Development and Characterization of Human iPSC-Derived Mesenchymal Progenitor Cells
Introduction
Mesenchymal progenitor cells (MPCs), derived from human pluripotent stem cells (hPSCs), are playing a key role in driving novel therapies for tissue repair and regenerative medicine. These cells offer several advantages over traditional mesenchymal stromal cells (MSCs), including a more consistent, scalable, and versatile source of progenitor cells. MSCs, originally identified in the adherent fraction of bone marrow stroma (1970) , are self-renewing and multipotent, capable of differentiating into osteoblasts, adipocytes, and supportive stromal cells. MSCs were first described as colony-forming unit fibroblasts (CFU-Fs) due to their ability to form single-cell-derived colonies. In their native environment, they play key roles in regulating early hematopoiesis and maintaining the bone marrow’s haemosupportive stroma.
The application of MSCs in therapy faces limitations such as donor variability, restricted availability, and the decline of potency with age or prolonged culture. Human induced pluripotent stem cell (iPSC)-derived MPCs have emerged as a compelling alternative, providing a renewable, reproducible source of cells with the ability to differentiate into mesenchymal lineages, including adipocytes, osteocytes, and chondrocytes. This not only makes MPCs a powerful tool for therapeutic applications but also offers a reliable platform for studying cellular differentiation processes.
Understanding the Terminology: MPCs vs. MSCs
The distinction between MPCs and MSCs is rooted in both biology and nomenclature. MSCs, often referred to as mesenchymal stem cells, are recognized for their multipotency, but their classification as true stem cells, especially in terms of unlimited self-renewal, has been questioned. Leading figures like Pamela Robey (NIH/NICDR) have advocated for the use of the term "mesenchymal stromal cells" to more accurately reflect that these cells may not meet the criteria of true stem cells (The Stem Cell Podcast, 2018 ). In contrast, MPCs, particularly those derived from pluripotent stem cells, retain the capacity for unlimited self-renewal while they remain in the undifferentiated state. However, once differentiated into specific lineages such as osteocytes or adipocytes, they lose this self-renewal capability, reflecting their commitment to specific cell fates.
This precise nomenclature is essential for accurately describing the nature of the cells used in both research and clinical applications. iPSC-derived MPCs are referred to as progenitor cells because they exhibit robust multipotency and retain self-renewal potential before differentiation, yet their defined lineage commitment distinguishes them from undifferentiated pluripotent stem cells. As PSC-derived MPCs continue to gain prominence in clinical therapies, this shift in terminology underscores the importance of clearly defining the characteristics and limitations of these cells.
Differentiation and Characterization of iPSC-Derived MPCs
The differentiation of iPSC-derived MPCs into specific mesenchymal cell types is a key focus of research and therapeutic development. This differentiation is typically assessed using well-established staining techniques. Adipogenic differentiation is evaluated through Oil Red O staining, which detects lipid droplets within adipocytes by Day 30. Osteogenic differentiation is confirmed using Alizarin Red S staining to highlight calcium deposits in osteocytes by Day 21. For chondrogenic differentiation, Alcian Blue and Nuclear Fast Red staining are employed to identify glycosaminoglycans and cell nuclei within chondrocytes by Day 21.
Advances in differentiation protocols have further improved the efficiency and reproducibility of generating MPCs from hPSCs. These protocols utilize specific culture conditions and growth factors that drive hPSCs into mesenchymal lineages. For example, osteogenic differentiation is enhanced by adding dexamethasone, ascorbate, and β-glycerophosphate to the culture medium (Jaiswal et al., 1997 ), while adipogenic differentiation can be stimulated with a cocktail of insulin, dexamethasone, and IBMX (Pittenger et al., 1999 ). Flow cytometric analysis of iPSC-derived MPCs reveals that by Day 21, these cells express mesenchymal markers such as CD73, CD90, and CD105, alongside the perivascular marker CD146. Importantly, these MPCs lack the expression of hematopoietic markers CD34 and CD45, as well as the endothelial marker CD144, further confirming their mesenchymal identity.
Clinical Applications and Trials of iPSC-Derived MPCs
The clinical potential of PSC-derived MPCs is vast, with ongoing research focusing on their application across a range of therapeutic areas. Cynata Therapeutics, a leader in stem cell technology, has been pioneering MPC-based treatments, particularly in the treatment of chronic, non-healing diabetic foot ulcers. Their randomized, controlled trial (NCT05165628 ) is evaluating the safety, tolerability, and efficacy of an MPC-based dressing, CYP-006TK. This approach aims to harness the regenerative capacity of MPCs to promote healing and reduce ulcer size, offering a potentially significant improvement over current standard treatments.
