Therapeutic compositions comprising pooled, culture-expanded human umbilical cord derived mesenchymal stromal cells

Abstract

The disclosure provides a therapeutic composition comprising pooled, culture-expanded human umbilical cord-derived mesenchymal stromal cells (hucMSCs) derived from at least four and up to twelve donors, which can be reproduced with batch to batch consistency. These cells exhibit consistent marker expression profiles, enhanced and consistent secretion of therapeutic factors, and potent immunomodulatory activities after at least ten to thirty population doublings. The composition, formulated with a pharmaceutically acceptable carrier, is designed for clinical applications addressing inflammatory, immune, and degenerative conditions.

Description

Therapeutic Compositions Comprising Pooled, Culture-Expanded Human Umbilical CordDerived Mesenchymal Stromal Cells Applicant / Assignee:INmune Bio Inc.CROSS-REFERNCE TO RELATED APPLICATIONS[1] This application claims benefit of U.S. Provisional Application Serial Number 63 / 556,999, filed February 23, 2024, titled “COMPOSITIONS COMPRISING POOLED hucMSCs FOR THERAPEUTIC USES,” and[2] further claims benefit of U.S. Provisional Application Serial Number 63 / 742,382, filed January 6, 2025, titled “COMPOSITIONS COMPRISING POOLED hucMSCs FOR THERAPEUTIC USES”; and[3] further claims benefit of U.S. Provisional Application Serial Number 63 / 756,606, filed February 10, 2025, titled “THERAPEUTIC COMPOSITIONS COMPRISING POOLED, CULTURE-EXPANDED HUMAN UMBILICAL CORD DERIVED MESENCHYMAL STROMAL CELLS”[4] the entire contents of each of which is hereby incorporated by reference for all purposes.Technical Field[5] The present invention relates to the field of regenerative medicine and cell therapy. Specifically, it pertains to therapeutic compositions comprising pooled, culture-expanded human umbilical cord-derived mesenchymal stromal cells (hucMSCs) that exhibit enhanced therapeutic efficacy and batch-to-batch consistency. These compositions are designed for use in treating inflammatory, immune, and degenerative conditions, as well as for promoting tissue repair and regeneration.Background Art[6] Mesenchymal stromal cells (MSCs) have gained significant attention in the field of medicine. These versatile cells have been extensively investigated for their therapeutic potential in various challenging medical conditions. Notably, they have shown promise in treating type 1 diabetes mellitus, systemic lupus erythematosus, rheumatoid arthritis, and Crohn’s disease due to their remarkable ability to differentiate into multiple cell types and modulate the immune response. MSCs can be derived from a diverse range of tissues, including but not limited to: umbilical cord, placenta, adipose tissue, bone marrow, gingiva, and dental pulp. However, it’s important to recognize that MSCs obtained from different tissue sources exhibit distinct characteristics. These variations manifest in their expression profiles of surface markers and biological functions, particularly in response to stimuli such as pro-inflammatory mediators. [7] The diversity observed among mesenchymal stromal cells (MSCs) complicates the comparison of therapeutic effects across different MSC medicinal products when applied in clinical settings. To establish a comprehensive definition of MSCs, the International Society for Cell & Gene Therapy (ISCT) has outlined a minimal set of standards. According to the ISCT, MSCs must meet three main criteria. First, they must exhibit plastic adherence when maintained under standard culture conditions. This means MSCs should attach to tissue culture plastic in vitro, a hallmark of their behavior in controlled environments. Second, MSCs must express a specific set of surface markers. They should express CD73, CD90, and CD105 on at least 95% of the cell population, while lacking expression of hematopoietic and endothelial markers, specifically CD34, CD45, CD14 or CD11b, CD19 or CD79α, and HLA-DR, with expression levels of these negative markers below 2%. Third, MSCs must demonstrate multipotent differentiation potential in vitro, meaning they can differentiate into osteoblasts (bone-forming cells), adipocytes (fat cells), and chondroblasts (cartilage-forming cells) when exposed to appropriate differentiation conditions. These standards aim to ensure the consistency of MSC characterization across laboratories and provide a foundation for their clinical and research applications.[8] However, these criteria have sparked ongoing debates in the field, in part, for potentially failing to sufficiently identify MSCs with adequate distinction and consistency. Each MSC product is considered unique, influenced not only by tissue origin but also by factors like culture methods and additional modulations. Consequently, products containing MSCs exhibit varying potency, gene expression profiles, growth phenotypes, and differentiation potentials.[9] To further complicate development of MSC -derived products, each donor of cells from which the MSC therapeutic product is made varies, as distinct sources will naturally express distinct biological characteristics. Summary of InventionTechnical Problem

[10] While the use of mesenchymal stromal cells (MSCs) to treat a variety of indications has gained some positive effect in certain pre-clinical and clinical trials, others have shown no- or negative effects. There remains significant uncertainty as to the therapeutic value of products containing MSCs as a therapeutic option. Although the ISCT has created standards aiming to provide some improvement in consistency between MSC -based products, there remains significant variability between products containing MSCs, especially when viewed batch-to-batch. Some problems include inability to sustain potency and consistency after multiple population doublings, variability of MSC product surface marker expression characteristics, and intensity of surface marker expression, both as-between various products and for the same product when viewed batch-to-batch. Other problems with conventional MSC products include limited number of cell divisions ultimately resulting in senescent cells after a finite number of population doublings. For example, adult derived stromal cells are often shown to be senescent after only about ten population doublings. This results in a maximum batch size from a single donor MSC product, and subsequent batches derived from a new donor are likely to reflect a change in potency characteristics, leading to batch-to-batch variability.Solution to Problem

[11] In various aspects, pooled, culture-expanded human umbilical cord -derived mesenchymal stromal cells (hucMSCs) for therapeutic uses are described. The pooled, culture-expanded hucMSCs are superior to conventional MSC products because they are more consistent batch to batch, presenting less variability, including less variability associated with surface markers CD73, CD90, CD105, CD14 / CD11b, CD34, CD45, CD79α / CD19, and HLA-DR, and additionally, with respect to surface markers CD10, CD29, CD44, CD142, CD146, CD166, CD200, CD271, and MSCA-1, as well as the expression intensity of these markers. The pooled, culture-expanded hucMSCs herein also secrete therapeutic factors more consistently when compared to single-donor MSC-based products.

[12] Whilst MSC-based products from single donor umbilical cords show high variability in surface marker expression and across multiple potency assays, the effect of pooling at least four of these individual umbilical cord products generates a more homogenous product and this homogeneity is maintained between batches generated from different pools of at least four donors.

[13] In an exemplary embodiment, a therapeutic product includes pooled, culture-expanded hucMSCs comprising cells from at least two (preferably four) and up to twelve umbilical cord donors; wherein surface markers CD29, CD44, CD73, CD90, and CD105, are positively expressed on a surface of at least 97% of cells of said pooled, culture-expanded hucMSCs, wherein surface marker CD166 is positively expressed on a surface of at least 85% of cells of said pooled, culture-expanded hucMSCs, wherein surface marker CD146 is positively expressed on a surface of between 30% and 75% (inclusive) of cells of said pooled, culture-expanded hucMSCs, and wherein said pooled, culture-expanded hucMSCs are essentially free of surface markers CD11b, CD14, CD19, CD34, CD36, CD45, CD79α, and HLA-DR; which therapeutic product can be repeatedly and consistently produced batch to batch independent of donor cord selection. The therapeutic product further exhibits consistent secretion of therapeutic factors for providing reliable therapeutic potency. Other advantages and characteristics are described herein.Advantageous Effects of Invention

[14] Batch to batch consistency of the MSC product is required to harmonize and render predictable the therapeutic effect of the product across multiple patients within a population, and across multiple patient populations, as well as between multiple doses of the MSC product with the same patient. The improvement of reduced variability of surface marker expression and therapeutic factor secretion that is achieved by the pooled, culture-expanded hucMSCs described herein, provides a corresponding batch-to-batch consistency that is expected to transfer reliability of therapeutic efficacy across patients and doses.

[15] There may be synergistic and other effects associated with the pooled, culture-expanded hucMSCs described herein, including not only consistency of surface marker expression, but also increased rate of doublings and expanded number of useful passages before cells become senescent, and enhanced therapeutic effectiveness.

[16] Additionally, there are manufacturing benefits, including shorter time required to reach intended population doublings, fewer passages, and less time and cost to manufacture.

[17] Moreover, the hucMSCs can be tuned to express optimized expression profiles, or to exhibit desired potency traits such as secretome profile or chemokine honing ability, among other characteristic features, as would be desirable for specific applications or disease indications.Brief Description of Drawings

[18] Detailed discussion of embodiments directed to one of ordinary skill in the art are set forth in the specification, which makes reference to the appended figures, in which:

[19] FIG.1 shows MSC product characteristics at CPD10 for single- and pooled- MSC products according to the examples herein.

[20] FIG.2 shows MSC product characteristics at CPD30 for single- and pooled- MSC products according to the examples herein.

[21] FIG.3 shows mean fluorescence intensity of surface markers at CPD10 for single- and pooled- MSC products according to the examples herein.

[22] FIG.4 shows mean fluorescence intensity of surface markers at CPD30 for single- and pooled- MSC products according to the examples herein.

