Correlation Between IL‐3 Receptor Expression and Growth Potential of Human CD34 ⁺ Hematopoietic Cells from Different Tissues

Abstract

CD123 (␣-subunit of IL-3 receptor) expression on primitive and committed human hematopoietic cells was studied by multicolor sorting and single-cell culture. The sources of cells included fetal liver (FLV), fetal bone marrow, umbilical cord blood, adult bone marrow and mobilized peripheral blood. Three subsets of CD34 + cells were defined by the levels of surface CD123: CD123 negative , CD123 low , and CD123 bright . Coexpression of lineage markers showed that a majority of CD34 + CD123 bright cells were myeloid and B-lymphoid progenitors, while erythroid progenitors were mainly in the CD34 + CD123 negative subset. The CD34 + CD123 low subset contained a heterogeneous distribution of early and committed progenitor cells. Single CD34 + cells from the CD123 subsets were cultured in a cytokine cocktail of stem cell factor, interleukin 3 (IL-3), IL-6, GM-CSF, erythropoietin, insulin-like growth factor-1, and basic fibroblast growth factor. After 14 days of incubation, a higher cloning efficiency (CE) was observed in the CD34 + CD123 negative and CD34 + CD123 low fractions (37 ± 23% and 44 ± 23%, respectively) than in the CD34 + CD123 bright fraction (15 ± 21%). Using previously published criteria that colonies containing dispersed, translucent cells (dispersed growth pattern, DGP) were derived from primitive cells and that colonies composed solely of clusters were from committed cells, early precursors were distributed evenly in the CD34 + CD123 negative and CD34 + CD123 low subsets. When CD38 and CD90 (Thy-1) were used for further characterization of CD34 + cells from FLV, CE increased from 37 ± 23% in CD123 negative to 70 ± 19% in CD123 negative CD38 -and from 44 ± 23% in CD123 low to 66 ± 19% in CD123 low CD38 -. No significant increase in CE or DGP progenitors was observed when CD34 + cells were sorted by CD90 and CD123. We concluded that: A) high levels of CD123 were expressed on B-lymphoid and myeloid progenitors; B) early erythroid progenitors had little or no surface CD123, and C) primitive hematopoietic cells are characterized by CD123 negative/low expression.

Prospective isolation of human clonogenic common myeloid progenitors

“…IL-3 is an early cytokine that supports proliferation and differentiation of HSCs as well as primitive multipotent and myeloid progenitors (27)(28)(29), whereas the receptor tyrosine phosphatase CD45, and presumably its CD45RA isoform, can negatively regulate at least some classes of cytokine-receptor signaling (30). Several previous studies have been communicated concerning the surfaceantigen expression of early myeloid progenitor cells in humans (3,6,(9)(10)(11)(12). It was shown that CD34 ϩ CD45RA ϩ cells are devoid of erythroid and enriched for granulocyte and macrophage progenitor cells (6,10).…”

“…It was shown that CD34 ϩ CD45RA ϩ cells are devoid of erythroid and enriched for granulocyte and macrophage progenitor cells (6,10). Also, different levels of IL-3R␣ expression on CD34 ϩ cells reportedly are associated with preferential lineage readout, as IL-3R␣ Ϫ cells are enriched for erythroid, IL-3R␣ lo cells for multipotent, and IL-3R␣ ϩ cells for granulocyte͞macrophage colony-forming cells (9). However, in those studies early lymphoid cells, lineage low or positive cells, and͞or HSCs and multipotent progenitors within the CD34 ϩ fraction were not excluded by using negative or positive markers, likely resulting in heterogeneous populations.…”

“…These findings together with data from other groups indicate that the commitment to either the lymphoid or myeloid lineage and downstream to the granulocyte͞macrophage and megakaryocyte͞erythro-cyte lineages are exclusive events. In humans, multiple reports describe the enrichment of progenitor populations in bone marrow, mobilized peripheral blood, thymus, cord blood, fetal bone marrow, and fetal liver (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). However, to the best of our knowledge, thus far only early lymphoid lineage-restricted progenitors have been isolated and evaluated rigorously for all their developmental capacity on a clonal basis: An adult human bone marrow lineage-negative (lin Ϫ ) CD34 ϩ CD38 ϩ CD10 ϩ cell gives rise to B, natural killer (NK), and dendritic cells on a clonal level and to T cells at high frequencies (7), and a cord blood CD34 ϩ CD38 Ϫ CD7 ϩ cell gives rise to B, NK, and dendritic cells (8), but both are devoid of myeloerythroid differentiation capacity.…”

Mathematical modeling reveals differential effects of erythropoietin on proliferation and lineage commitment of human hematopoietic progenitors in early erythroid culture

“…To identify the less mature erythroid progenitors, MEP, at the point of lineage restriction from CMP, prior to GPA expression, we examined the change during culture of the previously defined markers interleukin-3 receptor (IL-3R/CD123) and Fmslike tyrosine kinase 3 (Flt-3/CD135), which are present at low levels on CMP and GMP and absent on MEP isolated from human bone marrow and cord blood. 27,31,32 Surface levels of both markers increase over the first two days in culture and then decrease to Day 4 on the bulk population, so they do not provide useful markers for early lineage restriction in erythroid culture ( Figure 1B). The erythroid marker CD36 was originally identified as a cell surface marker of erythroblasts 33 and later shown to be detectable prior to the appearance of the erythroid differentiation marker GPA.…”

Nephrin Forms a Complex with Adherens Junction Proteins and CASK in Podocytes and in Madin-Darby Canine Kidney Cells Expressing Nephrin

“…Cells were sorted by FACS 33 to produce cell populations and pure subclones with different levels of nephrin expression. Sorting was …”