We present cell cycling and functional evidence that the CD34+CD38- immunophenotype can be used to define a rare and primitive subpopulation of progenitor cells in umbilical cord blood. CD34+CD38- cells comprise 0.05% +/- 0.08% of the mononuclear cells present in cord blood. Cell cycle analysis with the fluorescent DNA stain 7- aminoactinomycin D showed that the percentage of CD34+ cells in cycle directly correlated with increasing CD38 expression. CD34+CD38- cord blood cells were enriched for long-term culture-initiating cells (LTCIC; cells able to generate colony-forming unit-cells [CFU-C] after 35 to 60 days of coculture with bone marrow stroma) relative to CD34+CD38- cells. In an extended LTCIC assay, CD34+CD38- cells were able to generate CFU-C between days 60 and 100, clearly distinguishing them from CD34+CD38+ cells that did not generate CFU-C beyond day 40. When plated as single cells, onset of clonal proliferation was markedly delayed in a subpopulation of CD34+CD38- cells; clones (defined as = 100 cells) appeared after 60 days of culture in 2.9% of CD34+CD38- cells. In contrast, 100% of CD34+CD38+ cells formed clones by day 21. Although the CD34+CD38- immunophenotype defines highly primitive populations in both bone marrow and cord blood, important functional differences exist between the two sources. CD34+CD38- cord blood cells have a higher cloning efficiency, proliferate more rapidly in response to cytokine stimulation, and generate approximately sevenfold more progeny than do their counterparts in bone marrow.
Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for genecorrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34 + cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.Adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) became an early disease candidate for studies of gene therapy based on the results of allogeneic bone marrow transplant. Transplantation of bone marrow from a fully HLA-matched, unaffected sibling donor allows SCID to be cured completely 1,2 . Patients develop a protective immune © 1998 Nature Publishing Group Correspondence should be addressed to D.B.K. NIH Public AccessAuthor Manuscript Nat Med. Author manuscript; available in PMC 2013 September 19.Published in final edited form as:Nat Med. 1998 July ; 4(7): 775-780. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript system of donor-derived T lymphocytes, despite the absence of significant numbers of donor-derived cells in other hematopoietic lineages 3 . This observation has been interpreted as indicating that the normal T-lymphoid cells have a selective advantage in SCID patients and repopulate the immune system from a small number of engrafted, genetically-normal donor hematopoietic stem cells. The selective advantage for normal T and B cells in SCID patients has been recently re-confirmed in two SCID patients (one with ADA-deficient SCID and one with the X-linked form) who had spontaneous improvements in their immune function [4][5] . Gene-correction of a small number of autologous hematopoietic stem cells could similarly result in the development of a functional immune system in SCID gene therapy recipients.In May 1993, we treated three newborns with ADA-deficient SCID with a single infusion for each of retroviral vector-transduced autologous umbilical cord blood CD34 + cells. Initial observations during the first 18 months after this intervention showed the persistent presence of leukocytes in their peripheral blood and bone marrow which contained and expressed the inserted normal human ADA cDNA (ref.6). This observation was important because it provided convincing evidence that human hematopoietic stem cells could be transduced with retroviral vectors and re-engrafted in their hosts without administration of...