[23] FIG.5 shows mean fluorescence intensity of surface markers consistency at CPD30 for single- and pooled- MSC products according to the examples herein.

[24] FIG.6 shows pooled MSCs products of the examples required less time to reach CPD30, which translates into less time and costs associated with manufacturing.

[25] FIG.7 illustrates less variance by passage number between pooled MSCs products versus single donor MSCs according to the examples.

[26] FIG.8 shows less variance with respect to secretome in the pooled MSCs batches versus the single-donor MSCs according to the examples.

[27] FIG.9 shows actual and predicted VEGF secretion in the single and pooled MSCs products of the examples, indicating that pooling provides a mechanism to harmonize expected secretion of various therapeutic factors, making for consistent products batch-to-batch independent of donor selection.

[28] FIG.10 shows improved potency of the pooled MSCs products of the examples compared to single-donor MSCs.

[29] FIG.11 shows results of a scratch test indicating the pooled MSCs products provide improved wound healing function compared to negative control.

[30] FIG.12 further shows results of a scratch test indicating the single-donor MSCs products provide improved wound healing function compared to negative control, but substantially less improvement compared to pooled MSCs.

[31] FIG.13 shows a reduction in T-Cell suppression in the pooled MSCs products of the examples compared to the single-donor MSCs products.

[32] FIG.14 shows a list of disease indications that may benefit from a pooled, culture expanded hucMSCs product, and key surface markers of cells the product should be enriched for and markers for cells to avoid.

[33] FIG.15 shows the ideal characterization and surface marker expression of cells in a pooled, culture-expanded hucMSCs composition for general therapeutic uses according to embodiments herein.DESCRIPTION OF EMBODIMENTS

[34] Reference now will be made in detail to embodiments, one or more examples of which are illustrated in the drawings or set forth in the appended descriptions. Each example is provided by way of explanation of the embodiments, not limitation of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments without departing from the spirit and scope of the present disclosure. For instance, features illustrated or described as part of one embodiment or example can be used with another embodiment or example to yield a still further embodiment. Thus, it is intended that aspects of the present disclosure cover such modifications and variations.

[35] Example aspects of the present disclosure are directed to a therapeutic composition comprising pooled, culture-expanded human umbilical cord -derived mesenchymal stromal cells (hucMSCs), said pooled, culture-expanded hucMSCs comprising cells from at least four and up to twelve donors; wherein surface markers CD29, CD44, CD73, CD90, and CD105, are positively expressed on a surface of at least 97% of cells of said pooled, culture-expanded hucMSCs, wherein surface marker CD166 is positively expressed on a surface of at least 85% of cells of said pooled, culture-expanded hucMSCs, wherein surface marker CD146 is positively expressed on a surface of between 30% and 75% (inclusive) of cells of said pooled, culture-expanded hucMSCs, and wherein said pooled, culture-expanded hucMSCs are essentially free of surface markers CD11b, CD14, CD19, CD34, CD45, CD79α, and HLA-DR; which therapeutic product can be repeatedly and consistently produced batch to batch independent of donor cord selection. The therapeutic product further exhibits consistent secretion of therapeutic factors for providing reliable therapeutic potency. Other advantages and characteristics are described herein.

[36] For purposes herein, the following terms are given the respective meanings:

[37] “essentially free of,” when used in connection with the expression or absence of one or more surface marker proteins on a cell, including but not limited to surface marker proteins CD11b, CD14, CD19, CD34, CD45, CD79α, and HLA-DR, means, for each surface marker protein, less than or equal to two percent (2%) of cells of a given population express the respective surface marker proteins;

[38] “therapeutic composition” means a substance, other than food, used in the prevention, alleviation, treatment, or cure of disease in a subject;

[39] “pooled, culture expanded” means combined and expanded, and more specifically in the context of the enclosed examples, means cells from at least two distinct donors combined together, and the pooled or combined cells are expanded in ex vivo cell culture at least one and up to fifty passages;

[40] “umbilical-cord -derived MSCs” means mesenchymal stromal cells that are obtained from an umbilical cord of a human donor at full term gestation, typically from Wharton’s jelly (substantia gelatinea funiculi umbilicalis), and their culture-expanded progeny; and

[41] “donor” means a human being from which an umbilical cord is obtained.

[42] It is known that cell-based therapies can carry the potential risk of transmitting infectious diseases. The quality and function of mesenchymal stromal cells (MSCs) can be influenced by factors such as the donor’s age, health condition, and the method of delivery. Therefore, a rigorous donor screening process is essential before collecting samples. This screening includes a thorough physical examination, detailed medical history assessment, and testing for infectious diseases. Additionally, a serological test for infectious diseases should be conducted at a reasonable time after umbilical cord donation to exclude any potential window phase of viral infections.

[43] Mesenchymal stromal cells for therapeutic applications are required to be produced in controlled environments following current Good Manufacturing Practice (cGMP) guidelines. The critical raw materials and reagents used in MSC culturing generally include fetal bovine serum (FBS), Tryple, and culture medium. Other materials can be used and are generally known by one having skill in the art.

[44] Human umbilical cord mesenchymal stromal cells (hucMSCs) are generally derived from Wharton’s jelly of a human umbilical cord. This specialized tissue contains primitive and pluripotent cells. hucMSCs are typically produced as follows:

[45] Isolation:

[46] The umbilical cord is collected after full-term childbirth. The umbilical cord is typically sectioned and dissected in segments about 5mm in length. Cells are isolated from the Wharton’s jelly using enzymatic digestion or other methods.

[47] Culture and Expansion:

[48] The isolated hucMSCs are cultured in a controlled environment. They are expanded through multiple passages to obtain sufficient cell numbers.

[49] Characterization:

[50] The cells are characterized based on specific expression of surface markers (such as, for e.g., CD73, CD90, CD105) using techniques like flow cytometry. Their differentiation potential into various cell types (adipocytes, osteoblasts, and chondrocytes) may also be assessed. MSCs are generally plastic adherent cells, which positively express the surface markers CD73, CD90, CD105 on their surface and do not express the surface markers CD34, CD45, and HLA-DR.

[51] Quality Control:

[52] The hucMSCs undergo rigorous quality control to ensure safety and efficacy. This includes testing for infectious diseases, sterility, and other relevant parameters. In addition, characteristics can be determined both for individual cord -derived and pooled cells, including characteristics such as surface marker expression profile, intensity of surface marker expression by median fluorescence intensity ratio (MFIR) or other measure, and potency such as, inter alia, IDO activity using in vitro assays.

[53] Storage and Cryopreservation:

[54] The hucMSCs can be cryopreserved for future use. Proper storage conditions are maintained to preserve cell viability.

[55] Pooled mesenchymal stromal cells (MSCs) are produced by combining cells from multiple individual donor cords. Pooled, culture-expanded hucMSCs are generally produced as follows:

[56] Isolation and Characterization:

[57] MSCs are isolated from different donor cords. Prior to pooling, the MSCs from single-donor seed stocks are each characterized based on their properties, such as, but not limited to, surface marker expression, surface marker intensity of expression, therapeutic factor secretion, differentiation potential, among other things. The cells are selected based on these characteristics and the desired properties of the pooled product. Computational models can be utilized for selecting individual MSCs seed stocks for pooling with the goal of producing a pooled product that exhibits desired characteristics.

[58] For example and not limitation, the indication treating osteoarthritis is expected to require a pooled MSC product that demonstrates strong anti-inflammatory properties and good differentiation to chondrocytes in support of cartilage repair. Therefore, characteristics like secretion of therapeutic factors such as interleukin-10 (IL-10) and prostaglandin E2 (PGE2), and strong chondrocyte differentiation potential, would be ideal for use in identifying optimal single-donor seed stocks to combine in a pooled product intended to treat osteoarthritis. MSCs with high expression of surface markers such as CD73, CD90, CD44, CD146, and HLA-G tend to have a more potent immunosuppressive phenotype and are likely to secrete higher levels of IL-10 and PGE2, especially in inflammatory environments such as those in osteoarthritis. Therefore, selecting from available single-donor seed stocks those with such characteristics will provide the optimal combination for pooling.

[59] Other indications may require optimization of other characteristics, such as therapeutic factor secretion, phenotype differentiation potential, immunomodulatory effects, and the like.

[60] Pooling Strategy:

[61] The pooled MSCs are obtained by combining cells from multiple donor cords, as mentioned above, by selecting from single-donor seed stocks those with optimal indication-specific characteristics. This strategy minimizes donor-to-donor heterogeneity at least with respect to the desired characteristics and ensures consistency in biological properties of the pooled MSC containing product.

[62] When selecting single-donor mesenchymal stromal cells for pooling to create a therapeutic product, several key surface markers and their corresponding biological effects must be considered. The core identity of MSCs, as defined by the ISCT, is confirmed through positive expression of CD73, CD90, and CD105. These cells should also lack markers such as CD14, CD19, CD34, CD45, and HLA-DR to ensure purity and avoid contamination with hematopoietic or endothelial cells.

