DCBs are analyzed for their gene expression related to pro and anti inflammatory properties/angiogenesis / disease specificity, using Real time PCR. Figures 8a to 8g show the gene expression profile of individual and pooled samples.

(iv) Secretome analysis:

The conditioned media collected from MSCs of each DCB samples are analyzed for the presence of cytokines and growth factors by ELISA method. DCB showing similar secretomes are selected for pooling. For example, to treat CLI, the individuals that show high level of VEGF secretion are pooled together. Figure 9 shows the secretome analysis of individual and pooled samples.

(v) Immunosuppression: The Mixed Lymphocyte Reaction (MLR) is a functional assay which measures the proliferative response of lymphocytes from one donor (the responder) to lymphocytes from another donor (the stimulator). MSCs are known to be immunosuppressive and their ability to suppress an immune reaction is tested by one way or two way MLR. For stimulation assays, MSCs treated with 10 mg/ml Mitomycin C (Sigma-Aldrich) for about 2.5 h are used as stimulator cells and allogeneic peripheral blood mononuclear cells (PBMC) isolated by density gradient centrifugation are used as responder cells. MSCs are seeded in 96-well plates (Corning) and allowed to attach overnight. Responder PBMC are then added to each well, at a ratio of about 1 :2.5 (MSCs: Responder) and the cultures are maintained for about 5 days, at which point, they are pulsed with 5-bromo-2-deoxyuridine (BrdU) for the final 24 h. Cell proliferation is measured using a fluorimetric immunoassay kit (Calbiochem) for the quantification of BrdU incorporation, according to the manufacturer's instructions. One-way mixed lymphocyte reactions (MLR) at the corresponding stimulator: responder ratio are used as positive controls for PBMC proliferation in response to alloantigen. For immunosuppression assays, Mitomycin C-treated MSCs are seeded in 96-well plates and allowed to attach overnight. A one-way MLR at a ratio of about 1 :2.5 is then added to each well. Lymphocyte proliferation is measured at the end of 5 days, as described above. A oneway MLR cultured in the absence of MSCs is considered as the 100% proliferation control. Four individual donors are analyzed in each group. All treatments are performed in triplicate wells in RPMI 1640 medium (Invitrogen) supplemented with about 10% FBS (Hyclone), about 2 mM glutamine (Invitrogen) and about 0.05 mM β-mercaptoethanol (Sigma-Aldrich).

WJ-MSCs exhibit hypo immunogenicity and arrest the growth of T cell proliferation in dose dependent manner when co cultured with peripheral blood mononuclear cells (PBMCs). Also WJ-MSCs suppress mixed lymphocyte reaction (MLR) where PBMCs from mismatched donors are co cultured with the MSCs. (fig 7 Figure 7 shows the immunosuppressive properties of individual samples.

Based on these characterization experiments and results, the final selection of DCBs to be pooled is decided based on the disease indications.

Table 3 gives the list of gene expression and secretome analysis which is considered for each indication/ disease condition to establish the pooled WCB. Table 3 : List of gene expression and secretome analysis considered for various indications/ disease conditions

Example 3:

Pooling of WJ-MSCs of characterized DCBs and establishment of indication specific WCBs

Based on the results observed from the above analysis (Example 2), the DCBs at passage 2 (P2) are selected and segregated for pooling. The selected DCBs are further expanded till passage 4 (P). The WJ-MSCs from selected DCBs are then pooled in equal proportion to prepare indication specific working cell banks at passage 4. The three WCB pools prepared based on the characterization of the MSC from DCB are RS, OPQ and QRS. The RS pool, N, R and S are female cords with high population doubling time (mean = 29.24); in the pool OPQ, the O, P and Q are male cords and have low doubling time (mean = 25.72). QRS is a mixed pool, where the individual DCB from male and female donors having low and high population doublings are pooled. Apart from population doubling time, other characteristics as described in Example 2 are also considered for pooling.

