STUDIES OF HUMAN PLURIPOTENTIAL HEMATOPOIETIC STEM-CELLS (CFU-GEMM) INVITRO

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 CFU-GEMM were found to be present in equal but low frequencies in blood (B) and bone marrow (M) mononuclear cell populations. When the culture system was modified by the substitution of Iscove''s modified Dulbecco''s medium for .alpha.-MEM [minimal essential medium] and the addition of mercaptoethanol, a significant enhancement of mixed colony formation occurred, and an approximately 3- to 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 requires only relatively low permissive concentrations of Ep, in contrast to the high Ep requirement for optimal BFU-E [erythropoietic burst forming cell] growth in vitro. CFU-GEMM is a distinct multipotential stem cell class the assay of which may prove useful in the study of human blood dyscrasias.