[63] Markers associated with immunomodulatory potential are crucial for therapeutic applications. High expression of CD146 (MCAM) correlates with increased secretion of anti-inflammatory cytokines like IL-10 and prostaglandin E2 (PGE2) and enhances homing to inflamed tissues. Similarly, CD54 (ICAM-1) facilitates interaction with immune cells, supporting anti-inflammatory responses. Other immunomodulatory markers, such as HLA-G and PD-L1 (CD274), are linked to the suppression of T-cell and natural killer cell activity and enhanced secretion of anti-inflammatory molecules like IL-10. CD39 and CD73, which contribute to extracellular adenosine production, further promote anti-inflammatory and tissue repair effects.

[64] For regenerative potential, markers such as CD105 (Endoglin) enhance angiogenesis and tissue regeneration through TGF-β signaling, while CD44 supports extracellular matrix interactions, cellular adhesion, and migration, all of which are critical for tissue repair. STRO-1 and CD271 (NGFR) are associated with high multipotency and tissue regeneration capabilities, including cartilage and bone repair. Homing and migration are supported by CXCR4, which responds to the SDF-1 (CXCL12) gradient, and CD146, which aids in perivascular adhesion.

[65] The strategy for pooling donor MSCs should include functional assays to assess anti-inflammatory cytokine secretion, such as IL-10 and PGE2, in response to inflammatory stimuli, as well as evaluating paracrine effects like VEGF and TGF-β production. Donors may be selected based on high expression of regenerative and immunomodulatory markers such as CD146, HLA-G, and ICAM-1, while excluding cells with high senescence markers or low telomerase activity. Batch consistency should be maintained by standardizing marker expression levels and potency assays across pooled batches, and immune tolerance must be confirmed by ensuring low levels of HLA-DR and other immunogenic markers. This comprehensive approach ensures the creation of a pooled MSC product with robust therapeutic potential.

[66] A mesenchymal stromal cell (MSC) product with increased expression of CD166 (Activated Leukocyte Cell Adhesion Molecule, ALCAM) could provide several therapeutic benefits due to its critical role in cell adhesion, migration, and interactions with the extracellular matrix (ECM). CD166 enhances MSC homing and engraftment, enabling better adhesion to vascular endothelium and migration to sites of injury. This improved ability to localize and integrate into damaged tissues is particularly beneficial in conditions such as myocardial infarction, stroke, and chronic wounds. Additionally, CD166 is involved in immune cell interactions, which could amplify the immunomodulatory effects of MSCs. This makes such a product well-suited for treating autoimmune and inflammatory diseases, including rheumatoid arthritis, Crohn’s disease, and systemic lupus erythematosus (SLE).

[67] Increased CD166 expression may also enhance MSC-mediated tissue regeneration by improving cell-ECM interactions, which are critical for the repair and maintenance of connective tissues. This could prove especially valuable in cartilage repair for osteoarthritis, bone regeneration in fractures or osteoporosis, and tendon or ligament repair. Moreover, CD166 may influence the paracrine activity of MSCs, boosting their secretion of bioactive molecules like growth factors, cytokines, and extracellular vesicles. These factors play an essential role in promoting tissue repair, reducing inflammation, and modulating the local microenvironment, particularly in chronic inflammatory conditions and ischemic injuries.

[68] Furthermore, MSCs with increased expression of CD166 may show better retention and integration at injury sites due to enhanced cell-cell and cell-ECM interactions. This is particularly important in therapies where MSC retention is a limiting factor, such as cardiac and musculoskeletal applications. CD166 also has roles in the nervous system, where it contributes to neural development and regeneration. This suggests that MSCs with increased CD166 expression could be highly effective in neuroprotective and neuroregenerative therapies, such as those targeting stroke, spinal cord injuries, and neurodegenerative diseases like Alzheimer’s or Parkinson’s. Lastly, the involvement of CD166 in cell signaling and migration makes these MSCs suitable for tumor-homing applications, where they could be engineered to deliver therapeutic agents directly to cancer sites.

[69] In summary, MSCs with increased CD166 expression offer enhanced homing, adhesion, immunomodulation, and tissue regeneration capabilities, making them a promising option for a wide range of therapeutic applications. These include regenerative medicine for cartilage, bone, and soft tissue repair, treatments for autoimmune and inflammatory diseases, neuroregenerative therapies, and even targeted cancer therapies. The broad utility and improved functionality of CD166-expressing MSCs highlight their potential as a versatile and highly effective therapeutic product.

[70] A mesenchymal stromal cell (MSC) product with increased expression of CD146 (Melanoma Cell Adhesion Molecule, MCAM) could provide significant therapeutic benefits due to its critical roles in vascular repair, tissue regeneration, and immunomodulation. CD146 is closely associated with the perivascular identity of MSCs and enhances their angiogenic potential by promoting the secretion of pro-angiogenic factors such as VEGF, FGF, and angiopoietins. This makes CD146-enriched MSCs particularly effective in treating conditions involving impaired blood flow or ischemia, such as peripheral artery disease, chronic wounds, myocardial infarction, and stroke. Furthermore, CD146 enhances MSC homing and migration to sites of injury or inflammation by facilitating adhesion to endothelial cells and transendothelial migration. This capability improves the efficacy of cell therapies targeting systemic or localized tissue damage, including ischemic injuries and autoimmune diseases.

[71] CD146 is also linked to enhanced MSC multipotency, particularly in osteogenic, chondrogenic, and adipogenic differentiation. This suggests that MSCs with elevated CD146 expression have superior regenerative capacity, making them ideal for repairing bone, cartilage, tendons, and ligaments. These properties are highly beneficial in treating conditions such as osteoarthritis, fractures, osteoporosis, and soft tissue injuries. Additionally, CD146 expressing MSCs demonstrate potent immunomodulatory effects, as they better suppress T-cell proliferation, regulate macrophage polarization, and modulate the activity of other immune cells like dendritic cells and natural killer cells. These enhanced immunoregulatory properties make CD146-enriched MSCs suitable for treating autoimmune disorders such as rheumatoid arthritis, Crohn’s disease, and multiple sclerosis, as well as graft-versus-host disease (GVHD).

[72] Another benefit of increased CD146 expression is improved MSC interaction with the extracellular matrix, which enhances their retention and integration at target sites. This is particularly important in therapies where durable cell engraftment is critical for efficacy, such as in cardiac and musculoskeletal repair. CD146 expressing MSCs may also exhibit anti-fibrotic effects by modulating fibroblast activity and promoting ECM remodeling, making them a promising option for treating fibrotic diseases, including liver fibrosis, pulmonary fibrosis, and chronic kidney disease. Furthermore, CD146 is implicated in neural development and repair, suggesting that MSCs with increased CD146 expression could have enhanced efficacy in neuroregenerative therapies for conditions like spinal cord injuries, stroke, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

[73] Finally, CD146 expressing MSCs demonstrate enhanced paracrine activity, secreting higher levels of bioactive molecules, including growth factors, cytokines, and extracellular vesicles, which are crucial for promoting repair and modulating inflammation. These capabilities make CD146-enriched MSCs highly versatile and effective for a range of therapeutic applications, including regenerative medicine, immune modulation, and the treatment of ischemic and fibrotic diseases. In summary, MSCs with increased CD146 expression provide a robust platform for addressing diverse clinical challenges, from vascular and neural repair to autoimmune and degenerative diseases.

[74] A therapeutic MSC product with increased expression of Mesenchymal Stem Cell Antigen-1 (MSCA-1), also known as tissue nonspecific alkaline phosphatase (TNAP), could offer several therapeutic advantages across a range of clinical applications. MSCA-1 is strongly associated with the osteogenic differentiation potential of MSCs, suggesting that such a product would have enhanced capacity to promote bone formation and repair. This could make it particularly valuable in treating bone fractures, non-union fractures, osteoporosis, and other degenerative bone diseases. Additionally, MSCs with higher MSCA-1 levels may exhibit superior regenerative capabilities, enhancing their ability to repair tissues such as cartilage, tendons, and ligaments, as well as facilitating wound healing in chronic or complex injuries.

[75] Beyond their regenerative potential, these cells may offer improved immunomodulatory and anti-inflammatory effects. MSCs are known for their ability to modulate immune responses, and increased MSCA-1 expression might further enhance these properties, making the product beneficial for autoimmune conditions like rheumatoid arthritis, Crohn’s disease, and multiple sclerosis, as well as other inflammatory disorders. Furthermore, MSCA-1 expressing MSCs have been implicated in supporting vascularization, as they can secrete pro-angiogenic factors like VEGF. This capability could aid in revascularization for ischemic injuries, such as myocardial infarction, and promote healing in ischemic wounds.

[76] Another potential benefit of enhanced MSCA-1 expression is improved MSC viability and longevity, particularly in challenging environments characterized by hypoxia or inflammation. This could ensure better survival and engraftment of the cells in vivo, leading to more robust therapeutic outcomes. Additionally, MSCA-1 expressing MSCs may exhibit enhanced integration with biomaterial scaffolds, improving outcomes in tissue engineering applications for bone and cartilage regeneration. These cells might also secrete higher levels of bioactive molecules, such as cytokines, growth factors, and extracellular vesicles, amplifying their ability to modulate the local microenvironment, promote repair, and reduce inflammation.

