Summary:Human fetal livers (FL), between 16 and 24 weeks of gestation, were studied for their potential as a source of hematopoietic stem cells for prenatal and postnatal transplantation. In this report we give a quantitative evaluation of human FL as a source of candidate stem cells, and develop a protocol for the isolation of these cells free of microbial contaminants and almost free of mature T cells. Human FLs contained a median 1.9 ؋ 10 9 viable cells and a mean of 1. 2,3 Some promising results were reported with the transplantation of FL cells in humans, either before or after birth.1,4-8 However, factors limiting the use of first trimester FL cells became apparent. Among these are the difficulties in harvesting liver at early gestational ages, the relatively low number of cells available from a single first trimester FL and the risk of microbial contamination of the FL tissues. Nonetheless, we have reported recently on a procedure for obtaining first-trimester FL that addresses the ethical concerns regarding such an endeavor and we also demonstrated that after minimal processing these FLs were free of microbial and specific viral contaminants. Thus, a bank of cryopreserved first-trimester FLs was established, but its utility is limited to pre-natal transplantation because of the small size of the samples. 9 We hypothesized that mid-trimester FLs, between the gestational ages of 16 and 24 weeks, represented an abundant source of HSCs that could be made safe for transplantation. It was the goal of this study to determine the potential yield of progenitors and HSCs that could be harvested from mid-trimester FLs and to estimate the risk of graftversus-host disease from mature T and NK cells. In this study we also define a protocol for the processing of human fetal HSC. The aim was to define a method of processing FLs that ensures the highest yield possible of committed progenitors and HSCs without compromising the quality of the grafts. The magnetic cell separation system, Isolex 50, was chosen for this task. The quality of the processed grafts was measured in terms of bacterial and fungal contamination in addition to the enumeration of T cells, progenitors and HSCs. Materials and methods AntibodiesThe following phycoerythrin (PE)-labeled, or fluorescein isothiocyanate (FITC)-labeled, monoclonal antibodies
We examined the potential of human fetal bone marrow (FBM) as a source of haematopoietic stem cells for transplantation. The median number of cells obtained between 20 and 24 weeks' gestation was 1·9 × 109 and a median 1·17 × 108 of these cells expressed CD34. Flow cytometry was also used to estimate the content of three different candidate stem cell populations in the tissues older than 20 weeks' gestation. A median 8·8 × 105 CD34++CD38− cells, 1·37 × 106 CD34++CD4+ cells and 2·20 × 106 CD34++CD90+ cells were detected. The content of colony‐forming units culture (CFU‐C) in the FBM ranged from 2·8 × 104 to 6·0 × 106 per fetus. The CFU‐C content could be expanded 50‐fold by culture for 1 week in serum‐deprived medium and the growth factors kit ligand and granulocyte–macrophage colony‐stimulating factor. Positive selection of FBM CD34+/++ cells was achieved using the Baxter Isolex 50 device. An average purity of 82% and yield of up to 19% of CD34+/++ cells was achieved. T cells were depleted by 99·84%. Analysis of candidate stem cell populations and primitive CFU‐C suggested a preferential enrichment of these cells over the total population of CD34+/++ cells. All FBM samples were found to be free of microbial contamination at the time of harvest and after selection of CD34+/++ cells. Thus, FBM is a safe source of stem cells. The large number of progenitors and candidate stem cells that can be obtained from FBM makes it suitable for in utero and possibly postnatal transplantation.
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