Mid-trimester fetal livers are a rich source of CD34+/++ cells for transplantation

Abstract

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

Long-term expansion of transplantable human fetal liver hematopoietic stem cells

“…Accumulated evidence suggests that fetal liver (FL) or umbilical cord blood (CB) or both represent alternative and possibly more "universal" sources of "early" HSCs and possess a better proliferative potential and also a preimmune status that may be important in mismatched transplantation situations. [3][4][5][6][7] However, both sources are compromised by the relatively small number of cells available. Therefore, the future clinical potential of FL and CB would be strongly enhanced if methods allowing a reliable HSC expansion, perhaps to a degree as little as 20-to 100-fold, without a loss of their engraftment ability, could be developed.…”

Transplantation of a fetus with paternal Thy-1+CD34+cells for chronic granulomatous disease

“…The fetus we transplanted received 1 × 10 7 Thy-1 + CD34 + cells at 14 weeks gestation. The frequency of these cells in fetal liver, the predominant hematopoietic organ at this age, has been measured at 0.16% 20 and the average number of liver cells at 14 weeks gestation is approximately 2 × 10 8 . 39 Thus, the number of Thy-1 + CD34 + cells in the liver at the time our transplant was performed was 3 × 10 5 .…”

Preparation and analysis of fetal liver extracts

“…The presence of extra-hematopoietic cells expressing CD34 in fetal liver cell extracts has largely been overlooked in most studies on fetal hematopoietic stem cells, and they may have contributed to an overestimation of the hematopoietic stem cell content of fetal livers. 15 The exact number of hematopoietic stem cells necessary for a transplant in utero has not been established yet. For children and adults, the minimal stem cell number required for rapid hematopoietic reconstitution is between 1 and 3 ϫ 10 6 CD34 ϩ cells/kg.…”

“…Their surface marker expression, [8][9][10][11][12][13][14][15][16] growth and differentiation in clonogeneic culture, 8,11,15,[17][18][19] and ability to reconstitute hematopoiesis in vivo in graft experiments, [20][21][22][23][24][25][26] have been reported.…”