Studies of human pluripotential hemopoietic stem cells (CFU-GEMM) in vitro

Abstract

An in vitro clonal assay for a class of human hemopoietic progenitors (CFU-GEMM) with several characteristics of pluripotential stem cells has been previously described. In the presence of medium conditioned by leukocytes stimulated with phytohemagglutinin (PHA-LCM) and erythropoietin (Ep), CFU-GEMM give rise to mixed hemopoietic colonies containing granulocytic, erythroid, monocyte-macrophage, and megakaryocytic elements. In initial studies we found that CFU-GEMM were present in equal but low frequencies in blood (B) and bone marrow (M) mononuclear cell populations. However, when the culture system was modified by the substitution of Iscove's modified Dulbecco's medium for alpha-MEM and the addition of mercaptoethanol, a significant enhancement of mixed colony formation occurred, and an approximately 3- 4-fold difference in the frequency of CFU-GEMM between B and M emerged. Replating studies showed the formation of secondary differentiated hemopoietic colonies and at least a limited capacity for self-renewal of CFU-GEMM. The in vitro growth of normal CFU-GEMM was highly dependent on hemopoietin(s) present in PHA-LCM. In vitro detection of CFU-GEMM, however, requires only relatively low permissive concentrations of Ep, in contrast to the high Ep requirement for optimal BFU-E growth in vitro. These and other data described demonstrate CFU-GEMM to be a distinct multipotential stem cell class whose assay may prove useful in the study of human blood dyscrasias.

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

“…At the end of the incubation the supernatants were collected, harvested, and used at a final concentration of 7%. These CM preparations contain both BPA and GEMM-stimulating activity, as already demonstrated by Ash et al (1981).…”

Hemopoietic marrow function in chronic neutropenia of blacks: Cure of aplastic anemia by allogeneic marrow transplantation from a neutropenic sibling donor

“…After obtaining informed consent, blood and/or marrow aspirates were drawn from either the patient and appropriate family members or normal control subjects. Heparinized marrow samples were subjected to Ficoll-Hypaque gradient centrifugation [26] to obtain mononuclear cell fractions.…”

Growth factors that stimulate human granulocyte—macrophage colony formation produced by the cell line CM‐S

“…To measure CM-S autostimulatory activity the procedure of Ash et al (1981) was used with minor modifications. Exponentially growing cells were harvested by centrifugation, washed three times with Iscove's modified Dulbecco's medium (IDM), and resuspended a t the desired concentration in fresh IDM enriched with I,asparagine 3 x lop4 M, L-glutamine 2.8 x lop3 M, Napyruvate 1 x M; 2-mercaptoethanol 5 x M, 30% fetal calf serum, and 0.9% (wiv) final concentration of methylcellulose (A-4M, Premium Methocel, Dow Chemicals).…”