Thrombopoietin, kit Ligand, and flk2/flt3 Ligand Together Induce Increased Numbers of Primitive Hematopoietic Progenitors From Human CD34+Thy-1+Lin− Cells With Preserved Ability to Engraft SCID-hu Bone

Abstract

CD34+Thy-1+Lin− cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34+Thy-1+Lin− cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34+Thy-1+Lin− cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34hiPKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34+Thy-1+Lin− cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34hi PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34hiPKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34+Thy-1+Lin− cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.

Relationship between Thy‐1 expression and cell‐cycle distribution in human bone marrow hematopoietic progenitors

“…Candidate HSCs have been characterized as CD34 + CD38 À and further subclassified based on the presence or absence of HLA-DR and Thy-1(CDw90) or on efflux of Rhodamine-123 dye and other dyes. Several investigators have reported that a subset of adult CD34 + bone marrow (BM) cells expresses Thy-1 antigen and that the CD34 + Thy-1 + subset also contains most longterm culture-initiating cells and cobble-stone areaforming cells [8][9][10][11][12][13][14]. Thus, the Thy-1 + subset among HSCs might represent valuable target cells for gene transfer.…”

Hematopoietic Repopulating Ability of Cord Blood CD34+Cells in NOD/Shi‐scidMice

“…Clinically transplantable hematopoietic stem cells (HSC) should prove to have a long-term repopulating ability. Until recently, most of the human HSC studies aimed at clinical application have used in vitro assays for CD34 + cells, colonyforming cells in clonal culture, cobblestone area-forming cells, and long-term culture-initiating cells [1][2][3][4][5], but these surrogate assays have been shown not to correctly reflect stem cell activity. However, the recent development of assays measuring the ability to reconstitute human hematopoiesis in ontologically or genetically immunodeficient animals has enabled investigators to evaluate the stem cell activity [6][7][8].…”

Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptoticBcl-2family genes

“…It is likely that synergistic cytokines induce distinct survival-signaling pathways. Previous studies have indicated that KL and FL have synergistic properties that lead to increased survival in human and mouse stem cells and early progenitors [6,7,[37][38][39]. The results offer an appealing explanation to these mechanisms.…”

“…To maintain the immature pool of stem cells and progenitors, mechanisms must be activated that suppress apoptosis and permit the cells to respond to other signals for further maturation. Several studies have indicated that certain cytokines are essential to promote survival, including kit ligand (KL; also stem cell factor or steel factor), flt3 ligand (FL), thrombopoietin (Tpo), interleukin-3 (IL-3), IL-6, and IL-11 [1][2][3][4][5][6][7].…”