Hematopoietic stem cells

Abstract

Hematopoietic stem cells (HSCs) have the capacity to self-renew and the potential to differentiate into all of the mature blood cell types. The ability to prospectively identify and isolate HSCs has been the subject of extensive investigation since the first transplantation studies implying their existence almost 50 years ago. Despite significant advances in enrichment protocols, the continuous in vitro propagation of human HSCs has not yet been achieved. This chapter describes current procedures used to phenotypically and functionally characterize candidate human HSCs and initial efforts to derive permanent human HSC lines.

FIG. 1

Flow cytometric characterization of human CD133⁺ cord blood cells expressing high levels of ALDH activity. (A) Negative control: Human cord blood cells highly enriched for CD133 expression (>95% CD133⁺) exhibiting low side scatter (SSC^lo) stained with BAAA (using the ALDEFLUOR^® reagent) in the presence of DEAB, a potent ALDH inhibitor, show background levels of BAA fluorescence (ALDH^dim). Events to the left of the ALDH^dim gate represent dead cells with no BAA fluorescence. (B) Human cord blood cells highly enriched for CD133 expression (>95% CD133⁺) exhibiting low side scatter (SSC^lo) stained with BAAA (using the ALDEFLUOR^® reagent) in the absence of DEAB showing that almost all of the cells expressed high levels of ALDH activity (ALDH^br). (C) The vast majority of ALDH^brCD133⁺ cells coexpress the CD34 HSC surface antigen. (D–F) Flow cytometric analysis indicates that cells within the more primitive CD133⁺CD34⁺CD38^lo subpopulation express higher levels of ALDH activity than cells within the CD133⁺CD34⁺CD38^hi subpopulation. (D) Gating strategy for CD133⁺CD34⁺CD38^hi and CD133⁺CD34⁺CD38^lo subpopulations. (E) CD133⁺CD34⁺CD38^hi cells are enriched for cells with the lowest levels of BAA fluorescence within the ALDH^br gate. (F) CD133⁺CD34⁺CD38^lo cells are enriched for cells with the highest levels of BAA fluorescence within the ALDH^br gate. Flow cytometry data was acquired on a FACSAria instrument (BD Biosciences) and analyzed with WinList 3D v6.0 pre-release software (Verity Software House).

FIG. 2

Human platelet production in peripheral blood of Rag2^−/−γ_c⁻-hTPO mice transplanted with candidate human HSCs. (A) Sublethally irradiated (350 cGy) Rag2^−/−γ_c⁻-hTPO and Rag2^−/−γ_c⁻mice were transplanted with 5 × 10⁵ human CD34⁺ cord blood cells. Human platelets were detected in the peripheral blood of all Rag2^−/−γ_c⁻-hTPO mice but not Rag2^−/−γ_c⁻ mice by staining with an anti-human CD41a monoclonal antibody and gating on low forward and side scatter (platelet population gate). Shown are representative examples. Flow cytometry data was acquired on a FACSCalibur instrument and analyzed with CellQuest software (BD Biosciences). (B) Summary of the analysis of human CD41a⁺ platelets within the platelet population in the peripheral blood of individual Rag2^−/−γ_c⁻-hTPO mice 4 to 8 weeks after transplantation with 5 × 10⁵ human CD34⁺ cells plus (dark bars) or minus (white bars) 1 × 10⁶ CD34⁻Lin⁺ accessory cells.

FIG. 3

Multilineage human hematopoietic engraftment in the peripheral blood of Rag2^−/−γ_c⁻-hTPO mice transplanted with candidate human HSCs. (A) Sublethally irradiated (350 cGy) mice were transplanted with 5 × 10⁵ human CD34⁺ cord blood cells plus (+) or minus (−) 1 × 10⁶ CD34⁻Lin⁺ accessory cells. Shown is a summary of the percentages of CD45⁺ human cell engraftment in the peripheral blood of transplanted Rag2^−/−γ_c⁻ (open circles) and Rag2^−/−γ_c⁻-hTPO (closed circles) mice 6 weeks after transplantation. Each circle represents data for an individual mouse and the horizontal lines indicate the mean levels of human cells. (B) Flow cytometric analyses showing percentages of human CD45⁺CD19⁺ B cells and CD45⁺CD33⁺ myeloid cells in the peripheral blood of a representative Rag2^−/−γ_c⁻-hTPO mouse at 6 weeks post-transplantation. Flow cytometry data was acquired on a FACSCalibur instrument and analyzed with CellQuest software (BD Biosciences).

FIG. 4

Loss of human hematopoietic repopulating potential during short-term in vitro culture of CD34⁺ cord blood cells. The potential of human CD34⁺ cord blood cells to engraft in the bone marrow of NOD/SCID mice was compared for cells cultured in vitro for 1 or 4 days in X-VIVO-15 serum-free medium supplemented with 10% BIT 9500 serum substitute, 100 μM β-mercaptoethanol, 100 ng/ml SCF, 20 ng/ml TPO and 100 ng/ml Flt3 ligand. The cells (1.5 × 10⁶) were harvested, mixed with 1 × 10⁶ CD34⁻Lin⁺ accessory cells and transplanted into sublethally irradiated (250 cGy) NOD/SCID mice. Twelve weeks after transplantation, the mice were euthanized and bone marrow cells collected for flow cytometric analysis. Human cells in the mouse bone marrow were detected after staining with anti-human CD45-PE-Cy5 (BD Biosciences Pharmingen, Catalog number 555484) monoclonal antibody. Transplantation of mice with CD34⁺ cord blood cells after 1 day of in vitro culture resulted in ~5% (0.5–50%) human hematopoietic cell engraftment (closed circles). The ability to repopulate NOD/SCID mouse bone marrow was significantly reduced (0–8%, mean: 0.2%) when the CD34⁺ cord blood cells were cultured in vitro for 4 days (open circles).