The pooled cells (WCBs) are plated and expanded till P6 to prepare final product. At P6, pooled samples are assayed for certain selected characteristics as mentioned in the DCB characterization at P2 along with a few additional assays. MSC of final composition (at P6) is further analysed/characterized for clinical potency. The said characterization of final product at P6 is described below: (i) Growth kinetics of pooled MSCs

The average Cumulative Population Doubling (CPD) of all the three pools (NRS, OPQ and QRS) is 16.05 ± 0.384. Similarly, the average PD and PDT at passage 6 is 4.22 ±0.496622 and 34.43667± 3.79961 respectively. Average total cell number at the end of Passage 5 for the pooled and upscaled cells is 1.81X10¹¹ ± 0.49X10¹¹. Figure 3(a) shows the proliferation rate of individual and pooled samples. Tables 4a and 4b gives the result of growth kinetics of the pooled samples.

Table 4a: Growth kinetics of individual samples

Table 4b: Growth kinetics of pooled samples

From the tables 4a and 4b, it is evident that pooling of the MSCs reduces the variability and maintains uniformity in the growth kinetics of individual samples. (ii) Gene expression

RNA extracted from individual and pooled WJ-MSCs are reverse transcribed and the gene expression is analyzed using Real time PCR. Figure 8a to 8g shows the gene expression profiles of individual and pooled samples. Figure 8a shows that the expression pattern of pooled MSCs averages out the individual values, but in some the pooling is advantageous - as like for IL-lb, the pooled samples have lesser expression of this pro inflammatory gene. Further, figures 8(e) and 8(f) summarizes the gene expression profile of pooled samples (NRS, OPQ and QRS) wherein it is shown that pooling overall brings down the expression of all genes in female (NRS) and male (OPQ) pools whereas the mixed pool (QRS) have mixed results and mostly averages out the individuals. From figure 8(g), it is seen that on comparison with the individual donor cells, pooled samples have normalized expression profiles, and are thus better in clinical applications vis-a-vis individual donor cells.

(iii) Secretome analysis:

The conditioned media collected from the individual WJ-MSC from DCB and pooled WJ-MSCs from WCBs are analyzed for the various cytokines such as HGF and TGF- βΐ . Figure 9 shows the secretome analysis of individual and pooled samples. It is found that pooling increases the HGF secretion and in case of TGF- βΐ, the pooled sample averages out the individual values. On a whole, it is observed from the comparison of the individual versus pooled cells that the characteristics of the pooled samples are normalized and therefore can be accordingly employed for further applications.

(iv) Immunosuppression

The immunosuppressive properties of WJ-MSCs are maintained even after pooling and even outweigh the individual immunosuppressive capacities. For example, at passage 6 and at about 1 :2.5 ratio of MSCs to responder (i.e. PBMCs isolated from another donor), the pooled MSCs show higher immunosuppression than the individuals. Thus, pooling affects the immunosuppression and the WCBs and final cell product prepared are highly immunosuppressive. Figure 7 shows the immunosuppressive properties of individual samples at P4 and P6. Figure 9 shows the secretome analysis of individual and pooled cells (N, R, S and NRS; O, P, Q and OPQ and Q, R, S and QRS) condition media collected at passage 6, with respect to a 'control' sample. This shows that the pooling of the MSCs normalizes the expression of cytokines- HGF and TGF-β at P6. (v) Trilineage potential of WJ-MSCs

The pooled WJ-MSCs form adipocytes, osteocytes and chondrocytes as similar to individual MSCs and thus their differentiation capacity is not affected by the pooling as shown in the figure 5. (vi) Surface marker characterization

Similar to that of individual MSCs, the pooled MSCs are positive for CD 90, 73 and 105 but negative for hematopoietic marker and class II MHCs. Figure 4 shows surface marker characterization of pooled cells. Table 5 provides the average of expression of various markers in all three pools.

