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.
Forraz
Nicolas
N
King-George Laboratory, St. George's Hospital Medical School and Kingston University, London, UK.
Pettengell
Ruth
R
McGuckin
Colin P
CP
D000954:Antigens, Surface / Q000276:immunology; D015415:Biomarkers; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000187:drug effects / Q000276:immunology; D019070:Cell Lineage / Q000276:immunology*; D002465:Cell Movement / Q000187:drug effects / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D036101:Cord Blood Stem Cell Transplantation / Q000379:methods*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology*; D006801:Humans; D007231:Infant, Newborn; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D013234:Stem Cells / Q000166:cytology / Q000276:immunology*; D013995:Time
D000954:Antigens, Surface; D015415:Biomarkers; D003470:Culture Media; D016207:Cytokines