Direct comparison by limiting dilution analysis of long-term culture- initiating cells in human bone marrow, umbilical cord blood, and blood stem cells

Abstract

Limiting-dilution analysis of long-term culture-initiating cells (LTCIC) is a quantitative method of estimating hematopoietic stem cell activity in clinical samples. We compared the numbers of LTCIC in bone marrow (BM), umbilical cord blood, and blood progenitor cells (obtained from patients with solid tumors at leukapheresis after mobilization with induction chemotherapy and filgrastim administration), using a two- stage long-term culture system and a limiting-dilution technique, scoring cobblestone areas of greater than 15 hematopoietic cells weekly for up to 8 weeks. Samples were obtained from 30 normal BMs, 20 human umbilical cords, and 32 leukapheresis products. Direct comparison of LTCIC in the three sources showed that the median proportions of cells generating hematopoietic foci from unfractionated mononuclear cells at 5 and 8 weeks, respectively, were 1:13,314 and 1:33,949 for BM, 1:12,506 and 1:34,546 for umbilical cord blood, and 1:10,302 and 1:12,891 for leukapheresis product. The estimated proportions of LTCIC from unfractionated mononuclear cells and CD34+ cells were similar in experiments with leukapheresis product. Leukapheresis product was superior to umbilical cord blood and cord blood to BM at 5 and 8 weeks of culture (P = .01). In two-stage long-term cultures, more colonies per flask and CD34+ cells were found in assays of leukapheresis product than in BM or umbilical cord blood cultures (P = .0005). Results obtained by this simplified limiting-dilution analysis correlated well with standard long-term cultures and can be used as a measure of the stem cell population. These data suggest that the incidence of putative stem cells in leukapheresis product and umbilical cord blood are at least comparable with that of BM.

Characterization of the Adherent Cells Developed in Dexter‐Type Long‐Term Cultures from Human Umbilical Cord Blood

“…Interestingly, when Dexter-type long-term cultures (D-LTC) are established from human UCB, HSPC proliferation is only transiently sustained. Indeed, several studies have shown that, compared to BM D-LTC, progenitor cell levels in UCB D-LTC are significantly lower throughout the entire culture period and reach undetectable levels several weeks earlier [9][10][11]. These findings have been explained by the fact that, in UCB D-LTC, a confluent stromal adherent cell layer always fails to form, leaving HSPC without a competitive hematopoietic microenvironment and a source of stimulatory cytokines [12].…”

Impact of obstetric factors on cord blood donation for transplantation

“…However, data suggest that low haemopoietic cell content of CB donations may limit their use to smaller recipients (Wagner et al, 1996). In-vitro evidence suggests that the frequency of primitive haemopoietic cells, such as long-term-culture initiating cells (LTC-IC), in CB is comparable to that of normal adult bone marrow (Pettengell et al, 1994), although CB LTC-IC have distinctive proliferative advantages compared to marrow LTC-IC (Hows et al, 1992).…”

Cobblestone Area‐Forming Cells in Human Cord Blood Are Heterogeneous and Differ from Long‐Term Culture‐Initiating Cells

“…Some centers routinely adherent cell deplete MNCs before seeding feeder layers for LTC-IC assays, a method adopted for LTC-IC analysis of bone marrow samples to deplete any thirdparty stromal cells. Petengell et al [19] compared CAFC and LTC-IC frequencies in CB adherent cell-depleted MNCs and found that although most had a lower LTC-IC than CAFC frequency, there was no instance of the dramatic differences reported for our CB samples. It was possible therefore that the majority of false CAFC were removed by adherence.…”