Initial CD34+ cell‐enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon Ex vivo expansion

Abstract

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal-based serum-free culture system to evaluate the effect of different initial CD34(+) cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34(+) and CD34(+) CD90(+) expression, we have identified early activation of CD34 expression on CD34(-) cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34(+) cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34(+)/CD34(+) CD90(+) cell yield (High: 14 ± 1.0/3.5 ± 1.4-fold; Medium: 22 ± 2.0/3.4 ± 1,0-fold; Low: 31 ± 3.0/4.4 ± 1.5-fold) after a 7-day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34(+) cell recovery for each strategy, on overall CD34(+) cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34(+) cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10(6) cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex-vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost-effective expansion of HSC for cellular therapy.