[77] Overall, an MSC product with increased MSCA-1 expression would hold significant promise for a variety of therapeutic areas, including orthopedic conditions, cardiovascular repair, wound healing, autoimmune disorders, and tissue engineering. By leveraging the enhanced regenerative, immunomodulatory, and paracrine properties of MSCA-1 expressing MSCs, such a product could provide a highly effective solution for diverse clinical challenges. In our examples herein, a surprising increase of MSCA-1 in the pooled product provides an unexpected and therapeutically advantageous benefit of the pooled MSCs, which was replicated both at the tenth cumulative population doubling (CPD10) and CPD30 for all of the four-donor cord pooled products.

[78] An MSC product with increased expression of CD271 (low-affinity nerve growth factor receptor, LNGFR) offers significant therapeutic advantages due to the unique properties associated with CD271 expressing MSCs. These cells exhibit enhanced stemness and differentiation potential, retaining a more primitive phenotype that allows for superior multipotency and regeneration of bone, cartilage, and soft tissues. This makes them particularly effective in treating conditions such as bone fractures, osteoporosis, cartilage damage, and osteoarthritis. Additionally, CD271 expressing MSCs demonstrate robust immunomodulatory properties, with greater capacity to suppress T-cell proliferation, regulate macrophage polarization, and modulate dendritic cell and natural killer (NK) cell activity. These enhanced effects are highly beneficial in managing autoimmune diseases like rheumatoid arthritis, Crohn’s disease, and multiple sclerosis, as well as preventing graft-versus-host disease (GVHD) in transplant recipients.

[79] CD271 expressing MSCs also exhibit improved migratory and homing abilities, facilitated by the expression of chemokine receptors and adhesion molecules that guide them to sites of inflammation, injury, or tissue damage. This enhanced migration is critical for effective therapies targeting systemic or localized damage, such as ischemic injuries, wound healing, and inflammatory conditions. Furthermore, CD271 expressing MSCs are less prone to senescence during ex vivo expansion, enabling scalable manufacturing of high-quality cell products while maintaining their therapeutic potential. Their neuroprotective properties are particularly noteworthy, as CD271 is a receptor for nerve growth factor (NGF), making these cells effective in promoting neural repair, reducing neural inflammation, and supporting recovery in conditions like stroke, spinal cord injuries, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

[80] In addition to their regenerative and immunomodulatory capabilities, CD271 expressing MSCs possess strong anti-fibrotic effects by modulating fibroblast activity and preventing excessive extracellular matrix deposition. This makes them valuable for treating fibrotic diseases, including liver fibrosis, pulmonary fibrosis, and chronic kidney disease. They also exhibit enhanced angiogenic potential, secreting higher levels of pro-angiogenic factors such as VEGF and bFGF, which promote vascularization and tissue repair in ischemic injuries and chronic wounds. The paracrine activity of CD271 expressing MSCs, including the secretion of bioactive molecules, further amplifies their ability to modulate the microenvironment, reduce inflammation, and promote tissue repair.

[81] In summary, MSC products with increased CD271 expression are highly versatile, offering enhanced stemness, immunomodulation, migratory capacity, and paracrine activity. These properties make them ideal for a wide range of therapeutic applications, including regenerative medicine for orthopedic and vascular conditions, immune modulation for autoimmune and inflammatory diseases, and neuroregeneration for neurological disorders. With their robust regenerative, anti-inflammatory, and anti-fibrotic capabilities, CD271 expressing MSCs represent a powerful tool in advancing cell-based therapies.

[82] Considering the foregoing, an MSC product demonstrating batch-to-batch consistent expression of one or more surface markers selected from CD166, CD146, MSCA-1, and CD271, in addition to the common surface markers expressed by MSCs, would provide a benefit across multiple therapeutic and manufacturing related areas.

[83] Now, in order to consistently manufacture an MSC -based therapeutic product, it may be beneficial to first characterize surface marker expression, MFIR of surface markers, ex vivo factor secretion, and the like, from a number of single umbilical cord -derived MSC seed stocks, then using computational methods select from the single umbilical cord -derived MSC seed stocks to pool in ratios estimated to yield the desired pooled population characteristics. Based on our data, the therapeutic product should comprise pooled MSCs being culture expanded between ten and thirty population doublings, achieving a heterogenous population and yet retaining sufficient stemness prior to reaching senescence.EXAMPLES

[84] Example 1 – Aseptic, Allogeneic, Pooled hucMSCs

[85] An off-the-shelf, culture-expanded mesenchymal stromal cell (MSC) therapeutic composition manufactured from pooled umbilical cord tissues and formulated for direct supply and intravenous administration or injection without washing or re-formulation was prepared. The therapeutic product is characterized as 3x106 hucMSCs per milliliter suspended in at least 50% w / v Dulbecco’s phosphate-buffered saline (DPBS), up to 10% w / v dimethylsulfoxide (DMSO), and up to 50% w / v human or recombinant albumin solution.

[86] The therapeutic composition forms a suspension of allogeneic MSCs sourced from umbilical cord tissue from four pooled umbilical cord donors. Umbilical cord tissue is supplied from consenting donors who have been screened for cord blood donation. Cord tissue is manually dissected before enzymatic dissociation to release MSCs. MSCs are isolated by plastic adherence and expanded in xeno-free culture conditions in closed-system bioreactors for up to 50 cumulative population doublings, preferably CPD10 to CPD30, to create the therapeutic composition.

[87] After the culture, the cells are harvested and can be cryopreserved before being adequately packaged, labelled, stored in vapor phase nitrogen and shipped to the clinical unit when prescribed. All batches are tested at release for identity, purity, sterility, viability, potency and absence of detectable mycoplasma and endotoxin before certification.

[88] The therapeutic composition consists of a sterile, yellowish cell suspension cryopreserved in 50ml freezing bags which are combined as necessary to make up the defined dose for an individual patient. The final cryopreserved product is supplied in clear and individually labelled overwrapped, 50ml freezing bags with a fill volume of 10ml or 15ml cell suspension per bag and a minimum concentration of 3.0x106 hucMSCs per ml. In this example, the cells in the therapeutic product were passaged eight to nine times, although more or fewer may be desirable and can be optimized with routine characteristics-measuring assays. Alternatively, the therapeutic composition may be filled in 250mL, 500mL, 750mL, 1000mL or other size bags. In this example, 50mL bags were used based on the stated fill volume.

[89] Eight individual umbilical cord MSC seed stocks (labeled “S-1” through “S-8”) were prepared, and of the eight individual umbilical cord seed stocks three pooled stocks (“P-1” through “P-3”) were prepared. Pooled seed stock P1 comprised a combination of S-1, S-5, S-6, and S-8. Pooled seed stock P2 comprised a combination of S-1, S-2, S-7, and S-8. Pooled seed stock P3 comprised a combination of S-3, S-4, S-6, and S-7. Based on our prior experiments, it was determined that poolingof at least four single donor seed stocks is desirable. Each pooled batch in these examples consists of four donor cords. All single donor and pooled quad products were phenotyped by multiparametric flow cytometry using qualified panels of fluorochrome-conjugated antibodies after 10 and 30 cumulative population doublings (CPDs). Cells were analyzed for expression of CD10, CD29, CD44, CD73, CD90, CD105, CD142, CD146, CD166, CD200, CD271, MSCA-1 and MHC class I (HLA-ABC) whilst testing for non-lineage commitment with a cocktail of CD3, CD14, CD19, CD45, and HLA-DR. The surface marker characteristics are described in Tables 1 (10 CPD) and Table 2 (30CPD), respectively.

[90] While not shown, all pooled products were essentially free of surface markers CD11b, CD14, CD19, CD34, CD45, CD79α, and HLA-DR.

[91] Moreover, while cells in various MSC products express common surface markers, they can do so at different intensities of expression. Tables 3 and 4 illustrate the median fluorescence intensity ratio (MFIR) associated with the various MSC products described above.

[92] All single donor and pooled products showed consistent expression of the common identity markers (CD73, CD90 and CD105) with no significant differences in intensity of expression. Using an extended identity panel aimed at investigating known subsets of MSCs, we found no significant differences in surface marker expression of CD10 / CD29 / CD44 / MHC class I, however there was a clear difference in expression of CD166 / CD146 / MSCA-1 / CD271 and CD200, most notably MSCA-1, which had increased expression in pooled products at both CPD10 and CPD30. Moreover, as shown in Tables 3 and 4, it is important to note that pooled products showed reduced heterogeneity of expression of surface markers CD29, HLA-ABC, CD44, CD146, CD10, CD166, CD142, CD200, CD271 between batches, leading to a more reproducible product. As one of the problems attributed to conventional MSC-based products is inconsistency or heterogeneity, there is a significant improvement with an MSCs product that has less heterogeneity between manufactured batches.

[93] The range of surface marker expressions for select surface markers identified at CPD 30 for both single cord and pooled products is shown in FIG.5 (Table 5), wherein pooled products (P1 to P3) show a significantly tighter range than single-cord counterparts (S-1 to S-8), indicating an improvement in consistency of the intensity of surface marker expression, derived from mean florescence intensity ratio, when considered batch-to-batch.

