Unicellular-unilineage erythropoietic cultures: Molecular analysis of regulatory gene expression at sibling cell level

In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage spe cificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by dau ghter cell analysis at cellular and molecular level. In the culture system reported here, (I)the growth factor (GF) stimulus induces cord blood (CB) p rogenitor cells to proliferate and differentiate/mature exclusively along t he erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie , nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc) , ie, the number of divisions performed by each cell is monitored. Single-c ell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was a pplied to this culture system to investigate gene expression of diverse rec eptors, markers of differentiation, and transcription factors (EKLF, GATA-1 , GATA-2, p45 NF-ES, PU.1, and SCL/Tal1) at discrete stages of erythropoiet ic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/T al1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Ta ll, EKLF NF-EP, and GATA-1 that preceeded expression of EpoR. In late stage s of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cel l RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+)CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allow ed us to semiquantitate the gradual downmodulation of CD34 mRNA from progen itor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneo us populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development. (C) 1999 by The American Society of Hematology.