Furthermore, Cynata is also exploring the role of iPSC-derived MPCs in treating severe respiratory conditions, including COVID-19-related complications. The MEseNchymal coviD-19 Trial (MEND) (NCT04537351 ) is assessing the early efficacy of intravenously administered MPCs in patients with respiratory failure. The potential of MPCs to modulate acute inflammatory responses and promote recovery in critically ill patients underscores their broader utility in regenerative medicine.
In addition to these specific trials, the use of MPCs is being investigated in a wide array of conditions. For example, hESC-derived MPCs are currently being studied for their safety in treating interstitial cystitis (NCT04610359 ), moderate to severe intrauterine adhesions (NCT04232592 ), and primary ovarian insufficiency (NCT03877471 ). The versatility of iPSC-derived MPCs is further demonstrated in trials targeting autoimmune diseases, reproductive health, and regenerative therapies, all of which highlight the broad therapeutic potential of these cells.
Summary
iPSC-derived MPCs represent a significant step forward in regenerative medicine, offering solutions to several limitations faced by traditional MSCs. While MSCs have been useful in various therapies, they often come with challenges like donor variability, limited expansion capacity, and inconsistent outcomes due to differences in donor quality. iPSC-derived MPCs, by contrast, offer a reproducible and scalable source of cells, which can be generated in a patient-specific manner, reducing the risk of rejection. With unlimited self-renewal in the undifferentiated state, iPSC-derived MPCs provide a reliable platform for tissue regeneration, disease modeling, and therapeutic applications. As translational research and clinical trials continue to evaluate the safety and efficacy of iPSC-derived MPCs, their role in advancing regenerative medicine becomes increasingly evident, paving the way for more consistent, scalable treatments for a wide variety of medical conditions.
Human iPSC-Derived Mesenchymal Progenitor Cells from STEMCELL Technologies
STEMCELL Technologies has developed cryopreserved Human iPSC-Derived Mesenchymal Progenitor Cells (MPCs) (Catalog #200-0781) for research use. These cells offer a reproducible and versatile tool for applications including tissue engineering, disease modeling, drug discovery, and other exploratory areas of regenerative medicine. Derived from the highly characterized Healthy Control Human iPSC Line, SCTi003-A (Catalog #200-0511), these MPCs demonstrate multipotency, as shown by their ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. They express key mesenchymal markers CD73, CD90, and CD105, providing a robust platform for researchers to explore mesenchymal biology and potential therapeutic strategies in preclinical models.
Advantages:
Cell Line Information - SCTi003-A
Human iPSC-Derived MPCs were differentiated from the Healthy Control Human iPSC Line, SCTi003-A (Catalog no. 200-0511); a cell line derived from peripheral blood mononuclear cells (PBMCs) from a 48-year-old female donor. Extensive quality control procedures are undertaken in our iPSC manufacturing process to ensure optimal product performance and reproducibility. SCTi003-A is karyotypically stable, demonstrates trilineage differentiation potential, expresses markers of the undifferentiated state, and was reprogrammed using a non-integrating reprogramming technology. The cell line is used as a healthy control for a multitude of pluripotent stem cell research applications including downstream differentiation to lineage-specific cell types and organoids.
Extensive quality control procedures are implemented at every stage of STEMCELL’s iPSC manufacturing process (Table 1). Our commercial iPSC quality assessments and release criteria have been developed based on recommendations and guidance from the Standards for Human Stem Cell Use In Research (ISSCR, 2023).
SCTi003-A is manufactured with mTeSR? Plus (Catalog no. 100-0276) and is fully compatible with STEMdiff? cell culture media products, allowing for standardized high-quality maintenance and differentiation to various cell types such as cardiomyocytes, neurons, astrocytes, and microglia.
SCTi003-A was derived from an αβ T cell and has undergone VDJ rearrangement.
The SCTi003-A certificate of analysis (COA) includes information relating to the product, cell line, recommended culture conditions, donor information, and detailed results for morphology, viability and recovery, cell line identity, sterility testing, mycoplasma testing, viral screening, parent cell lineage determination, chromosome analysis, 20q status, copy number variants, donor ancestry, genetic variants, TP53 and BCOR status, undifferentiated status, and pluripotency.
