Ex VivoCulture of Cord Blood CD34⁺Cells Expands Progenitor Cell Numbers, Preserves Engraftment Capacity in Nonobese Diabetic/Severe Combined Immunodeficient Mice, and Enhances Retroviral Transduction Efficiency

Abstract

Ex vivo culture of hematopoietic stem/progenitor cells could potentially improve the efficacy of human placental/umbilical cord blood (CB) in clinical hematopoietic stem cell (HSC) transplantation and allow gene transduction using conventional retroviral vectors. Therefore, we first examined the effects of a 7-day period of ex vivo culture on the hematopoietic capacity of CB CD34+ cells. Medium for the ex vivo cultures contained either serum and six recombinant human hematopoietic growth factors (GFs), including Flt-3 ligand (FL), Kit ligand (KL = stem cell factor), thrombopoietin (Tpo), interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6), or a serum-free medium containing only FL, KL, and Tpo. After culture under both ex vivo conditions, the total numbers of viable cells, CD34+ cells, colony-forming cells (CFCs), and long-term culture initiating cells (LTC-ICs) were increased. In contrast, the severe combined immunodeficiency (SCID) mouse engrafting potential (SEP) of cultured cells was slightly decreased, as compared with fresh cells. Nevertheless, cultured human CB CD34+ cells were able to generate engraftment, shown to persist for up to 20 weeks after transplantation. We next tested the efficacy of retroviral transduction of cultured cells. Transduced cultured human cells were able to engraft in NOD/SCID mice, as tested 4 weeks after transplantation, and EGFP+CD34+ cells and EGFP+ CFCs were isolated from the chimeras. Thus, although additional improvements in ex vivo culture are still needed to expand the numbers and function of human HSCs, the current conditions appear to allow gene transduction into hematopoietic SCID engrafting cells, while at least qualitatively preserving their in vivo engraftment potential.

Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

“…27 After thawing, the CD34 ϩ cells were cultured and activated for 2 to 4 days to stimulate cell proliferation, 27 before gene transduction by the 4 standard retroviral vectors. 1 Colonies resembling human ES/iPS cells emerged approximately day 16 after gene transduction from CB samples and approximately day 21 from adult BM CD34 ϩ cells.…”

Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells

“…We first used human cord blood stem cells (CBSCs) because they have higher engraftment capacity in this mouse model than PBSCs or BM CD34 ϩ cells. [12][13]16,[50][51][52] Cyropreserved CB CD34 ϩ cells were cultured overnight and transduced twice with DR.GFP or EF.GFP at the MOI of 60 in the next 2 days. Two days after the last transduction, GFP and CD34 expression of the transduced cells was examined by FACS analysis.…”

“…[1][2][3][4][5][6][7][8][9][10] However, RV transduction requires division of the target cells while most of primitive HSPCs are mitotically quiescent. Although significant progress has been made in improving conditions for ex vivo HSPC culture and transduction, RV design and production, and RV-mediated transgene expression, [11][12][13][14][15][16] RVs still possess intrinsic limitations. For example, a preactivation lasting for 2 to 3 days is required for efficient RV transduction of HSPCs.…”

Efficient retrovirus-mediated transduction of primitive human peripheral blood progenitor cells in stroma-free suspension culture

“…Comparable results, showing general improvement of gene transfer techniques, were obtained in other recent studies in NOD/SCID mice or in non-human primates. 50,51 That IL-3-treated hematopoietic stem cells are capable of repopulating bone marrow has been demonstrated in murine and in large-animal models. 17,19,28,47,[49][50][51][52][53] Several reports, however, indicate that engraftment of hematopoietic cells exposed to IL-3 is relatively poor.…”

“…50,51 That IL-3-treated hematopoietic stem cells are capable of repopulating bone marrow has been demonstrated in murine and in large-animal models. 17,19,28,47,[49][50][51][52][53] Several reports, however, indicate that engraftment of hematopoietic cells exposed to IL-3 is relatively poor. [22][23][24] In a recent study, stimulation of murine bone marrow with IL-3 and IL-11 reduced transduction of long-term repopulating cells whereas SCF, FL and TPO led to efficient stem cell transduction.…”