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666 Loss of TGF-β Signaling in Bone Marrow Mesenchymal Progenitors Promotes Adipocyte over Osteoblast Differentiation but Does Not Disrupt the HSC Niche

Hematopoiesis and Stem Cells: Microenvironment, Cell Adhesion and Stromal Stem Cells
Program: Oral and Poster Abstracts
Type: Oral
Session: 506. Hematopoiesis and Stem Cells: Microenvironment, Cell Adhesion and Stromal Stem Cells: Hematopoietic Stem Cell Niche
Monday, December 7, 2015: 4:00 PM
W304EFGH, Level 3 (Orange County Convention Center)

Grazia Abou Ezzi, PhD1*, Teerawit Suparkorndej2*, Bryan Anthony2*, Jingzhu Zhang, BS3, Shilpi Ganguly4*, Roberto Civitelli5* and Daniel C. Link2

1Department of Medicine, Division of Oncology, Section of Stem Cell Biology, Washington University School of medicine, Saint Louis, MO
2Department of Medicine, Division of Oncology, Section of Stem Cell Biology, Washington University School of Medicine, Saint Louis, MO
3Department of Internal Medicine, Division of Oncology, Section of Stem Cell Biology, Washington University School of Medicine, St Louis, MO
4Washington University School of Medicine, Saint Louis, MO
5Int Med - Bone & Mineral Diseases, Washington University School of Medicine, Saint Louis, MO

Hematopoietic stem cells (HSCs) reside in specialized microenvironments (niches) in the bone marrow. Several mesenchymal stromal cells have been implicated in hematopoietic niches, including osteoblasts, pericytes, CXCL12-abundant reticular (CAR) cells, and mesenchymal stem cells (MSCs). Members of the transforming growth factor (TGF) superfamily, in particular TGF-β, have a well-documented role in regulating osteoblast development. However, the contribution of TGF family member signaling to the establishment and maintenance of hematopoietic niches is largely unknown. Here, we characterize the role of transforming growth factor-β (TGF- β) signaling in mesenchymal stromal cells on the HSC niche. 

TGF-β receptor 2 (encoded by Tgfbr2) is required for all TGF-β signaling.  To selectively disrupt TGF-β signaling in bone marrow mesenchymal stromal cells, we generated Osx-Cre Tgfbr2fl/fl mice.  Osx-Cre targets most bone marrow mesenchymal stromal cells (including osteoblasts, CAR cells, MSCs, pericytes, and adipocytes) but not endothelial cells or hematopoietic cells.  Osx-Cre Tgfbr2fl/fl mice are severely runted and most die by 4 weeks of age.  We analyzed mice at 3 weeks, when the mice appeared healthy.  Osteoblast number was severely reduced in Osx-Cre Tgfbr2fl/fl mice, as assessed by histomorphometry and immunostaining for osteocalcin. Accordingly, microCT analysis demonstrated reduced tissue mineral density and cortical thickness of long bone and marked trabecularization of long bones in diaphyseal regions.  Surprisingly, marrow adiposity, as measured by osmium tetroxide staining with microCT, was strikingly increased in Osx-Cre Tgfbr2fl/fl mice.  CAR cells are mesenchymal progenitors with osteogenic and adipogenic potential in vitro. To assess CAR cells, we generated Osx-Cre Tgfrb2fl/fl x Cxcl12gfp mice.  Surprisingly, CAR cell number was significantly increased.  However, despite the increase in CAR cells, the number of CFU-osteoblast (CFU-OB) in Osx-Cre Tgfbr2fl/fl mice is nearly undetectable.  Together, these data suggest that TGF-b signaling contributes to lineage commitment of mesenchymal progenitors.  Specifically, our data suggest that TGF-β signaling suppresses commitment to the osteoblast lineage, while increasing adipogenic differentiation. 

We next asked whether alterations in bone marrow stromal cells present in Osx-Cre Tgfbr2fl/fl mice affect HSC number or function.  The increase in marrow adipocytes and loss of osteolineage cells is predicted to impair HSC maintenance, while the increase in CAR cells might augment HSCs.  Osx-Cre Tgfrb2fl/fl mice have modest leukopenia, but normal red blood cell and platelet counts.  Bone marrow and spleen cellularity are reduced, even after normalizing for body weight.  The frequency of phenotypic HSCs (defined as Kit+ lineage- Sca+ CD34- Flk2- cells) is comparable to control mice.  To assess HSC function, we performed competitive repopulation assays with bone marrow from Osx-Cre Tgfrb2fl/fl or control mice.  Surprisingly, these data show that the long-term multi-lineage repopulating activity of HSCs from Osx-Cre Tgfrb2fl/fl mice is normal.  Moreover, serial transplantation studies suggest that the self-renewal capacity of HSCs is normal.  Thus, despite major alterations in mesenchymal stromal cell populations, the HSC niche is intact in Osx-Cre Tgfrb2fl/fl mice. 

Collectively, these data show that TGF-b signaling in mesenchymal progenitors is required for the proper development of multiple stromal cell populations that contribute to hematopoietic niches.  Studies are underway to assess the impact of post-natal deletion of Tgfbr2 in mesenchymal stromal cell on hematopoietic niches.  Since drugs that modulate the activity of TGF-b are in development, this research may suggest novel approaches to modulate hematopoietic niches for therapeutic benefit.

Disclosures: No relevant conflicts of interest to declare.

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