As an example, the certificate of analysis for SCTi003-A Lot # 2205404000 can be found here .
To learn more about SCTi003-A on the hPSCreg? website, click here .
Note - iPSC-Derived MPCs were manufactured from the SCTi003-A working cell bank (WCB).
Table 1. Characterization Assessments Are Performed at Various Stages throughout the iPSC Manufacturing Process.
Master cell banks are tested for identity, adventitious agents, genomic integrity and stability, survival, undifferentiated state, and pluripotency. Working cell banks and commercial vials are tested for a subset of these characterization criteria.
Donor Information
STEMCELL collects donor demographic information ethically, using consent forms and protocols approved by either an Institutional Review Board (IRB), the Food and Drug Administration (FDA), the U.S. Department of Health and Human Services, and/or an equivalent regulatory authority. Donations are performed in the United States in compliance with applicable federal, state, and local laws, regulations, and guidance. Healthy donors must be over the age of 18, weigh at least 120 lb, have a body mass index (BMI) between 18.5–24.9, demonstrate no use of tobacco products, and be in good general health. Additionally, donors in our healthy pool are pre-screened using a health questionnaire aimed at excluding any donors with diseases, blood disorders, or other health concerns.
Table 2 details attributes that were determined for the SCTi003-A donor. Age, diagnosis, ethnicity and/or race, and tobacco use were self-declared by the SCTi003-A donor. Sex, ancestry, height, weight, BMI, blood type, HLA haplotype, and pathogenic genetic variants were calculated using various methods detailed in the table legend.
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Table 2. iPSC Line SCTi003-A Is Derived from a Healthy Female Donor.
Demographic, health, and genetic characteristics of the SCTi003-A donor were compiled based on self-reported information and whole-exome sequencing. Sex was determined by karyotype. Ancestry was calculated by EthSEQ analysis from whole-exome sequencing data. HLA haplotype was determined by next-generation sequencing, sequence-base typing, and sequence-specific oligonucleotide probes as needed to obtain the required resolution. Other genetic variants were determined from whole-exome sequencing using ClinVar analysis. Blood type (ABO/Rh blood group) was determined by next-generation sequencing. Height, weight, and BMI were calculated at the donation facility.
Morphology and Expansion of iPSC-Derived Mesenchymal Progenitor Cells
Human iPSC-Derived MPCs display a typical spindle-shaped, fibroblast-like morphology consistent with mesenchymal lineage cells. Upon thawing and plating in MesenCult?-ACF Plus Medium (Catalog #05445), these cells adhere quickly and show consistent growth, reaching ~80% confluency within four days, as shown in Figure 1A. This high-quality morphology is indicative of their readiness for passaging and further expansion.
The proliferative capacity of these MPCs is reflected in their rapid doubling time, averaging approximately one day across three passages post-thaw, as illustrated in Figure 1B. This rapid expansion demonstrates their robustness and suitability for studies requiring large quantities of cells. In Figure 1C, the cumulative expansion data highlight the cells' ability to maintain stable growth over multiple passages, which is critical for applications that demand reliable and scalable production of MPCs.
These attributes make iPSC-Derived MPCs an ideal tool for various research applications, including tissue engineering, drug discovery, and regenerative medicine studies. Their consistent expansion capacity allows researchers to reliably produce enough cells for exploratory studies in these areas, providing a robust and physiologically relevant platform for investigating mesenchymal biology.
Figure 1. Human iPSC-Derived Mesenchymal Progenitor Cells Show High-Quality Morphology and Robust Expansion for Multiple Passages.
(A) Representative microscopy image showing cryopreserved Human iPSC-Derived Mesenchymal Progenitor Cells thawed and maintained in MesenCult?-ACF Plus Medium for 4 days and ready for passaging at ~80% confluency. (B) Cells show robust expansion post-thaw. Data points show the average doubling time over 3 passages post-thaw from 3 technical replicates. C) Human iPSC-Derived Mesenchymal Progenitor Cells were thawed and maintained in MesenCult?-ACF Plus Medium. Cells were passaged every 3-4 days post thaw and cell expansion was analyzed as cumulative cell number over 4 passages from 2 technical replicates.
