Hematopoietic stem cells residing within mouse bone-marrow were first discovered by Till and McCulloch in the 1960s and indicated that (1) hematopoiesis could be studied as a quantitative science, (2) clonal hematopoietic cells in the marrow existed that could give rise to mixed myeloid progeny (granulocytes, macrophages, red cells, megakaryocytes), (3) some of these cells could self-renew and (4) in the spleens of these mice, cells existed that could also make lymphocytes. All colony-forming activity of human bone marrow (BM) cells is found in the CD34+ progenitor cell fraction. Clinical transplantation studies that used enriched CD34+ cells from bone-marrow, umbilical cord and peripheral blood mononuclear cells (PBMCs) indicated the presence of HSCs with long-term tissue reconstitution ability. Below are commonly used in vitro cell culture protocols and assays used to isolate, expand, differentiate and characterize human CD34+ hematopoietic stem cell populations from various tissues.
HSCs can be isolated using flow cytometry based on surface marker expression. Primitive multipotent hematopoietic progenitors have phenotypes with CD34+/CD38-/CD45RA-/CD71- expression. Other positive markers for hematopoietic progenitors include CD133+, CD90+ (Thy-1), ALDH+ and Sca-1+. Other negative marker for hematopoietic progenitors include mature blood lineage (Lin-) markers: CD2-, CD3-, CD19-, CD41-, CD16-, CD14-, and CD15-. The ideal sample is fresh, anticoagulated blood or tissue samples. The preparation details vary, depending on the specific tissue source. If samples cannot be processed within 48 hours, they should be frozen.
Figure 1. Human hematopoietic stem cell markers analyzed by flow cytometry. Flow cytometric characterization of the bench-scale HSC expansion from a variety of donors reveals that the Stemline™ Hematopoietic Stem Cell Expansion Medium generates a significant expansion of both committed (Blue, CD34-/CD15+/CD41+) and early (Red, CD34+/CD15-/CD41-) hematopoietic progenitor cells.
Figure 2. Morphology of human CD34+ hematopoietic progenitor cells. Suspension culture of bone-marrow derived isolated human CD34+/CD38-hematopoietic stem cells (A, 10X and B, 40X).
The colony forming unit (CFU) cell assay, or CFC assay, is used to study the proliferation and differentiation of hematopoietic progenitors by their ability to form colonies in a semisolid medium such as methylcellulose or agar. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. This assay is useful for assessing myeloid (granulocytic, monocytic, erythroid, and megakaryocytic cells) but not lymphoid lineage differentiation.
Figure 3. Colony formation unit (CFU) cell assay results of human CD34+ hematopoietic progenitor cells. A) Human CD34+ hematopoietic stem cells isolated from normal, healthy human bone marrow were cultured in either Stemline™ HSC media in methylcellulose with Epo or competitor media in methylcellulose with Epo. Cultures were scored after 12 - 16 days of culture using an inverted microscope. Cultures were scored for burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte, -monocyte (CFU-GM) and colony-forming unit-granulocyte, - erythrocyte, -macrophage, and megakaryocyte CFU-GEMM). B) Differentiated human CD34+ hematopoietic progenitor cells showing a human granulopoietic colony containing clonogenic precursors of granulocytes and macrophages (CFU-GM) and human erythroid progenitor colony (BFU-E). The CFU-GM colonies demonstrate a relatively homogeneous morphology and typically have a concentrated central core of cells surrounded by a less dense halo of cells. The BFU-E colonies are frequently referred to as having a grape-like morphology and range in size from a single, large cluster containing several hundred erythroblasts to 14 or more clusters containing thousands of cells (40X magnification).