Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

Common Connective Tissue Progenitor Cells are the Alternative Solution to the Failing Mesenchymal Stem Cells in Regenerative Medicine Angelo D. Monaco, OMS-I; Korinna Toth, OMS-I; Zoltan Hajdu, MD Edward Via College of Osteopathic Medicine, Carolinas Campus, Spartanburg, SC Introduction Results Results

Figure 4: In vivo CCTPCs

• The stromal mesenchymal stem cells (MSCs) were originally observed and described by Friedenstein: plastic adherent, spindle-shaped cells of the bone marrow, which can differentiate into osteogenic, fibrogenic and adipogenic lineages. • The in vitro differentiation potential were reported by numerous labs way broader then initially described. This gave the original momentum for developing stem cell therapies. However, the human in vivo results came out very disappointing. • In our lab, we have identified the hematopoietic-derived common connective tissue progenitor cells (CCTPCs), which differentiate into multiple connective tissues across the body. Objective: to compare the in vitro and in vivo behavior of MSCs and CCTPCs for their potential use in regenerative medicine. Cell Cultures: • Murine and human bone marrow mononuclear cells were analyzed for viability and CD45 (hematopoietic) expression then plated in T25 flasks until the first cell attachments were observed (approx. 2 weeks). • The free-floating cells were replated while the adhered cells were fed and passaged. This was repeated for 12 times. • Cells were continuously analyzed: pictures of the cultures (for morphology) and immunohistochemistry (for phenotyping) were performed at each manipulation. Mouse bone marrow transplants: • Lethally irradiated wild type mice (Ly=5.1) were tail vein injected with hematopoietic stem cells isolated from EGFP/Ly-5.2 transgenic mice. • Bone marrow and other tissue samples were collected from the chimeric mice starting 1 month and up to 2 years post transplant. Methods

Figure 1: Identification of MSCs and CCTPCs in cultured bone marrow cells. A B C

B

D

A

C

CD45 CD34 CD133

One-year post-transplantation virtually all bone marrow cells are GFP + (A, green). The mouse mitral valve (B – low mag, C-high mag) shows fusiform-shaped CCTPCs (red: CD45, green: GFP). Adult human mitral valve showing CCTPCs (red: CD45, green: CD133). Blue: YoPro1 (nuclei).

Immunohistochemistry on cultured, cytocentrifuged human bone marrow mononuclear cells. A – CD45 - MSCs (adherent), B – CD45 - (arrows) and CD45 + free floating cells, C – CD45 + /CD34 + /CD133 + CCTPCs (arrows). Green on A,B: YoPro1 (nuclei). Red on B: CD45 (hematopoietic). Figure 2: Morphology of the cultured bone marrow cells. A B C D 3 days After a month of culturing the bone marrow cells, the initially adhering cells (MSCs) are virtually the same as the initially free floating cells (CCTPCs) (A, D). B,C – CD45 labeling of the CCTPCs as they take a fusiform shape in culture. Figure 3: In vitro differentiation potential of the MSCs and CCTPCs. Hsp47 Alkaline Phosphatase Alcian Blue Oil Red O MSCs CD45 + /CD133 + /C D34 + Both cell populations have the same in vitro differentiation potential toward fibrogenic, osteogenic, chondrogenic and adipogenic lineages. MSCs 1 day

Discussion

• The human bone marrow contains only about 1% of MSCs, which is not enough for autologous stem cell therapies. • The CD45 + hematopoietic bone marrow cells give rise to the MSCs. • Only CD45 + hematopoietic cells contribute to different tissues in vivo, we never seen bone marrow-derived parenchymal cells.

CCTPCs

Conclusion

MSCs are only an artificial cell population, the CCTPCs are their real alternative to use in regenerative medicine.

References

This project was supported by the NIH COBRE SC Biocraft Pilot Project (ZH), the VCOM Research Eureka Accelerator Program (ZH). The authors wish to express special thanks to Kayla Afkinich for critical reading of our abstract. Acknowledgements 1. Friedenstein et al.: Heterotopic bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation. 6(2): 230-247. 1968. 2. Cagliani et al.: Immunomodulation by mesenchymal stromal cells and their clinical applications. J Stem Cell Regen Biol. 3:1-26. 2017. 3. Jackson et al.: Adult mesenchymal stem cells: Differentiation potential and therapeutic applications. J Postgrad Med. 53:121-127. 2007. 4. Ogawa et al.: Hematopoietic stem cells are pluripotent and not just “hematopoietic”. Blood Cells Mol Dis. 51(1): 3 -8. 2013. 5. Bucala et al.: Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1(1): 71-81. 1994.

2025 Research Recognition Day

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