ABSTRACT
The molecular basis for the unique proliferative and self-renewal properties that hierarchically distinguish human stem cells from progenitors and terminally differentiated cells remains largely unknown. We report a role for the Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) as an indispensable regulator of self-renewal in human stem cells and show that a functional dependence on Mcl-1 defines the human stem cell hierarchy. In vivo pharmacologic targeting of the Bcl-2 family members in human hematopoietic stem cells (HSCs) and human leukemic stem cells reduced stem cell regenerative and self-renewal function. Subsequent protein expression studies showed that, among the Bcl-2 family members, only Mcl-1 was up-regulated exclusively in the human HSC fraction on in vivo regeneration of hematopoiesis. Short hairpin RNA-knockdown of Mcl-1 in human cord blood cells did not affect survival in the HSC or hematopoietic progenitor cell fractions in vitro but specifically reduced the in vivo self-renewal function of human HSCs. Moreover, knockdown of Mcl-1 in ontogenetically primitive human pluripotent stem cells resulted in almost complete ablation of stem cell self-renewal function. Our findings show that Mcl-1 is an essential regulator of stem cell self-renewal in humans and therefore represents an axis for therapeutic interventions.
Subject(s)
Cell Proliferation , Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Blotting, Western , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Female , Fetal Blood/drug effects , Fetal Blood/metabolism , Flow Cytometry , Humans , Indoles , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins c-bcl-2/genetics , Pyrroles/pharmacology , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , beta 2-Microglobulin/physiologyABSTRACT
Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make them vulnerable to transformation. As hES cells with cancer-cell characteristics share properties with normal hES cells, such as self-renewal, teratoma formation and the expression of pluripotency markers, they may be misconstrued as superior hES cells with enhanced 'stemness'. We characterize two variant hES cell lines (v-hESC-1 and v-hESC-2) that express pluripotency markers at high levels and do not harbor chromosomal abnormalities by standard cytogenetic measures. We show that the two lines possess some features of neoplastic progression, including a high proliferative capacity, growth-factor independence, a 9- to 20-fold increase in frequency of tumor-initiating cells, niche independence and aberrant lineage specification, although they are not malignant. Array comparative genomic hybridization reveals an amplification at 20q11.1-11.2 in v-hESC-1 and a deletion at 5q34a-5q34b;5q3 and a mosaic gain of chromosome 12 in v-hESC-2. These results emphasize the need for functional characterization to distinguish partially transformed and normal hES cells.