Distinct Requirements for Optimal Growth and In Vitro Expansion of Human CD34+CD38− Bone Marrow Long-Term Culture-Initiating Cells (LTC-IC), Extended LTC-IC, and Murine In Vivo Long-Term Reconstituting Stem Cells

Abstract

Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34+CD38− phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34+CD38− BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34+CD38− progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.

Self-Renewal of Multipotent Long-Term Repopulating Hematopoietic Stem Cells Is Negatively Regulated by FAS and Tumor Necrosis Factor Receptor Activation

“…We have recently demonstrated that Lin − Sca1 + c-kit + cells cultured under serum-free conditions in the presence of K36, undergo self-renewing cell divisions and preserve their ability to short- and long-term multilineage reconstitute lethally irradiated mice 2425. Thus, Lin − Sca1 + c-kit + cells were cultured under such self-renewing conditions in the absence or presence of TNF-α and/or Jo2.…”

“…TGF-β has been shown to accelerate depletion of HSCs under in vitro culture conditions which themselves result in loss of HSC activity 5051, making it difficult to distinguish between effects on self-renewal and other mechanisms. Recent developments have now made it possible to study HSC self-renewal and its regulation in vitro 24252627. Through such studies, a number of growth stimulatory cytokines have been shown to promote HSC self-renewing cell divisions.…”

“…All animal procedures were performed with consent from the local ethics committee at Lund University. Lineage-depleted (Lin − ) BM cells were isolated from normal 6–14-wk-old C57BL/6 mice (CD45.2), congenic B6.SJL-Ptprc a Pep b /BoyJ (CD45.1), or Fas-deficient (lpr) mice inbred to C57BL/6 background (B6.MRL-Fas<lpr&gt mice; CD45.2) according to previously described protocols 24253031. When phenotyping or sorting Lin − Sca1 + ckit + CD34 − HSCs, donor mice were always 12–14 wk old, as it has been shown that HSCs from younger mice are mostly CD34 + 3233.…”

“…We have recently, through HSC division tracking and serial transplantation experiments, demonstrated that murine Lin − Sca1 + c-kit + long-term reconstituting stem cells can undergo multiple self-renewing divisions in vitro 2425. Others and we have demonstrated that this self-renewal process is positively affected by cytokines such as IL-3, IL-6, IL-11, c-kit ligand, and thrombopoietin 24252627. Thus, this allowed us to investigate how TNF-α and Fas might potentially affect HSC self-renewal.…”

Cellular‐based therapies to prevent or reduce thrombocytopenia

“…Toward this, great deals of efforts were devoted to develop cytokine cocktails and culture protocols that support the expansion of HSCs and HPCs ex vivo to increase the cell dose available for the transplantation. [21][22][23][24][25][26] This strategy rapidly gained popularity given that the proliferative potential of UCB HPCs far exceeds that of their adult counterpart (reviewed in Mayani and Lansdorp 27 ). The governing principle behind this strategy is that ex vivo expansion of HPCs should accelerate hematopoietic recovery based on the infusion of a greater quantity of HPCs and the fact that these cells have already begun the process of differentiation, thus reducing the length of time required for mature cells to be released to the periphery.…”