The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4 −/− HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.

A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ∼90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a–regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division.

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The function of microRNAs (miRNAs) in hematopoietic stem cells (HSCs), committed progenitors, and leukemia stem cells (LSCs) is poorly understood. We show that miR-29a is highly expressed in HSC and down-regulated in hematopoietic progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors results in acquisition of self-renewal capacity by myeloid progenitors, biased myeloid differentiation, and the development of a myeloproliferative disorder that progresses to acute myeloid leukemia (AML). miR-29a promotes progenitor proliferation by expediting G1 to S/G2 cell cycle transitions. miR-29a is overexpressed in human AML and, like human LSC, miR-29a-expressing myeloid progenitors serially transplant AML. Our data indicate that miR-29a regulates early hematopoiesis and suggest that miR-29a initiates AML by converting myeloid progenitors into self-renewing LSC.

Hematopoietic stem cells (HSCs) have robust proliferative potential, as they can undergo extensive expansion to quickly restore hematopoiesis after transplantation or histological injury. However, under steady state, HSCs proliferate at a very low rate and most HSCs are kept in the G 0 phase of the cell cycle ( 1 ). Disruption of HSC quiescence leads to premature exhaustion of the stem cell pool and causes hematological failure under stress conditions ( 2, 3 ). Thus, HSC self-renewal and quiescence have to be fi nely balanced to maintain a stable HSC pool that is capable of producing blood cells for the lifetime of the organism. Although numerous transcription factors and cell cycle molecules have been identifi ed to regulate HSC self-renewal, it is not understood how nuclear regulatory factors adjust the HSC self-renewal rate to accommodate hematopoiesis under homeostatic and cytopenic conditions. It has been reported that HSCs are relocated from the osteoblastic niche to vascular zones in the BM after myeloablation ( 4 ). The translocation of HSCs is accompanied with an increase in HSC proliferation, suggesting that signals emanating from the BM niche where HSCs reside determine the balance between quiescence and selfrenewal of HSCs.The chemokine CXCL12 is the major chemoattractant for HSCs ( 5 ). It is expressed at a high level by osteoblasts, endothelial cells, and by a subset of reticular cells scattered throughout the BM ( 6, 7 ). Inactivation of CXCL12 or its receptor CXCR4 impairs the translocation of HSCs from the fetal liver to the BM during embryogenesis ( 8 -11 ), and direct ablation of CXCR4 signaling or indirect modulation of CXCL12 level by proteases results in mobilization of primitive hematopoietic cells and compromises their engrafting activity ( 4, 12 -14 ). This suggests an important role for CXCR4/ CXCL12 in BM retention of primitive hematopoietic cells. Additional eff ects of CXCR4 on HSCs are still not fully understood, and studies evaluating its regulatory role in the cell cycle yielded contradictory results ( 7, 15 ). To better understand the function of CXCR4 in HSCs, we deleted the Cxcr4 gene during adult hematopoiesis. We found that the compartment of primitive hematopoietic cells (Flt3 Ϫ Lin Ϫ Sca-1 + c-Kit + cells) was stably maintained in the BM in the absence of CXCR4 and sustained longterm hematopoiesis. These CXCR4-defi cient primitive hematopoietic cells proliferated vigorously and outcompeted the coexisting WT counterpart in the same host. CXCL12 directly inhibited the cell cycle of WT, but not Cxcr4 Ϫ / Ϫ , primitive hematopoietic cells. Thus, our results demonstrate a critical role of CXCR4 in restraining HSCs in the quiescent state. The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by t...