Integrin-α3 Is a New Marker of Long-Term Hematopoietic Stem Cells in Ex Vivo Expanded Cells

Hematopoietic stem cell (HSC) transplantation constitutes one of the most effective therapeutic strategies to treat numerous hematological diseases. Cord blood (CB) is one of the most attractive donor sources of stem cells for this procedure due to its rapid availability, HLA mismatches tolerance and low associated risk of chronic graft-versus-host disease. However, these advantages are offset by the limited cell dose in CB units, which can contribute to delayed hematopoietic engraftment following transplantation. Mastering ex vivo HSC expansion is therefore of great interest for clinical purposes and for genetic manipulation. HSCs can be functionally defined as either long-term (LT-HSC), providing life-long hematopoiesis and characterized by delayed engraftment pattern, or short-term repopulating stem cells (ST-HSC), providing early and transient hematopoietic recovery. Major hurdles hindering the study of these cell populations is the current inability to evaluate their content in cultured samples and the lack of understanding of the molecular mechanisms regulating stem cell self-renewal ex vivo. Those issues highly benefited from the discovery by our laboratory of the small molecule UM171, which promote HSC expansion ex vivo, as well as from the identification of EPCR as one of the most reliable surface markers for cultured HSCs.

We now describe the identification of Integrin-α3 (ITGA3) as a novel marker for cultured HSCs. ITGA3 expression was found to be sufficient to split the primitive EPCR⁺CD90⁺CD133⁺CD34⁺CD45RA^- HSC population in two functionally distinct fractions presenting only short-term (ITGA3^-) and both short-term and long-term (ITGA3⁺) repopulating potential. ITGA3⁺ cells, as opposed to the ITGA3^- fraction, exhibited robust multilineage differentiation potential and serial reconstitution ability in immunocompromised mice. This combination of markers identifies repopulating HSCs in culture by FACS beyond what is currently possible with other approaches, with a frequency of LT-HSC found in the ITGA3⁺ population estimated at 1:38 in day 7 UM171 expanded CB-cells. Moreover, lentiviral-mediated ITGA3 knockdown was shown to compromise the LT repopulating activity of cultured HSC in vivo. Gene expression profiling revealed striking molecular similarity between ITGA3⁺ and ITGA3^- cells, showing overrepresentation of genes involved in fundamental hematopoietic programs known to govern HSC specification and function in both of these populations. However, ITGA3⁺ and ITGA3^- subsets clearly clustered separately by principle component analysis, indicating broad differences in gene expression. Concordantly with their primitive phenotype, stem cell markers and cell quiescence are gene sets enriched in ITGA3⁺ cells, while progenitor markers, DNA replication, M/G1 and G2/M checkpoints, mRNA processing, reduction of hypoxia and Myc targets were significantly downregulated in these cells.

Altogether, our results indicate that ITGA3 is a reliable marker for cultured HSCs, improving the accuracy of prospective HSC identification in culture. Deciphering the function of genes upregulated in primitive ITGA3⁺ HSCs will represent an invaluable resource for dissecting the genetic programs that govern hematopoietic stem cells biology.