Long-term culture-initiating cells (LTC-IC) are hematopoietic progenitors able to generate colony-forming unit-cells (CFU) after 5 to 8 weeks (35 to 60 days) of culture on bone marrow (BM) stroma and represent the most primitive progenitors currently detectable in vitro. We have recently reported that long-term cultures initiated with CD34+CD38- cells from BM or cord blood are able to continue generating CFU for at least 100 days, ie, beyond the standard LTC-IC period. In this report, single-cell cultures from cord blood and retroviral marking of cord blood and BM were used to study whether the subpopulation of CD34+CD38- cells able to generate CFU beyond 60 days (“extended long-term culture-initiating cells” or ELTC-IC) are functionally distinct from LTC-IC in terms of timing of initial clonal proliferation and generative capacity. All cord blood LTC-IC formed clones of greater than 50 cells by day 30. In contrast, cord blood ELTC-IC proliferated later in culture, 50% forming clones after day 30. Although efficient retroviral marking of LTC-IC was seen (25% to 45%), marking of ELTC-IC was inefficient (< 1%), consistent with a more quiescent progenitor population. There was a positive correlation between time of clonal proliferation and generative capacity. ELTC-IC generated threefold to fourfold more progeny than did LTC-IC (P < .002). These studies show that there is a functional hierarchy of progenitors in long-term culture which correlates with their level of quiescence. By extending the LTC-IC assay, a more primitive progenitor may be studied that may be functionally closer to the human long-term repopulation stem cell in vivo.
Flt3 is a class III tyrosine kinase receptor expressed on primitive human and murine hematopoietic progenitor cells (HPC). In previous studies using stroma-free short term assays, Flt3 ligand (FL) has been shown to induce proliferation of HPC at proportions similar to or less than c-kit ligand (steel factor, SF). Using long term stromal cocultivation assays, we studied the effects of FL on proliferation and differentiation of a highly primitive and cytokine nonresponsive subpopulation of human HPC, CD34+cd38- cells. Cell Proliferation was significantly greater with FL than with SF, when used individually or in combinations with interleukin-3 (IL-3) and/or IL-6. The effect of FL was greater on bone marrow (BM) CD34+CD38- cells than the more cytokine responsive cord blood CD34+CD38- cells. Little or no effect was seen with FL on more mature CD34+CD38+ cells from either BM or cord blood. The frequency of colony-forming units (CFU) and high proliferative potential-colony forming cells (HPP-CFC) during early culture ( < or = 30 days) was increased by both SF and FL to similar levels. However, in the LTC-IC period (35 to 60 days) and extended long-term culture initiating cell (ELTC-IC) period ( > 60 days), the frequency of CFU and HPP-CFC was significantly greater in cultures containing FL than those without FL (P < .0025). Fluorescence-activated cell sorter analysis of cultures after 21 days showed a significantly higher percentage of cells remained CD34+ in the combination of FL, IL-3, IL-6, and SF (F/3/6/S) than in 3/6/S (0.78% +/- 0.52% v 0.21% +/- 0.29% respectively, mean +/- SD). Cloning efficiency of BM CD34+CD38- cells was significantly increased by the addition of FL to the combination of 3/6/S (mean 11.7% v 0.5%, P < .0001). These data show that FL is able to induce proliferation of CD34+CD38-cells that are nonresponsive to other early acting cytokines and to improve the maintenance of progenitors in vitro.
Homeobox genes encode transcription factors known to be important morphogenic regulators during embryogenesis. An increasing body of work implies a role for homeobox genes in both hematopoiesis and oncogenesis. We have analyzed the role of the homeobox gene, HOX B7, in the program of differentiation of the biphenotypic myeloid cell line, HL60. Induction of monocytic differentiation in HL-60 cells by vitamin D3 resulted in rapid expression of HOX B7 mRNA, but stimulation with phorbol ester or dimethyl sulfoxide (DMSO) did not. Constitutive overexpression of HOX B7 in the HL60 cell line inhibited the granulocytic differentiation associated with stimulation with DMSO or retinoic acid, but had no effect on the monocytic differentiation induced by vitamin D3. Normal human monocytes do not constitutively express HOX B7, nor are they able to be induced to do so by stimulation with colony-stimulating factor 1 (CSF-1) and gamma interferon (IFN gamma), or with vitamin D3 and lipopolysaccharide. Human bone marrow (BM) cells were found to express HOX B7 in response to granulocyte- macrophage CSF (GM-CSF) and antisense oligonucleotides directed against HOX B7 inhibited the formation of colonies derived from GM-CSF- stimulated BM. These data suggest a critical role for HOX B7 in myelomonocytic differentiation.
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