Immunophenotype of hematopoietic stem cells from placental/umbilical cord blood after culture

Abstract

Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 ± 0.9% CD34+ cells, 2.6 ± 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 ± 1.8-fold, but the number of viable CD34+ cells decreased by 46 ± 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 ± 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype

Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount™-BD and ISHAGE protocols

“…A great variation in the number of these cells in the cord blood has been reported in the literature (20,22,(24)(25)(26)(27)(28). Hao et al (22) observed a variation of 0.02 to 1.43% in the number of CD34+ cells in the total mononuclear cell population.…”

“…Keeney et al (36) showed that the use of 7AAD decreased the presence of CD34+ cells per microliter of blood by 50%, suggesting that these were non-viable cells. This is particularly important after thawing of samples from umbilical cord blood banks or culture of umbilical cord blood samples (17,26,37,38). Thus, it has been suggested that 7AAD should be routinely used for quantification of CD34+ cells in the ISHAGE protocol (36).…”

Microarray profiles of ex vivo expanded hematopoietic stem cells show induction of genes involved in noncanonical Wnt signaling

“…Cells were cultured under serum-free conditions, in FreeStyle 293 Expression Medium (Invitrogen, Carlsbad, CA, USA), supplemented with 1% human albumin and a cocktail of cytokines, based on previous reports (Pranke et al, 2005;Kelly et al, 2009): 50 ng/mL flt-3 ligand, 35 ng/mL thrombopoietin, 50 ng/mL stem cell factor, and 10 ng/mL interleukin-6.…”

“…However, the low number of HSC in UCB is directly related to increased risk of graft failure and delayed engraftment and immune reconstitution. Effective in vitro expansion of HSC has been long tried, with conflicting reports of success (Gilmore et al, 2000;McNiece et al, 2000;Pranke et al, 2005;Walenda et al, 2011;Auvray et al, 2012). The failure of these protocols may be due to the inability to reproduce in vitro the combination of factors that occurs in vivo, where simultaneous HSC proliferation and maintenance of long-term hematopoietic reconstitution is achieved (Astori et al, 2001;Blank et al, 2008).…”

Fetal heart extract facilitates the differentiation of human umbilical cord blood-derived mesenchymal stem cells into heart muscle precursor cells

“…Amniotic fluid contains a multi-potential stem cell population, but these cells were rejected when transplanted into a myocardial infarction rat model (Chiavegato et al 2007). Thus, human umbilical cord bloodderived mesenchymal stem cells (UCB-MSCs) which are young, healthy, easy to acquire, and potential immune modulators (Iafolla et al 2014;Pranke et al 2005;Wagner et al 2005) should be considered for differentiation into cardiomyocytes and treatment of cardiovascular disease.…”