Table 5: Average of expression of various markers in all three pools

(vii) Clonal expansion assay

To test the colony forming capacity of the MSCs, the cells are plated at a density of 100 cells per 35mm dish. Complete media change is carried out every third day. The cells are incubated for about 8 days, at which point they are fixed and stained with 0.1% Toluidene Blue in 1% Paraformaldehyde (all reagents from Sigma-Aldrich), to visualize the colonies. Stained colonies are manually counted. The assay for each sample is carried out in triplicates.

Pooled and upscaled cells in xenofree & serum free medium show good CFU-F numbers during prolonged expansion and retain the clonal formation efficacy throughout their expanded passages (figure 3b).

(viii) Karyotype analysis

Karyotype analysis of the pooled samples reveals no chromosomal abnormalities. Figure 6a shows the karyotype analysis of pooled samples.

(ix) STR analysis: Short tandem repeat (STR) analysis is a molecular biology method used to compare specific loci on DNA from different donors. A short tandem repeat is a microsatellite, consisting of a unit of two to thirteen nucleotides repeated hundreds of times in a row on the DNA strand. STR analysis measures the exact number of repeating units. This method differs from restriction fragment length polymorphism analysis (RFLP) since STR analysis does not cut the DNA with restriction enzymes. Instead, probes are attached to desired regions on the DNA, and a polymerase chain reaction (PCR) is employed to discover the lengths of the short tandem repeats. Number of repeats for particular allele is unique for each donor. In order to determine the composition and traceability STR analysis is performed for the individual and pooled samples. Figure 10 shows that the pooled sample has representation of all three donors. Therefore, the method of the present disclosure aims at establishing improved stem cell compositions targeting specific disease conditions wherein the method uses the concept of pooling of stem cells from individual donors based on stem cell characteristics. Cryopreservation of the composition:

The pooled cell population of WCB is cryopreserved in known commercial or in-house developed cryopreservation solution. The in-house cryo-formulation comprises of ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent. The ionic buffer is selected from a group comprising PlasmaLyte A, ringer lactate solution, sucrose, and combinations thereof; the cryoprotectant is selected from a group comprising dimethylsulfoxide (DMSO), glycerol, triglycerol, poly-L-lysine, and combinations thereof; the protein is selected from a group comprising human serum albumin, recombinant plant derived xenofree human serum albumin, and a combination thereof; the energy substrate is selected from a group comprising trehalose, hydroethyl starch, and a combination thereof; and the anti-aging agent is selected from a group comprising L-Glutamine, poly-L-lysine, ectoine, and combinations thereof.

Alternatively, the pooled cell population of WCB can be cryopreserved in commercial available cryopreservation/cryo-formulation solution selected from but not limited to animal protein-free, defined cryopreservation medium CryoStor™ CS2, CryoStor™ CS5, CryoStor™ CS10, from Biolife solution and other known cryopreservation solutions used in the art can be used without limitation. The P6 expanded indication- specific pooled cell population of 10 million to 25 million is cryopreserved in 1ml of the either in-house or commercially available cryopreservation solution.

ADVANTAGES OF THE POOLED MSC-BASED COMPOSITION OF THE PRESENT DISCLOSURE AND METHOD OF PREPARING THE SAME:

1. The culture conditions for culturing of the MSCs are optimized which maximizes the yield of MSCs from WJ of umbilical cord within a reasonable time frame. 2. The method for culturing MSCs are described in the present disclosure involves establishing well characterized indication specific cell banks referred to as working cells banks (WCBs). The indication specific working cell bank is used in developing the indication specific therapeutic cell products/compositions.

3. The pooled MSCs forming the final composition have high expression levels of bioactive factors such as cytokines and growth factors which are beneficial for the relevant indications thus making the compositions more efficacious for a specific disease condition.

4. Pooled MSCs show reduced variability with regards to properties such as proliferation rate, gene expression, immunosuppressive capacities etc., which are substantiated in the above examples by comparing individual samples vs. pooled samples.