[94] Example 2 – Efficient Manufacturing of Allogeneic, Pooled hucMSCs

[95] In addition to more consistent product batch-to-batch, more efficient manufacture, due to reduced population doubling time, was also discovered in pooled hucMSCs products. Manufacture of MSC drugs requires expansion of the cells in culture and the rate of expansion is calculated as the time (hrs) required for each cell in culture to divide; termed the population doubling time (PDT). Pooling 4 or more MSC reduces the PDT significantly and reduced the variability of cell culture, as shown below.

[96] Days to reach CPD10 / CPD30

[97] As shown in FIG.6, pooling single donor cord derived hucMSCs at completion of passage 1 significantly reduces the mean time to CPD10 from 7.0 days (SD 2.4) to 3.6 days (SD 1.0) for four-cord combinations. This initial difference led to an 8.0 day average difference in the total production time to 30 cumulated population doublings (CPD30) between the single donor cultures and the quadruplet pools, respectively. All of the quadruplet products reached 30CPD after 8 passages or fewer. In contrast, three of the single umbilical cord cultures required 9 passages.

[98] It was also noted that the range of days to CPD30 is tighter and more consistent for the four-cord pooled hucMSCs products compared to single donor MSC products.

[99] MSC must grow as adherent cells and, as they divide, they are monitored for cell density and, when the surface area is >80% covered (i.e. reach confluence), they require additional surface area to sustain the expansion. Each change of surface is called a “passage”. Pooled products showed significant reduction in the variability of the time to achieve confluence across all nine passages required to achieve 30 CPD.

[100] FIG.7 shows the coefficient of variance (%) indicating the variability of cells in single umbilical cord versus four-cord (“quad”) pooled batches. It was shown that cells from the first passage exhibited more variance than cells of subsequent passages. However, not shown, it was observed that after repeated passages the single umbilical cord cells became senescent, whereas the quad pooled cells remained robust and potent. Quad pooled products exhibited much less variability after multiple passages, where at passage 8 the pooled cells reached 30CPD and less than five percent variability. On the other hand, single umbilical cord cells required nine passages to reach 30CPD, more time, and exhibited higher variability compared to the quad pooled cells.

[101] Example 3: Secretome Profiles

[102] As shown in FIG.8, all products were also tested for cytokine secretion using ELISA immunoassay kits. Cell culture supernatants were analyzed for secretion of VEGF, TIMP1, TIMP2, IL-8, IL-6, and FGF- β for single donors and quads at CPD30. A reduction in variation was noted in quad donor pooled products compared to single donor products, with %CV reducing 81%, 41%, 34%, and 58% respectively for VEGF, TIMP2, TIMP1, and FGF- β.

[103] Furthermore, an increase in the amount of secreted cytokine was observed in quads versus single donors. By comparison, average VEGF increased from 121.6 pg / 105 cells to 257.5 pg / 105 cells (average 112% improvement in pooled products), IL-8 increased from 123.8 pg / 105 cells to 315.8 pg / 105 cells (average 155% improvement in pooled products), and IL-6 increased from 605.9 pg / 105 cells to 1144.4 pg / 105 cells (average 89% improvement in pooled products). For example, the VEGF secretion of individual seed stocks and corresponding pooled stocks in illustrated in FIG.9.

[104] Only 3 of 8 single donor MSC cultures produced detectable levels of VEGF. All of the quadruplet CORDStrom products contained at least one of the individual UC-MSCs products which secreted VEGF and they all continued to secrete the cytokine even after pooling; again confirming the improved reproducibility of the quad product by virtue of pooling individual UC-MSCs.

[105] IDO secretion by MSC is induced by proinflammatory cytokines which can be replicated in vitro by co-culture with gamma interferon and tumor necrosis factor. As shown below, single donor MSC products secreted significantly less IDO after cytokine stimulation than the four donor pooled products, and the variability of secretion between donors was greater for single donors than the pooled product (35.0% cv versus 4.85% cv).

[106] FIG.10 shows IDO activity (potency) as picograms kynurenine / cell. Here, pooled products yield at least 1.5 pg Kynurenine / cell, and average about 2.0 pg Kynurenine / cell. Secretion of these cytokines are known to be important in wound healing and other regenerative medicine applications, and mechanisms of action for MSC products due to secretion of pharmaco-active cytokines.

[107] Example 4: Scratch Test- Wound Healing

[108] One of the uses of pooled, allogeneic hucMSCs is the ability to enhance wound healing in multiple clinical settings. In contrast to single donor products, pooled hucMSCs made from cells of at least four donors showed increased potency in a conventional “scratch test”. In this test, human skin fibroblasts were grown to >80% confluence on the surface of culture plates and then subjected to a standardized scratch to mimic a skin wound. The dynamic re-growth of the skin fibroblasts was measured over 90 hours in an xCELLigence device where single donor MSC products or four donor pooled products were added in Transwell co-culture inserts. Thus, the effect of cytokines secreted from the single donor MSCs and pooled MSCs products on the rate of fibroblast re-growth could be measured.

[109] Regrowth of the fibroblast monolayer was achieved within 64hrs in the absence of MSC derived cytokines. In contrast, as shown in FIG.11, complete scratch regrowth was achieved in 49hrs with the pooled products, compared to an average of 64hrs with the untreated control, an improvement of 23.4%.

[110] For comparison, FIG.12 shows complete scratch regrowth was achieved in 57hrs with the single donor MSC products, compared to an average of 66hrs with the untreated control, representing a 13.6% improvement by single-donor MSCs, which is substantially less beneficial when compared to the 23.4% improvement with the pooled MSCs products.

[111] Example 5: T-Cell Suppression

[112] As shown in FIG.13 suppression of T cell proliferation by MSCs to determine the immunomodulatory capacity of single donor versus pooled MSCs was assessed with co-culture of the single and pooled MSCs and activated PBMCs, with results obtained by flow cytometry of CellTrace labelled lymphocytes. The average reduction of T cells against a PBMC only control was greater in pooled MSCs products than single donors at both CPD10 and CPD30, with a significantly lower standard deviation and coefficient of variance in pooled MSCs than single donor MSCs. This again indicates improved performance and efficiency of the pooled MSCs products, as well as reducing the variation noted with single donor products.

[113] Example 6: Allogeneic, Pooled, Culture-expanded MSC Products Tuned for Select Indications

[114] The following descriptions are directed to indication-specific MSC medicinal products comprising cells from four or more donor umbilical cords, wherein the seed stocks selected for pooling are those expressing select surface markers and with sufficient intensity as measured by MFIR, in accordance with various alternative embodiments of the invention, which may be referenced in conjunction with FIG.14. In addition to the common MSC markers, and optionally CD166 and CD146 as further described herein, the following indication-specific examples describe markers for tuning the desired hucMSCs product to meet the needs of various indications.

[115] Graft-versus-Host Disease (GvHD)

[116] For GvHD, MSCs must exert strong immunomodulatory effects while homing to inflamed tissues to prevent immune attacks on transplanted cells. CD200 and CD274 (PD-L1) help suppress excessive immune responses by inhibiting T-cell activation and enhancing regulatory T cells (Tregs), reducing cytokine storms. CD73 and CD146 further contribute to anti-inflammatory effects and vascular stability, respectively, helping to restore immune balance. CXCR4 is essential for MSC migration to inflamed tissues via SDF-1 signaling. Conversely, CD86 should be avoided, as it promotes immune activation instead of suppression, while CD36 is linked to fibrotic pathways that may worsen GvHD complications.

[117] Systemic Lupus Erythematosus (SLE)

[118] SLE is characterized by systemic autoimmunity that leads to organ damage. Enriching for CD200 and CD274 ensures suppression of autoreactive immune cells, while CD73 and CCR2 reduce systemic inflammation and improve tissue repair. CD146 enhances endothelial stability, protecting against lupus vasculopathy. Avoiding CD86 prevents unnecessary T-cell activation, and CD45 elimination ensures that hematopoietic contamination does not contribute to unwanted immune interactions.

[119] Multiple Sclerosis (MS)

[120] For MS, a disease involving neuroinflammation and demyelination, CD271 (LNGFR) is crucial due to its role in nerve protection and repair. CD146 aids in endothelial stability, ensuring proper blood-brain barrier function, while CXCR4 facilitates homing to inflamed neural tissues. CD73 provides local anti-inflammatory adenosine signaling, reducing microglial activation. Markers such as CD86 and HLA-DR should be avoided to minimize immune activation, which could exacerbate inflammation.

[121] Rheumatoid Arthritis (RA)

[122] RA requires MSCs that can suppress chronic joint inflammation and prevent cartilage destruction. CD200 and CD274 help regulate autoreactive immune responses, while CD73 reduces inflammation via adenosine pathways. CD271 and CXCR4 contribute to synovial repair and homing to inflamed joints. Avoiding CD36 is crucial since it promotes fibrosis, which could lead to synovial thickening, and CD86 should be excluded to prevent immune activation.

[123] Type 1 Diabetes (T1D)

[124] T1D is an autoimmune disorder destroying pancreatic beta cells. CD200 suppresses autoreactive T-cell responses, while CD73 and CD146 help modulate immune balance and prevent pancreatic inflammation. PDGFR-β (CD140b) aids in vascular stabilization, which is necessary to prevent further pancreatic islet damage. Avoiding CD36 and CD45 prevents unwanted fibrotic and hematopoietic contamination that could exacerbate the disease.