Flow Cytometric Characterization of Human iPSC-Derived Mesenchymal Progenitor Cells
Human iPSC-derived MPCs exhibit hallmark characteristics of mesenchymal cells, as demonstrated by their expression of key surface markers. Upon thawing and a single passage in MesenCult?-ACF Plus Medium (Catalog #05445), the MPCs maintain robust expression of CD73, CD90, and CD105—canonical markers for mesenchymal lineage. These markers are commonly used to identify MSCs in both research and clinical contexts.
The consistency of marker expression across five technical replicates, as shown in Figure 2A, illustrates the reliability and reproducibility of these cells. Each marker’s expression is quantified and represented with minimal variability, reflecting the quality control measures in place during manufacturing. CD73, CD90, and CD105 are expressed in over 95% of the cells, as shown by the representative flow cytometry plots (Figures 2B-D), confirming the mesenchymal identity of these cells.
Notably, this high expression of key mesenchymal markers aligns with the phenotypic criteria established by the International Society for Cellular Therapy (ISCT) (2006) for defining mesenchymal cells. By adhering to these widely accepted standards, these iPSC-Derived MPCs provide a reliable and physiologically relevant model for use in translational research and tissue engineering.
Figure 2. Human iPSC-Derived Mesenchymal Progenitor Cells Express Characteristic Markers.
Human iPSC-Derived Mesenchymal Progenitor Cells were thawed, cultured in MesenCult?-ACF Plus Medium? for one passage, characterized using flow cytometry for mesenchymal cell markers, and show high expression of? CD73, CD90, and CD105. (A) Percentage marker expression was quantified from the analyses of five technical replicates. Representative flow cytometry plots are displayed for (B) CD73, (C) CD90 and (D) CD105.
Multipotency of Human iPSC-Derived Mesenchymal Progenitor Cells: Differentiation into Adipocytes, Osteoblasts, and Chondrocytes
Human iPSC-Derived MPCs possess robust multipotency, demonstrated by their ability to differentiate into multiple cell types, including adipocytes, osteoblasts, and chondrocytes. This ability makes them a valuable resource for exploring cell lineage differentiation and potential applications in tissue engineering and regenerative medicine.
Using the MesenCult? product line from STEMCELL Technologies, iPSC-derived MPCs can be efficiently directed into specific lineages:
These results underscore the versatility and reliability of Human iPSC-Derived MPCs as a model system for exploring cell differentiation pathways. Their ability to differentiate into key skeletal lineages highlights their applicability in research fields such as skeletal tissue development, disease modeling, and regenerative strategies targeting bone, cartilage, and fat tissue repair. By providing a reproducible and defined source of mesenchymal progenitors, these cells are essential tools for advancing research in tissue-specific regeneration and translational studies.
Figure 3. Human iPSC-Derived Mesenchymal Progenitor Cells Can Differentiate into Adipocytes, Osteoblasts, and Chondrocytes
(A) Adipocytes were generated from Human iPSC-Derived Mesenchymal Progenitor Cells using MesenCult? Adipogenic Differentiation Kit. Adipogenic differentiation was assessed by Oil Red O stain at Day 30 post differentiation. (B) Osteoblasts were differentiated using MesenCult? Osteogenic Differentiation Kit. Osteogenic differentiation was assessed by Alizarin Red S stain at Day 21 post differentiation. (C) Chondrocytes were differentiated using MesenCult?-ACF Chondrogenic Differentiation Kit. Chondrogenic differentiation was assessed by Alcian Blue and Nuclear Fast Red Stain at Day 21 post differentiation.
Summary
STEMCELL Technologies has developed cryopreserved Human iPSC-Derived Mesenchymal Progenitor Cells (Catalog #200-0781), optimized for a variety of research applications in mesenchymal biology. These cells offer a robust, consistent, and highly characterized source of mesenchymal progenitors, providing an essential tool for tissue engineering, disease modeling, drug discovery, and exploratory research in regenerative medicine.
Human iPSC-Derived Mesenchymal Progenitor Cells are available now at a price of $799 USD per vial (1 million viable cells). Unlike iPSC lines from STEMCELL's iPSC Repository, an annual license fee is not required for the use of our differentiated cell types.
For more information about STEMCELL's iPSC lines and differentiated cells, refer to our Frequently Asked Questions on iPSCs .
For any other queries, click here to contact STEMCELL's iPSC Team or email us directly at [email protected] .