5. The present disclosure provides potent indication specific pooled WJ-MSC compositions, wherein the compositions are prepared by the present method for indication/disease selected from a group including but not limited to Rheumatoid arthritis, Diabetes Mellitus (DM), graft-versus-host disease (GVHD), Diabetic Foot Ulcer (DFU), Critical limb Ischemia (CLI), Acute myocardial infarction (AMI), Liver cirrhosis (LC), stroke and Multiple sclerosis (MS) or any combination thereof.

6. The MSC- based composition of the present disclosure is obtained by pooling of the cells, wherein cells from DCBs are pooled based on the results of characterization of the DCBs (at passage 2). This method of pooling is advantageous over random pooling (i.e., which is not based on any characterization studies), since pooling minimizes variability among the cells of the DCBs, with regards to properties such as proliferation rate, gene expression, cytokine secretion, immunosuppressive capacities etc., averages out individual values and normalizes the expression profiles of the pooled cells which thus make these cells better for clinical applications.

Claims

We Claim:

1. A method of preparing pooled mesenchymal stromal cell based composition, said method comprising acts of:

2) The method as claimed in claim 1, wherein the mesenchymal stromal cells are Wharton jelly derived mesenchymal stromal cells.

3) The method as claimed in claim 1, wherein the mesenchymal stromal cells are cultured in a xeno-free culture medium comprising Basal medium, 20% Proliferation Supplement, L-Glutamine, penicillin and streptomycin.

4) The method as claimed in claim 1, wherein the first predetermined passage is passage 2.

5) The method as claimed in claim 1, wherein the characterization in step (b) is carried out by performing analyses selected from a group comprising growth kinetics, gender analysis, gene expression, secretome analysis and immunosuppression analysis, or any combination thereof.

6) The method as claimed in claim 1, wherein the pooling in step (c) is performed by combining the mesenchymal stromal cells of characterized donor cell banks (DCBs) showing similar properties.

7) The method as claimed in claim 1, wherein the second predetermined passage is passage 4.

8) The method as claimed in claim 1, wherein the pooled mesenchymal stromal cell of the composition obtained in step (d) is characterized by performing analyses selected from a group comprising gender analysis, karyotype analysis, growth kinetics, gene expression analysis, secretome analysis, immunosuppressive analysis, trilineage potential analysis, surface marker characterization, tumorigenicity, STR analysis and clonal expression, or any combination thereof.

9) The method as claimed in claim 1, wherein the third predetermined passage is passage 6.

10) The method as claimed in claim 1, wherein the pooled mesenchymal stromal cell based composition is specific to an indication selected from group comprising but not limiting to Rheumatoid arthritis, Diabetes mellitus, graft-versus-host disease, diabetic foot ulcer, acute myocardial infarction, liver cirrhosis, stroke, multiple sclerosis and critical limb ischemia, or any combination thereof.

11) A mesenchymal stromal cell based composition comprising mesenchymal stromal cells and cryopreservation medium wherein said composition is prepared by the method as claimed in claim 1.

12) The composition as claimed in claim 11, wherein the cryopreservation medium is animal protein-free defined cryopreservation medium or a cryopreservation medium comprising ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent.

13) The composition as claimed in claim 11, wherein the composition is cryopreserved, and wherein said cryopreserved composition comprises mesenchymal stromal cells at a concentration ranging from about 10 million cells per ml to 25 million cells per ml of the medium.

14) A method of managing a disease, said method comprising act of administering the composition of claim 11 to a subject in need thereof.

15) The method as claimed in claim 14, wherein the disease is selected from a group comprising Rheumatoid arthritis, Diabetes mellitus, graft-versus-host disease, diabetic foot ulcer, acute myocardial infarction, liver cirrhosis, stroke, multiple sclerosis and critical limb ischemia, or any combination thereof.