In vitro maintenance of highly purified, transplantable hematopoietic stem cells

The cellular and molecular mechanisms that regulate the most primitive hematopoietic stem cell are not well understood. We have undertaken a systematic dissection of the complex hematopoietic microenvironment to define some of these mechanisms, An extensive panel of immortalized stromal cell lines from murine fetal liver were established and characterized. Collectively, these cell lines display extensive heterogeneity in their in vitro hematopoietic supportive capacity, In the current studies, we describe a long-term in vitro culture system using a single stromal cell clone (AFT024) that qualitatively and quantitatively supports transplantable stem cell activity present in highly purified populations, We show multilineage reconstitution in mice that received the equivalent of as few as 100 purified bone marrow and fetal liver stem cells cultured for 4 to 7 weeks on AFT024, The cultured stem cells meet all functional criteria currently ascribed to the most primitive stem cell population, The levels of stem cell activity present after 5 weeks of coculture with AFT024 far exceed those present in shortterm cytokine-supported cultures. In addition, maintenance of input levels of transplantable stem cell activity is accompanied by expansion of other classes of stem/progenitor cells, This suggests that the stem/progenitor cell population is actively proliferating in culture and that the AFT024 cell line provides a milieu that stimulates progenitor cell proliferation while maintaining in vivo repopulating activity. (C) 1997 by The American Society of Hematology.