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

Abstract

IntroductionThere is a growing clinical need for large numbers of human hematopoietic stem cells (HSCs) for transplantation or gene therapy. 1,2 In cancer patients in whom the probability of tumor cell contamination in either leukapheresis or bone marrow (BM) is high, the use of allogeneic cells is preferable. 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. 3-7 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. 8 Ex vivo expansion of CB and of adult nonobese diabetic/severe combined immunodeficiency (NOD/SCID)-repopulating cells (SRCs; Wang et al 9 ) has been demonstrated (eg, Piacibello et al 10 and Gammaitoni et al 11 ), but successful expansion of FL SRCs has not been reported so far.We recently published a simple and reproducible stroma-free liquid culture system allowing long-term (Ͼ 6 months) expansion of human hematopoietic cells contained within previously frozen crude FL cell suspensions. In these cultures, CD34 ϩ cells, primitive colony-forming progenitors, and cells possessing a putative stem cell phenotype were not only present, they also underwent a continuous amplification. 12 However, the maintenance and expansion of functional repopulating HSCs was not tested. This is critical given the dissociation between stem cell phenotype and in vivo repopulating function observed in hematopoietic cultures. [13][14][15] Here, we used the NOD/SCID mouse xenotransplantation assay to quantify FL SRCs and demonstrated that our culture conditions allowed a 10-to over 100-fold net SRC expansion following 28 days of culture. Study designCryopreserved human FL crude cellular suspensions were prepared from aborted fetuses (gestational weeks 12-17) as described, with the approval of the ethical committee of the Lausanne University Medical Faculty. 12,16 Total nucleated FL hematopoietic cells were expanded in RPMI 1640 supplemented with 8% human AB plasma, Flt-3 ligand (50 ng/mL), interleukin 6 (10 ng/mL), megakaryocyte growth and development factor (MGDF; 10 ng/mL), and stem cell factor (SCF; 50 ng/mL). Cultures were fed and maintained as described, 12 except that cells were grown in T25 flasks instead of 24-well plates. For NOD/SCID-repopulating assays, 6-to 8-week old NOD/LtSz-scid/scid (NOD/SCID) mice were sublethally irradiated (375 cGy using a 137 Cs source), and cells to be tested (at the doses indicated, in about 600 L RPMI) were injected into the lateral tail vein 4 to 24 hours later. Mice were killed after 6, 8, or 12 weeks, the BM harvested from femora, and human engraftment analyzed b...

Biological characteristics of stem cells from foetal, cord blood and extraembryonic tissues

“…Foetal liver is also a source of HSCs, and, despite their relatively small number, it is possible to generate sufficient numbers of cells for transplantation in four weeks (Rollini et al 2004), although, to our knowledge, it has not been proved that foetal liver HSCs can successfully transplant a human recipient. The phenotype of foetal liver HSCs changes with gestation age.…”

Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays

“…The output of LTC-IC and CFC per CRU can also be calculated, which allows to compare the intrinsic potential of CRU from ontogenically different sources (Holyoake et al, 1999). Use of these more or less specific end points explains the variation in the measures of engraftment, and in the extent of huSRC expansion in response to cytokines Gammaitoni et al, 2003;Rollini et al, 2004). However, if mice are analysed long enough after the graft, the total level of engraftment may be considered as a measure of the population of lymphomyeloid repopulating cells as was demonstrated in mice .…”

“…Nevertheless, one can argue that this is valid, since in the mouse or sheep, measuring the end points at 10 weeks or 4 months yield equivalent conclusions. (2) More problematic is the ontogeny-related decrease in the frequency and potential of human primitive progenitors, which precludes any long-term analysis of the function of huSRC/CRU from adult stem cells as opposed to fetal cells, and most successful studies demonstrating regeneration or amplification of huSRC/CRU have been carried with fetal human sources Gammaitoni et al, 2003;Rollini et al, 2004). (3) There is fluctuation in the homing properties, as mentioned above.…”

In vitro human embryonic stem cell hematopoiesis mimics MYB independent yolk sac hematopoiesis

“…Fetal, newborn and adult HSC are known to lose the capacity to self-renew within days of in vitro culture. [27][28][29] To study the emerging HPC in vitro, we isolated CD34 + CD43 -endothelial cells and studied the characteristics of hematopoietic cells generated from them through timelapse microscopy. We did not observe generation of MYBeGFP expressing cells that subsequently lose MYB expression in vitro.…”