[125] Amyotrophic Lateral Sclerosis (ALS)

[126] ALS is a neurodegenerative disorder requiring neuroprotection and inflammation control. CD271, CD146, and CXCR4 facilitate MSC homing to damaged motor neurons and protect against oxidative stress. CD73 provides additional neuroinflammation modulation. To avoid worsening the disease, CD86, HLA-DR, and CD36 should be excluded as they can promote neuroinflammation and fibrosis.

[127] Parkinson’s Disease (PD)

[128] PD is characterized by progressive loss of dopaminergic neurons. CD271, CD146, and CD105 contribute to neuronal survival and synaptic plasticity, while CXCR4 enhances homing to affected brain regions. CD73 reduces neuroinflammation and supports dopaminergic neuron maintenance. Avoiding CD36 and HLA-DR minimizes excessive immune activation and fibrosis, which can worsen disease progression.

[129] Alzheimer’s Disease (AD)

[130] AD is marked by neuroinflammation and amyloid plaque deposition, leading to cognitive decline. CD200 and CD146 help modulate microglial activation and prevent neuronal loss. CD73 and CXCR4 facilitate MSC migration and anti-inflammatory signaling, protecting against oxidative stress. CD271 supports neuroprotection and cognitive resilience. Avoiding CD36, HLA-DR, and CD45 prevents unnecessary immune activation and fibrosis.

[131] Stroke Recovery

[132] Stroke involves ischemic damage leading to neuronal death and functional deficits. CD146, CD271, and CXCR4 improve vascular repair, reduce neuroinflammation, and promote neuronal survival. CD73 enhances anti-inflammatory pathways, reducing secondary damage. CD105 supports angiogenesis and tissue regeneration. Avoiding CD36, HLA-DR, and CD45 is crucial to prevent inflammatory responses that can worsen recovery.

[133] Idiopathic Pulmonary Fibrosis (IPF)

[134] IPF involves excessive lung fibrosis, requiring MSCs with anti-fibrotic and regenerative capabilities. CD146, PDGFR-α, and CD271 support lung tissue repair, while CD73 helps control inflammation. Avoiding CD36 is critical to prevent exacerbation of fibrotic pathways.

[135] Chronic Obstructive Pulmonary Disease (COPD)

[136] COPD is marked by chronic lung inflammation and progressive airway remodeling. CCR7 and CD146 promote lung repair and epithelial regeneration, while CD73 and CXCR4 facilitate migration to damaged lung tissue. PDGFR-β supports vascular integrity, reducing the risk of pulmonary hypertension. CD36 and CD45 should be avoided to prevent excessive fibrosis and immune activation.

[137] Cirrhosis & Liver Fibrosis

[138] Liver fibrosis results from chronic liver damage, requiring MSCs that modulate fibrosis while promoting hepatocyte regeneration. CD146, PDGFR-α, CD200, CXCR4, and CD90 support liver repair by reducing fibrotic activity and enhancing angiogenesis. CD36 and CD45 should be excluded to prevent worsening fibrosis and unwanted immune interactions.

[139] Chronic Kidney Disease (CKD)

[140] CKD leads to progressive renal fibrosis and loss of kidney function. CD146, CD90, CXCR4, CD73, and PDGFR-β enhance MSC retention and repair in kidney tissue. CD36 and CD45 should be avoided as they contribute to fibrotic progression and immune activation, which can worsen CKD pathology.

[141] Epidermolysis Bullosa (EB)

[142] EB is a rare genetic disorder causing fragile skin prone to blistering. CD200, CD274, CD146, CXCR4, and CD49f enhance wound healing by promoting keratinocyte stability and reducing inflammation. CD36 and CD45 should be excluded to prevent excessive fibrosis and immune activation that may worsen wound healing.

[143] More specifically, the pooled, culture-expanded human umbilical cord-derived mesenchymal stromal cells (hucMSCs) therapeutic composition for treating epidermolysis bullosa (EB) is ideally characterized by a surface marker profile optimized for wound healing, immune modulation, extracellular matrix (ECM) stabilization, and angiogenesis. Single donor seed stocks should be selected such that at least a portion of the pooled, culture-expanded hucMSCs in the product express CD39, CD49f, CD73, CD90, CD105, CD146, CD200, CD271, CD274, CXCR4, and PDGFR-β, as these markers play critical roles in tissue repair and inflammation control. CD39 and CD73 contribute to immunosuppressive adenosine signaling, reducing inflammation in chronic wounds. CD49f (Integrin α6) enhances keratinocyte adhesion and migration, which is crucial for skin regeneration. CD90 and CD105 support fibroblast function and ECM remodeling, essential for dermal integrity. CD146 and CXCR4 facilitate vascularization and MSC homing to injured tissues, ensuring optimal cell integration and repair. CD200 and CD274 (PD-L1) help suppress excessive immune activation, preventing autoimmune-mediated skin deterioration. CD271 (LNGFR) provides highly regenerative MSC subpopulations with enhanced tissue repair capabilities, while PDGFR-β (CD140b) modulates fibroblast activation, ensuring proper ECM deposition without excessive scarring.

[144] In this optimized EB therapy, the pooled, culture-expanded hucMSCs are essentially free of CD11b, CD14, CD19, CD31, CD34, CD36, CD45, CD79α, CD86, and HLA-DR, as these markers are associated with hematopoietic, endothelial, inflammatory, or fibrotic activity, which could compromise therapeutic efficacy. CD11b, CD14, and CD19 are markers of monocytes / macrophages and B cells, which could lead to unwanted immune responses. CD31 (PECAM-1) and CD34 are endothelial and progenitor cell markers, which are not relevant for MSC-mediated tissue repair. CD36 is linked to pro-fibrotic activity, which could contribute to excessive scar formation, counteracting the therapeutic goal of promoting functional skin regeneration. CD45 and CD79α indicate hematopoietic lineage cells, which are undesirable in an MSC product. CD86 and HLA-DR are immune-activating markers that could trigger undesirable alloimmune responses, reducing the immune-privileged status of MSCs and limiting their therapeutic efficacy.

[145] In one aspect, the therapeutic composition described herein comprises a population of mesenchymal stromal cells (MSCs) optimized for the treatment of recessive dystrophic epidermolysis bullosa (RDEB), a genetic disorder characterized by insufficient collagen VII deposition at the dermal-epidermal junction (DEJ), leading to severe skin fragility, chronic wounds, and fibrosis. To enhance the therapeutic efficacy of MSCs in RDEB, the expression level of CD146 (MCAM) within the MSC population is specifically controlled within an optimal range of 40% to 60% CD146⁺ cells.

[146] The selection of this CD146 expression range is based on its critical role in cell homing, extracellular matrix (ECM) remodeling, and immunomodulation, which are essential mechanisms for improving collagen VII restoration and skin repair in RDEB patients. MSCs expressing CD146 exhibit enhanced migratory capacity, which facilitates targeted homing to sites of epithelial injury and inflammation. This is particularly advantageous in RDEB, where persistent wounding and dermal inflammation require MSCs capable of efficiently localizing to damaged skin and supporting re-epithelialization. The controlled level of CD146 expression also ensures that MSCs retain their pericyte-like properties, which contribute to vascular stabilization and dermal remodeling without excessive angiogenic activity.

[147] Furthermore, the moderate CD146 expression range of 40–60% is selected to optimize ECM repair by enhancing the secretion of key ECM components, including matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), fibronectin, and laminins, all of which are involved in the restructuring of the DEJ. Importantly, this level of CD146 expression also supports modulation of fibroblast activity, reducing fibrotic tissue remodeling, which is a major concern in chronic RDEB wounds. Excessive CD146 expression above 60% may lead to unregulated ECM degradation and excessive vascular remodeling, while CD146 levels below 40% may result in reduced homing efficiency and insufficient support for collagen VII deposition.

[148] By maintaining CD146 expression between 40% and 60%, the MSC therapeutic composition described herein provides a balanced approach to tissue regeneration, ensuring enhanced cell migration, ECM stabilization, and immune modulation while minimizing the risk of vascular instability or fibrosis. This precise control over MSC phenotype offers a novel and optimized strategy for improving clinical outcomes in RDEB patients, addressing both the structural deficiency of collagen VII and the inflammatory and fibrotic complications associated with the disease.

[149] To enhance the therapeutic efficacy of MSCs in RDEB, the composition is specifically formulated to contain 85–95% of MSCs expressing CD166 (ALCAM), a key adhesion molecule involved in cell anchoring, extracellular matrix (ECM) stabilization, and immune modulation.

[150] CD166 plays a critical role in MSC retention and interaction with epithelial cells and fibroblasts at the DEJ, improving MSC engraftment, tissue adhesion, and structural reinforcement in RDEB skin. High CD166 expression enhances MSC-mediated secretion of ECM components, including fibronectin, laminins, and integrins, which support collagen VII deposition and dermal stability. Additionally, MSCs within this optimized CD166 range exhibit enhanced immunosuppressive effects, including increased secretion of TGF-β, prostaglandin E2 (PGE2), and IL-10, reducing chronic inflammation and fibroblast overactivation, which are major contributors to wound progression and fibrosis in RDEB.

