Expansion on stromal cells preserves the undifferentiated state of human hematopoietic stem cells despite compromised reconstitution ability.
Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38-CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38-CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.
Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.
Mattias
M
Magnusson
Maria I
MI
Sierra
Rajkumar
R
Sasidharan
Sacha L
SL
Prashad
Melissa
M
Romero
Pamela
P
Saarikoski
Ben
B
Van Handel
Andy
A
Huang
Xinmin
X
Li
Hanna K A
HK
Mikkola
D015703:Antigens, CD / Q000378:metabolism; D017209:Apoptosis; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D049109:Cell Proliferation; D018920:Coculture Techniques / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D020869:Gene Expression Profiling; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism
D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D004268:DNA-Binding Proteins; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; C083400:PBX1 protein, human