Reversible switching of expression of c-kit and Pax-5 in immature hematopoietic progenitor cells by stromal cells.
OBJECTIVE
Bone marrow stromal cells provide the microenvironment for self-renewal and differentiation of hematopoietic stem/progenitor cells through complex cell-cell interaction. To elucidate the regulatory mechanisms of hematopoiesis by stromal cells, we established a novel stroma-dependent hematopoietic cell line and explored the phenotypic changes regulated by the two stromal cells.


METHODS
DFC-28 cells clonally established from long-term bone marrow culture of C57BL/6 mice were sustained by coculture on MSS62 cells (mouse spleen stromal cell line). When DFC-28 cells were transferred to TBR31-1 cells (mouse bone marrow stromal cell line), their phenotypic changes were analyzed by flow cytometry and reverse transcriptase polymerase chain reaction.


RESULTS
DFC-28 cells on MSS62 cells exhibited surface phenotypes of the immature hematopoietic progenitor cells (Lin(-)AA4.1(+)c-kit(+)Sca-1(-)). By stroma-replacement from MSS62 cells to TBR31-1 cells, DFC-28 cells were differentiated into very early B-lymphoid stage characterized by c-kit down-regulation and induction of BP-1 and B-lymphoid-associated genes (Pax-5, CD19, TdT, Rag-1, and Rag-2). In addition, the differentiation phenotypes reverted to the immature state characterized by c-kit induction and down-regulation of BP-1 and B-lymphoid-associated genes by replacing stroma back to MSS62 from TBR31-1. Interleukin-7 stimulation and conditioned medium of TBR31-1 cells were ineffective in converting the differentiation phenotypes of DFC-28 cells.


CONCLUSIONS
The results demonstrate that the differentiation phenotypes and growth potential of stroma-dependent hematopoietic progenitor cells we established could be reversibly controlled via direct contact with stromal cells in the microenvironment.
Okubo
Tadashi
T
Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Yanai
Nobuaki
N
Ikawa
Shuntaro
S
Obinata
Masuo
M
D000818:Animals; D001854:Bone Marrow Cells / Q000502:physiology; D002455:Cell Division; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004268:DNA-Binding Proteins / Q000235:genetics*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D051757:PAX5 Transcription Factor; D016133:Polymerase Chain Reaction; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D017154:Stromal Cells / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics*
D017077:Culture Media, Conditioned; D004268:DNA-Binding Proteins; D051757:PAX5 Transcription Factor; C497282:Pax5 protein, mouse; D014157:Transcription Factors; D019009:Proto-Oncogene Proteins c-kit