Engraftment in Nonobese Diabetic Severe Combined Immunodeficient Mice of Human CD34+ Cord Blood Cells After Ex Vivo Expansion: Evidence for the Amplification and Self-Renewal of Repopulating Stem Cells

Abstract

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

Sustained Long-Term Engraftment and Transgene Expression of Peripheral Blood CD34+ Cells Transduced with Third-Generation Lentiviral Vectors

“…Clonogenic Assays. Assays for granulopoietic, erythroid, megakaryocyte, and multilineage granulocyte-erythroid-macrophage-megakaryocyte colony-forming units were performed as previously described [11][12][13]. Colonies were scored after 14 days' culture, and EGFP ϩ (fluorescent) colonies were identified by fluorescence microscopy.…”

“…Stroma-free long-term expansion cultures were performed in 24-well plates for 5 weeks as reported [11][12][13][14]. Briefly, after transduction, 2 ϫ 10 4 MPB CD34 ϩ cells were cultured in quadruplicate flat-bottomed 24-well plates in 1 ml of IMDM ϩ 10% FCS with FST6.…”

Expression of the c-ErbB-2/HER2 proto-oncogene in normal hematopoietic cells

“…For hematopoietic progenitor in vitro expansion, purified CB CD34 ϩ cells derived from 13 CB samples were pooled, and 1 ϫ 10 6 cells/mL were cultured as previously reported [15] in the presence of 50 ng/mL Flt3 ligand (FL; kindly provided by Stewart D. Lyman, Immunex Corp, Seattle, WA), 20 ng/mL thrombopoietin (TPO), 50 ng/mL stem cell factor (SCF), and 10 ng/mL interleukin-6 (IL-6). TPO, SCF, and IL-6 were generous gifts from Kirin Brewery (Tokyo, Japan).…”

“…We explored the hypothesis that the level of c-ErbB-2 expression correlates to the proliferating state of the cells. To this purpose, CD34 ϩ cells were cultured in the presence of FL, TPO, SCF, and IL-6, the growth factor combination that was previously demonstrated to induce proliferation and self-renewal of CD34 ϩ cells [15,19]. Moreover, we compared c-ErbB-2 expression on PB MNC before and after G-CSF stimulation.…”

Decrease in Apoptosis and Increase in Polyploidization of Megakaryocytes by Stem Cell Factor During Ex Vivo Expansion of Human Cord Blood CD34 + Cells Using Thrombopoietin

“…At present, TPO is one of the most popular cytokines used for ex vivo expansion of cord blood (CB) HPCs [17]. In particular, Piacibello et al demonstrated that the simple combination of TPO and FL was capable of sustaining proliferation of CB HPCs for more than 6 months [18], and that the expanded cells were capable of repopulating nonobese diabetic/severe combined immunodeficient mice [19]. Using TPO and FL, we have also investigated myeloid differentiation from CB CD34 + cells [20].…”