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Turkish Journal of Biology

Abstract

Background/aim: Hematopoietic stem cells (HSCs) are essential for treating hematological disorders; however, their low abundance and limited proliferative capacity restrict their clinical use. This study investigated whether pifithrin-α (PFT-α), a small-molecule p53 inhibitor with known genoprotective properties, could enhance the ex vivo expansion of HSCs.

Materials and methods: Mouse lineage-negative (Lin⁻) cells were isolated by magnetic depletion, while human umbilical cord blood (hUCB) and mouse bone marrow (mBM) mononuclear cells were obtained using density gradient centrifugation. CD34⁺ cells were subsequently isolated by fluorescence-activated cell sorting using an antimouse CD34 antibody. The isolated cells were cultured in StemSpan SFEM II (STEMCELL Technologies Inc., Vancouver, BC, Canada) medium supplemented with mouse stem cell factor, thrombopoietin, and Flt3-L for 7 days. PFT-α was administered throughout the culture period. After 7 days, mouse cells were analyzed for Lin⁻Sca-1⁺c-Kit⁺ (LSK) and LSK CD34^low populations, whereas human HSCs were evaluated for CD34 and CD133 expression. Additional assessments included apoptosis analysis, cell cycle distribution, and in vivo engraftment in severe combined immunodeficient (SCID) mice.

Results: PFT-α treatment resulted in a threefold increase in HSC populations derived from mouse and hUCB samples (p < 0.05; p < 0.01) and a twofold increase in mBM-derived HSC populations (p < 0.05). Apoptosis levels tended to decrease, and most cells remained in the G0/G1 phase, consistent with the maintenance of stem cell characteristics. Expanded mouse HSCs successfully engrafted in SCID mice. PFT-α did not exhibit overt cytotoxic effects on human umbilical vein endothelial cells, human dermal fibroblasts, or MSCs under the experimental conditions.

Conclusion: PFT-α significantly enhanced the ex vivo expansion of HSCs and appeared to preserve early engraftment potential. These findings indicate that PFT-α may represent a promising candidate for enhancing ex vivo hematopoietic stem cell expansion for therapeutic applications. Further studies should investigate long-term repopulation ability, multilineage differentiation potential, optimized dosing regimens, and efficacy in disease-specific or transplantation models.

Author ORCID Identifier

NESLİHAN MERİÇ: 0000-0002-2878-5052

PINAR ÇOLAKOĞLU ERKAN: 0000-0002-2870-5614

FATİH KOCABAŞ: 0000-0001-8096-6056

DOI

10.55730/1300-0152.2977

Keywords

Pifithrin-α, hematopoietic stem cells, p53, mesenchymal stem cells, umbilical cord blood

First Page

327

Last Page

338

Publisher

The Scientific and Technological Research Council of Türkiye (TÜBİTAK)

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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