[151] Maintaining 85–95% CD166⁺ MSCs ensures that the therapeutic composition effectively modulates fibroblast activity to prevent excessive scarring while supporting controlled tissue remodeling. The presence of a small subpopulation (≤15%) of CD166-low MSCs provides paracrine signaling benefits, supporting angiogenesis and tissue regeneration without excessive adhesion that may hinder MSC migration across large wound areas. By balancing MSC adhesion, migration, and immune regulation, this provides a standardized and reproducible MSC product designed to improve collagen VII deposition, enhance wound healing, and prevent fibrosis in RDEB patients.

[152] Thus, by selecting for regenerative, immunomodulatory, and homing-enhancing surface markers while excluding those associated with immune activation, fibrosis, and non-MSC lineages, the pooled, culture-expanded hucMSC product is optimized for enhanced wound healing, reduced inflammation, and long-term tissue regeneration in epidermolysis bullosa patients.

[153] Systemic Sclerosis (SSc)

[154] SSc is characterized by excessive fibrosis affecting the skin and internal organs. CD271, PDGFR-α, CD146, CD73, and CD200 help regulate fibroblast activity and promote vascular repair. CD36 and CD45 should be avoided, as they contribute to excessive collagen deposition and immune dysregulation, worsening fibrosis.

[155] Duchenne Muscular Dystrophy (DMD)

[156] DMD is a progressive muscle-wasting disorder requiring MSCs that support muscle regeneration. CD146, PDGFR-β, CXCR4, CD73, and CD271 promote muscle repair while mitigating fibrosis. CD36 and CD45 should be avoided to prevent excess scar tissue formation that could impair muscle function.

[157] Hypoxic-Ischemic Encephalopathy (HIE)

[158] HIE results from oxygen deprivation, leading to severe neurological damage. CD146, CD271, CD73, CD105, and CXCR4 enhance neuroprotection, reduce inflammation, and support brain repair. CD36, HLA-DR, and CD45 should be excluded to minimize immune activation and fibrotic scarring.

[159] Wet Age-Related Macular Degeneration (Wet AMD)

[160] In wet AMD, excessive neovascularization leads to retinal damage. CD146 and CXCR4 help stabilize retinal vasculature, while CD73 and CD200 reduce retinal inflammation. PDGFR-β supports pericyte function, reducing leaky blood vessels. CD271 and CD105 aid neuroprotection and proper angiogenesis. Avoiding CD36 is crucial to prevent fibrosis, while CD86, HLA-DR, CD45, and CD31 should be excluded to reduce immune activation and pathological neovascularization.

[161] By carefully selecting seed stocks with MSC markers tailored to each disease indication, the resulting pooled, culture expanded MSC therapy can be optimized to enhance efficacy and minimize adverse effects, paving the way for more targeted and effective treatments. Prior to pooling, MSCs from an individual cord donor can be characterized for surface marker expression, intensity of expression (e.g. MFIR), differentiation potential, plastic adherence, and more. Based on indication-specific needs, characteristics of the single donor seed stocks can be assessed and four or more seed stocks pooled, prior to culture expansion, and expanded ex vivo up to CPD50, or preferably between CPD10 and CPD30, to manufacture a pooled, culture-expanded therapeutic product with enhanced potency and batch-to-batch consistency.

[162] Example 7 – hucMSCs Product with Balanced CD146 Expression

[163] In this example, the pooling strategy ensures that the final MSC therapeutic product maintains a CD166⁺ expression of at least 85% and a CD146⁺ expression up to 75%, and preferably between 30% and 75%, aligning with the desired therapeutic profile for treating complex inflammatory diseases. To achieve this, individual donor MSC seed stocks were first characterized for CD146 and CD166 expression levels, along with key MSC markers and doubling times. A computational model was then used to determine the optimal proportions of each donor’s MSC seed stock to pool together before expansion, ensuring that the final product consistently meets the defined marker expression criteria. The adjusted proportions distribute contributions evenly across the selected donor sources, preventing overrepresentation from any single source while maintaining a balanced growth rate and functional potency. These optimized ratios provide a robust foundation for subsequent culture expansion under controlled conditions, helping to preserve batch-to-batch consistency in the final therapeutic product. Next, an optimized expansion protocol will be designed to stabilize these marker expressions throughout culture, ensuring the therapeutic MSCs retain their desired characteristics upon administration.

[164] Maintaining CD146 expression up to 75%, and preferably between 30% and 75% in the MSC therapeutic product is essential to balance immunomodulation, migration, and vascular support while minimizing potential risks associated with extreme expression levels. High CD146 expression (above 75%) enhances vascular interaction and angiogenesis, which can be beneficial in ischemic conditions, but excessive levels may lead to uncontrolled vascular remodeling, pro-fibrotic activity, or even tumor-supportive properties in certain inflammatory environments. Additionally, highly migratory MSCs may exhibit reduced immunosuppressive potency, potentially limiting their effectiveness in treating immune-mediated diseases. Conversely, low CD146 expression (below 30%) correlates with weaker homing ability and reduced secretion of key immunosuppressive factors, such as TGF-β, PGE2, and IDO, which are critical for modulating inflammation and promoting tissue repair. CD146-low MSCs also show a higher tendency to differentiate into fibroblasts, which can compromise their regenerative potential and increase variability in therapeutic outcomes. By ensuring that 30–75% of cells express CD146, the final product retains adequate migratory capacity for tissue homing and repair, while preserving immunosuppressive and anti-inflammatory functions. This approach not only improves batch-to-batch consistency but also enhances the therapeutic safety and efficacy of MSCs for complex inflammatory diseases, ensuring the product remains suitable for a wide range of clinical applications without unwanted pericyte-like or fibroblastic differentiation.

[165] Example 8 - Sorting Protocol for MSC Selection via Seed Stock Characterization & Computational Pooling

[166] In another example, an MSC selection and pooling protocol ensures batch-to-batch consistency by first characterizing individual donor MSC seed stocks based on at least surface marker expression and median fluorescence intensity ratio (MFIR), followed by computational modeling to determine the optimal proportions of each donor seed stock to pool before culture expansion. Each donor-derived MSC seed stock is expanded up to five passages, preferably one to three, and analyzed via flow cytometry, measuring the percentage of cells expressing CD146 and CD166, as well as their respective MFIRs, which provide a normalized measure of marker intensity relative to background fluorescence. Additionally, key exclusion markers (CD45, CD34, CD11b) and functional parameters such as viability and doubling time may be recorded. Using these data, a computational optimization model determines the ideal mix of donor MSC stocks, ensuring the final pooled batch maintains CD146 expression between 30–75% and CD166 expression at ≥85%, while also standardizing MFI ratios to ensure a uniform phenotype across production runs. By balancing donor contributions, this method prevents dominance by any single donor while ensuring the desired immunomodulatory and regenerative properties are consistently met. The optimized pooled MSC stock is then expanded under controlled culture conditions to maintain its composition and therapeutic function. This approach enhances batch-to-batch reproducibility, improves scalability, and ensures a stable and potent MSC therapeutic product for inflammatory disease treatment.

[167] Example 9 - Optimized Expansion Protocol

[168] In another example, an optimized expansion protocol may be provided and designed to preserve the target MSC composition established through computational pooling, ensuring that, inter alia, CD146 and CD166 expression levels remain within the desired range throughout culture. To achieve this, low-density seeding (e.g., 3,000–5,000 cells / cm²) can be used to prevent early contact inhibition, which can lead to phenotypic drift. In this example, the cells are cultured in a low-glucose DMEM / F12 or Alpha-MEM base medium supplemented with 5% human platelet lysate (hPL) or 2% human serum albumin (HSA), 1 ng / mL TGF-β3, 5 ng / mL PDGF-BB, 5 ng / mL FGF-2, and 2 ng / mL VEGF-A, which help maintain pericyte-like characteristics and stabilize CD146 expression. Culture is performed under hypoxic conditions (e.g., 2–5% O₂) to delay senescence and support the retention of immunomodulatory and migratory properties. To further enhance stability, MSCs can be expanded on fibronectin- or collagen-coated plates or beads, which improve cell adhesion and CD166 retention. Passaging is conducted every 3–4 days, ensuring that cells do not exceed 80% confluency, as overgrowth can trigger a loss of CD146 expression. Additionally, a ROCK inhibitor (Y-27632, 10 µM) can be included for the first 24 hours post-thaw or post-passage to minimize stress-induced marker downregulation. Throughout expansion, periodic flow cytometry quality control (e.g., every 2–3 passages) may be performed to verify that CD146 expression remains within 30–75% and CD166 remains ≥85%, with any deviations prompting adjustments to culture conditions or early termination of the batch. This optimized expansion strategy can be provided to ensure a consistent, phenotypically stable MSC product that maintains its desired immunomodulatory and regenerative properties, improving the reliability of therapeutic outcomes.

