CD34+, kit+, rhodamine123low phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells

Abstract

We hypothesized that human hematopoietic cells displaying a CD34 + , kit + , rhodamine123 low phenotype would be highly enriched for cells with stem-like properties. To test this hypothesis, we employed fluorescence activated cell sorting (FACS) to isolate cells with this phenotype from normal light density marrow mononuclear cells (MNC). CD34 + , kit + , rhodamine123 low cells comprised from 0.05-0.01% of the total MNC population. They were small, had scant cytoplasm, and contained nuclei with dense, hyperchromatic chromatin and inconspicuous nucleoli. Additional immunophenotyping revealed that these cells were CD33 − , CD38 − , CD20 − , and glycophorin A − . When plated in semisolid cultures containing optimal concentrations of IL-3, GM-CSF, KL, EPO, IL-6, and IL-1 these cells did not form colonies. However, when cultured over irradiated stromal cells, cobblestone areas were observed to form after 3 weeks, and harvested cells were able to initiate long-term cultures. To further demonstrate that these cells were indeed stem like, we also tested their ability to engraft and mature in immunocompromised (SCID) mice. Irradiated (400 cGy) SCID mice were transplanted with 2 × 10 3 candidate stem cells which were then injected with recombinant human growth factors every other day. Two months post-transplant the animals were sacrificed. PCR and FACS analysis of marrow and spleen cell samples revealed the presence of cells expressing human CD45 consistent with engraftment of human stem cells and the establishment of murine-human chimerism. Moreover, MNC isolated from transplanted mice formed unambiguously human BFU-E, CFU-GM and B cell colonies when stimulated with the appropriate growth factors. Accordingly, we have identified a relatively rapid and simple mechanism for isolating primitive human hematopoietic cells with stem cell-like properties. We anticipate that this strategy will be useful for experimental and therapeutic applications that require human stem cells in quantity.

Improved purification of hematopoietic stem cells based on their elevated aldehyde dehydrogenase activity

“…This strategy consistently identified a small (~1%) but distinct population of ALDH + cells within the low-density fraction of human cord blood cells with low light sidescattering (SSC lo ) properties (a well-established feature of primitive hematopoietic cells). 31 Prior removal of the lin + cells gave a marked enrichment of the ALDH + population (16±2% of the total low-density lin -fraction, Figure 1C), as expected. 24,26 Notably, this was true even when we excluded the weakly fluorescent cells indicated by the dotted box in Figure 1C, which are typically included when the DEAB control is not applied.…”

Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors

“…CD34 is known to be expressed on committed and uncommitted hematopoietic precursor cells, small vessel endothelial cells and on some cells in nervous tissue (Lin et al, 1995;Ratajczak et al, 1998;Suda et al, 2000;Watt and Chan, 2000). Experiments using a cDNA clone have characterized CD34 as a sialomucin (Simmons et al, 1992;Suda et al, 2000).…”

Biological significance of the expression of HIV-related chemokine coreceptors (CCR5 and CXCR4) and their ligands by human hematopoietic cell lines

“…30,31 Cultures were performed in 96-well plates. The cells from the various hematopoietic cells lines (2 × 10 3 cells/ml) were resuspended in 1 ml of the culture medium without or in the presence of 5 ng (low dose) or 50 ng/ml (high dose) of the following chemokines: SDF-1␣, SDF-1␤, MIP-1␣, MIP-1␤, RANTES (R&D, Minneapolis, MN,USA).…”

“…30,31 Cells were infected at day 0 by BAL (R5 HIV) or HXB2 (X4 HIV). HIV strains (a gift of Dr F Gonzales-Scarano, University of Pennsylvania, Philadelphia, PA) were employed at a multiplicity of infection (MOI) of 0.02.…”