Characterization of a Lineage‐Negative Stem‐Progenitor Cell Population Optimized for Ex Vivo Expansion and Enriched for LTC‐IC

Current hematopoietic stem cell transplantation protocols rely heavily upon CD34+ cells to estimate hematopoietic stem and progenitor cell (HSPC) yield. We and others previously reported CD133+ cells to represent a more primitive cell population than their CD34+ counterparts. However, both CD34+ and CD133+ cells still encompass cells at various stages of maturation, possibly impairing long‐term marrow engraftment. Recent studies demonstrated that cells lacking CD34 and hematopoietic lineage markers have the potential of reconstituting long‐term in vivo hematopoiesis. We report here an optimized, rapid negative‐isolation method that depletes umbilical cord blood (UCB) mononucleated cells (MNC) from cells expressing hematopoietic markers (CD45, glycophorin‐A, CD38, CD7, CD33, CD56, CD16, CD3, and CD2) and isolates a discrete lineage‐negative (Lin−) cell population (0.10% ± 0.02% MNC, n = 12). This primitive Lin− cell population encompassed CD34+/− and CD133+/− HSPC and was also enriched for surface markers involved in HSPC migration, adhesion, and homing to the bone marrow (CD164, CD162, and CXCR4). Moreover, our depletion method resulted in Lin− cells being highly enriched for long‐term culture‐initiating cells when compared with both CD133+ cells and MNC. Furthermore, over 8 weeks in liquid culture stimulated by a cytokine cocktail optimized for HSPC expansion, TPOFLK (thrombopoietin 10 ng/ml, Flt3 ligand 50 ng/ml, c‐Kit ligand 20 ng/ml) Lin− cells underwent slow proliferation but maintained/expanded more primitive HSPC than CD133+ cells. Therefore, our Lin− stem cell offers a promising alternative to current HSPC selection methods. Additionally, this work provides an optimized and well‐characterized cell population for expansion of UCB for a wider therapeutic potential, including adult stem cell transplantation.