[169] The media composition and culture conditions used in the expansion of the mesenchymal stromal cell (MSC) product can be specifically designed to stabilize CD146 and CD166 expression, support cell proliferation, and preserve immunomodulatory and regenerative properties. The expansion process can employ low-glucose DMEM / F12 or Alpha-MEM as the basal medium, which is selected to prevent spontaneous differentiation while maintaining MSC multipotency and immunosuppressive function. Low-glucose conditions are particularly beneficial in delaying senescence and reducing glycolytic stress, ensuring a consistent and functional MSC phenotype. DMEM / F12 enhances pericyte-like properties, supporting vascular interactions, while Alpha-MEM provides an amino acid-rich environment that sustains MSC viability and longevity.

[170] To further regulate MSC behavior, the basal medium can be supplemented with human platelet lysate (hPL) or human serum albumin (HSA), which serve as a serum-free alternative to fetal bovine serum (FBS). These supplements provide a defined source of growth factors, including PDGF, VEGF, and EGF, while reducing batch variability inherent in FBS-containing cultures. The addition of TGF-β3 (1 ng / mL) may be critical for maintaining CD146 expression, as it preserves the pericyte-like phenotype of MSCs and prevents excessive differentiation. Similarly, PDGF-BB (e.g., 5 ng / mL) enhances CD166 expression, promotes cell adhesion, and supports MSC migration. FGF-2 (e.g., 5 ng / mL) is included to stimulate MSC proliferation, delay senescence, and enhance immunosuppressive effects, particularly through increased secretion of PGE2 and IDO. Additionally, VEGF-A (e.g., 2 ng / mL) supports MSC survival under inflammatory conditions, promotes endothelial interaction, and enhances angiogenic potential, making the final MSC product well-suited for inflammatory and vascular diseases.

[171] To further preserve CD146 and CD166 expression, MSCs may be expanded under hypoxic conditions (e.g., 2–5% O₂), which mimics the native physiological niche of perivascular MSCs. Hypoxia is known to delay senescence, maintain CD146 expression, and enhance immunomodulatory potency. Furthermore, low-density seeding (e.g., 3,000–5,000 cells / cm²) can be employed to prevent contact inhibition, which can lead to loss of CD146 and uncontrolled differentiation. Cells can be cultured on fibronectin- or collagen-coated surfaces, which provide an extracellular matrix (ECM) environment that enhances CD166 expression and cell adhesion, ensuring improved engraftment and function post-administration.

[172] Passaging is performed before 80% confluency to maintain a stable MSC phenotype and avoid excessive differentiation. MSCs can be further stabilized through the use of ROCK inhibitor (e.g., Y-27632, 10 µM) for 24 hours post-thaw or post-passage, which reduces cellular stress, prevents detachment-induced apoptosis, and preserves CD146 / CD166 expression levels. Throughout expansion, flow cytometry-based quality control (QC) assessments may be conducted every 2–3 passages, ensuring that CD146 expression remains within the target range of 30–75%, while CD166 expression is maintained at ≥85%. Any deviations trigger immediate adjustments in culture conditions or batch termination, ensuring product consistency.

[173] This optimized expansion protocol ensures that the final MSC composition maintains its regenerative, migratory, and immunomodulatory properties, improving batch-to-batch reproducibility while enhancing therapeutic efficacy. The combination of defined media, low oxygen tension, ECM support, and controlled passaging creates an optimized environment for MSC expansion, resulting in a stable and potent cell therapy product for the treatment of complex inflammatory diseases.

[174] Example 10 –Release Criteria

[175] To ensure the consistency, potency, and safety of the final mesenchymal stromal cell (MSC) product, a comprehensive Quality Control (QC) and release testing framework can be implemented throughout expansion and prior to product release. The MSC composition may be routinely analyzed for surface marker expression, with flow cytometry assessments conducted, for example and not limitation, every 2–3 passages to confirm that CD146 expression remains between 30–75% and CD166 expression remains at or above 85%, ensuring the maintenance of the desired immunomodulatory and regenerative phenotype. In addition to marker expression, the median fluorescence intensity (MFI) ratios for each of the common MSC markers plus CD146 and CD166 can be monitored to ensure that fluorescence levels remain consistent across production batches, preventing phenotypic drift. Viability assessments can be performed at each passage, with only cultures maintaining, for example, ≥90% viability progressing through expansion. Functional testing can include immunosuppressive assays, such as measuring indoleamine 2,3-dioxygenase (IDO) activity, prostaglandin E2 (PGE2) secretion, and TGF-β release, as indicators of MSC-mediated immune modulation. Migration and adhesion properties can also be evaluated via, for example, Transwell assays and colony-forming unit (CFU) efficiency tests to ensure the cells retain homing ability and self-renewal capacity. Additionally, the MSC composition can be assessed for sterility (bacterial, mycoplasma, and endotoxin testing), chromosomal stability (karyotyping), and mycoplasma contamination to ensure the final product meets clinical-grade safety and regulatory standards. Prior to release, final cryopreserved MSC lots should be tested post-thaw to confirm maintenance of viability and marker expression, ensuring that the therapeutic product remains stable, reproducible, and functionally potent for administration.Industrial Applicability

[176] The pooled, culture-expanded hucMSCs described herein are useful as therapeutic products in the treatment of various disease indications, as manufacturing products for manufacturing supernate / secretome, which can be used as a separate therapeutic product, and as research materials used to study biological mechanisms and related studies.Citation ListPatent Literature1. Phan, Toan Thang. US Pat. No. 11,821,0062. Svahn, Mathias Gösta. US Pat. Pub. No. 2021 / 00081193. Bader ,Peter et al. EP2975118A14. Christopherson, Kent W. US Pat. Pub. No. 2008 / 0118477Non-Patent Literature5. Xie Y, Liu S, Wang L, Yang H, Tai C, Ling L, Chen L, Liu S, Wang B. Individual heterogeneity screened umbilical cord-derived mesenchymal stromal cells with high Treg promotion demonstrate improved recovery of mouse liver fibrosis. Stem Cell Res Ther. 2021 Jun 22;12(1):359.

Claims

1. A therapeutic composition, comprising: pooled, culture-expanded human umbilical cord-derived mesenchymal stromal cells (hucMSCs), said pooled, culture-expanded hucMSCs comprising cells derived from umbilical cords of at least four donors; wherein donor-derived hucMSC populations, each derived from a respective donor-derived hucMSC seed stock, are selected, prior to pooling, from a plurality of donor-derived hucMSC seed stocks based upon characterization of one or more biological characteristics; andwherein the pooled, culture-expanded hucMSCs express surface markers CD44, CD73, CD90, and CD105.

2. The therapeutic composition of claim 1, wherein CD44, CD73, CD90, and CD105 are each positively expressed on at least 95% of the pooled, culture-expanded hucMSCs.

3. The therapeutic composition of claim 1, wherein the one or more biological characteristics comprise one or more of: surface-marker expression, intensity of surface-marker expression, therapeutic-factor secretion, differentiation potential, viability, doubling time, IDO activity and combinations thereof.

4. The therapeutic composition of claim 3, wherein the one or more biological characteristics comprise quantitative surface marker expression.

5. The therapeutic composition of claim 4, wherein the quantitative surface marker expression comprises one or more of: the percentage of cells expressing one or more surface markers, the intensity of surface marker expression, and combinations thereof.

6. The therapeutic composition of claim 3, wherein the one or more biological characteristics comprise secretion of one or more therapeutic factors.

7. The therapeutic composition of claim 6, wherein secretion of the one or more therapeutic factors is quantitatively measured, and wherein the one or more therapeutic factors comprise one or more of VEGF, IL-6, IL-8, PGE2, and TGF-β.

8. The therapeutic composition of any one of claims 1 to 7 for use in treating epidermolysis bullosa.

9. The therapeutic composition for use according to claim 8, wherein the epidermolysis bullosa is recessive dystrophic epidermolysis bullosa, and wherein 40% to 60% of the pooled, culture-expanded hucMSCs express CD146 and 85% to 95% of the pooled, culture-expanded hucMSCs express CD166.

10. A method for producing a pooled therapeutic composition, the method comprising:a) obtaining a plurality of donor-derived hucMSC seed stocks;b) quantitatively characterizing one or more biological characteristics of each donor-derived hucMSC seed stock;c) selecting donor-derived hucMSC seed stocks based upon said quantitative characterization;d) pooling the selected donor-derived hucMSC seed stocks; ande) expanding the pooled donor-derived hucMSC seed stocks to produce a therapeutic composition exhibiting one or more biological characteristics associated with a selected therapeutic indication.

11. The method of claim 10, wherein the biological characteristics comprise one or more of surface-marker expression, surface-marker intensity, therapeutic-factor secretion, differentiation potential, viability, doubling time, and indoleamine 2,3-dioxygenase activity.

12. The method of claim 11, wherein quantitatively characterizing comprises one or more of flow-cytometric measurement of surface-marker expression or intensity, measurement of viability or doubling time, and quantitative measurement of secretion or activity of one or more therapeutic factors.

13. The method of claim 10, wherein the donor-derived hucMSC seed stocks are selected to produce a pooled composition exhibiting one or more biological characteristics associated with the selected therapeutic indication.

14. The method of claim 10, wherein selecting the donor-derived hucMSC seed stocks comprises applying a computational model.

15. The method of claim 14, wherein the computational model determines respective proportions of the selected donor-derived hucMSC seed stocks based on quantitative measurements of one or more biological characteristics.  

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