In Vivo Rescue of the Hematopoietic Niche By Pluripotent Stem Cell Complementation of Defective Osteoblast Compartments.

Bone-forming osteoblasts play critical roles in supporting bone marrow hematopoiesis. Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSC), are capable of differentiating into osteoblasts. To determine the capacity of stem cells needed to rescue aberrant skeletal development and bone marrow hematopoiesis in vivo, we used a skeletal complementation model. Mice deficient in Runx2, a master transcription factor for osteoblastogenesis, fail to form a mineralized skeleton and bone marrow. Wild-type (WT) green fluorescent protein (GFP)+ ESCs and yellow fluorescent protein (YFP)+ iPSCs were introduced into Runx2-null blastocyst-stage embryos. We assessed GFP/YFP+ cell contribution by whole-mount fluorescence and histological analysis and found that the proportion of PSCs in the resulting chimeric embryos is directly correlated with the degree of mineralization in the skull. Moreover, PSC contribution to long bones successfully restored bone marrow hematopoiesis. We validated this finding in a separate model with diphtheria toxin A-mediated ablation of hypertrophic chondrocytes and osteoblasts. Remarkably, chimeric embryos harboring as little as 37.5% WT PSCs revealed grossly normal skeletal morphology, suggesting a near-complete rescue of skeletogenesis. In summary, we demonstrate that fractional contribution of PSCs in vivo is sufficient to complement and reconstitute an osteoblast-deficient skeleton and hematopoietic marrow. Further investigation using genetically modified PSCs with conditional loss of gene function in osteoblasts will enable us to address the specific roles of signaling mediators to regulate bone formation and hematopoietic niches in vivo. Stem Cells 2017;35:2150-2159.

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy.

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage. Postnatal removal of Gsα in the osteoblast lineage (P-Gsα(OsxKO) mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1-34) (80 µg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-Gsα(OsxKO) mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-Gsα(OsxKO) mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of Gsα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond Gsα and Gq/11 act downstream of PTH on osteoblast differentiation.

Loss of chloroplast protease SPPA function alters high light acclimation processes in Arabidopsis thaliana L. (Heynh.).

SPPA1 is a protease in the plastids of plants, located in non-appressed thylakoid regions. In this study, T-DNA insertion mutants of the single-copy SPPA1 gene in Arabidopsis thaliana (At1g73990) were examined. Mutation of SPPA1 had no effect on the growth and development of plants under moderate, non-stressful conditions. It also did not affect the quantum efficiency of photosynthesis as measured by dark-adapted F(v)/F(m) and light-adapted Phi(PSII). Chloroplasts from sppA mutants were indistinguishable from the wild type. Loss of SPPA appears to affect photoprotective mechanisms during high light acclimation: mutant plants maintained a higher level of non-photochemical quenching of Photosystem II chlorophyll (NPQ) than the wild type, while wild-type plants accumulated more anthocyanin than the mutants. The quantum efficiency of Photosystem II was the same in all genotypes grown under low light, but was higher in wild type than mutants during high light acclimation. Further, the mutants retained the stress-related Early Light Inducible Protein (ELIP) longer than wild-type leaves during the early recovery period after acute high light plus cold treatment. These results suggest that SPPA1 may function during high light acclimation in the plastid, but is non-essential for growth and development under non-stress conditions.

PTH Signaling in Osteoprogenitors Is Essential for B-Lymphocyte Differentiation and Mobilization.

Cells of the osteoblast lineage provide critical support for B lymphopoiesis in the bone marrow (BM). Parathyroid hormone (PTH) signaling in osteoblastic cells through its receptor (PPR) is an important regulator of hematopoietic stem cells; however, its role in regulation of B lymphopoiesis is not clear. Here we demonstrate that deletion of PPR in osteoprogenitors results in a significant loss of trabecular and cortical bone. PPR signaling in osteoprogenitors, but not in mature osteoblasts or osteocytes, is critical for B-cell precursor differentiation via IL-7 production. Interestingly, despite a severe reduction in B-cell progenitors in BM, mature B-lymphocytes were increased 3.5-fold in the BM of mice lacking PPR in osteoprogenitors. This retention of mature IgD(+) B cells in the BM was associated with increased expression of vascular cell adhesion molecule 1 (VCAM1) by PPR-deficient osteoprogenitors, and treatment with VCAM1 neutralizing antibody increased mobilization of B lymphocytes from mutant BM. Our results demonstrate that PPR signaling in early osteoblasts is necessary for B-cell differentiation via IL-7 secretion and for B-lymphocyte mobilization via VCAM1.

Loss of Gsα early in the osteoblast lineage favors adipogenic differentiation of mesenchymal progenitors and committed osteoblast precursors.

In humans, aging and glucocorticoid treatment are associated with reduced bone mass and increased marrow adiposity, suggesting that the differentiation of osteoblasts and adipocytes may be coordinately regulated. Within the bone marrow, both osteoblasts and adipocytes are derived from mesenchymal progenitor cells, but the mechanisms guiding the commitment of mesenchymal progenitors into osteoblast versus adipocyte lineages are not fully defined. The heterotrimeric G protein subunit Gs α activates protein kinase A signaling downstream of several G protein-coupled receptors including the parathyroid hormone receptor, and plays a crucial role in regulating bone mass. Here, we show that targeted ablation of Gs α in early osteoblast precursors, but not in differentiated osteocytes, results in a dramatic increase in bone marrow adipocytes. Mutant mice have reduced numbers of mesenchymal progenitors overall, with an increase in the proportion of progenitors committed to the adipocyte lineage. Furthermore, cells committed to the osteoblast lineage retain adipogenic potential both in vitro and in vivo. These findings have clinical implications for developing therapeutic approaches to direct the commitment of mesenchymal progenitors into the osteoblast lineage.