- Pluripotent stem cells as a source of osteoblasts for bone tissue regeneration BIOMATERIALS 2019; 196: 31–45
Pluripotent stem cells as a source of osteoblasts for bone tissue regeneration.
Appropriate and abundant sources of bone-forming osteoblasts are essential for bone tissue engineering. Pluripotent stem cells can self-renew and thereby offer a potentially unlimited supply of osteoblasts, a significant advantage over other cell sources. We generated mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) from transgenic mice expressing rat 2.3 kb type I collagen promoter-driven green fluorescent protein (Col2.3GFP), a reporter of the osteoblast lineage. We demonstrated that Col2.3GFP ESCs and iPSCs can be successfully differentiated to osteoblast lineage cells that express Col2.3GFP in vitro. We harvested GFP+osteoblasts differentiated from ESCs. Genome wide gene expression profiles validated that ESC- and iPSC-derived osteoblasts resemble calvarial osteoblasts, and that Col2.3GFP expression serves as a marker for mature osteoblasts. Our results confirm the cell identity of ESC- and iPSC-derived osteoblasts and highlight the potential of pluripotent stem cells as a source of osteoblasts for regenerative medicine.
View details for PubMedID 29456164
Transcriptome profiling of stem cell-derived osteoblasts identifies genes associated with osteoblast differentiation.
WILEY. 2017: S292
View details for Web of Science ID 000418869203131
Foxp3(+) regulatory T cells maintain the bone marrow microenvironment for B cell lymphopoiesis
Foxp3(+) regulatory T cells (Treg cells) modulate the immune system and maintain self-tolerance, but whether they affect haematopoiesis or haematopoietic stem cell (HSC)-mediated reconstitution after transplantation is unclear. Here we show that B-cell lymphopoiesis is impaired in Treg-depleted mice, yet this reduced B-cell lymphopoiesis is rescued by adoptive transfer of affected HSCs or bone marrow cells into Treg-competent recipients. B-cell reconstitution is abrogated in both syngeneic and allogeneic transplantation using Treg-depleted mice as recipients. Treg cells can control physiological IL-7 production that is indispensable for normal B-cell lymphopoiesis and is mainly sustained by a subpopulation of ICAM1(+) perivascular stromal cells. Our study demonstrates that Treg cells are important for B-cell differentiation from HSCs by maintaining immunological homoeostasis in the bone marrow microenvironment, both in physiological conditions and after bone marrow transplantation.
View details for DOI 10.1038/ncomms15068
View details for PubMedID 28485401
An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors.
Stem cell reports
Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.
View details for DOI 10.1016/j.stemcr.2017.01.015
View details for PubMedID 28238792
Prevention of breast cancer skeletal metastases with parathyroid hormone.
2017; 2 (17)
Advanced breast cancer is frequently associated with skeletal metastases and accelerated bone loss. Recombinant parathyroid hormone [teriparatide, PTH(1-34)] is the first anabolic agent approved in the US for treatment of osteoporosis. While signaling through the PTH receptor in the osteoblast lineage regulates bone marrow hematopoietic niches, the effects of anabolic PTH on the skeletal metastatic niche are unknown. Here, we demonstrate, using orthotopic and intratibial models of 4T1 murine and MDA-MB-231 human breast cancer tumors, that anabolic PTH decreases both tumor engraftment and the incidence of spontaneous skeletal metastasis in mice. Microcomputed tomography and histomorphometric analyses revealed that PTH increases bone volume and reduces tumor engraftment and volume. Transwell migration assays with murine and human breast cancer cells revealed that PTH alters the gene expression profile of the metastatic niche, in particular VCAM-1, to inhibit recruitment of cancer cells. While PTH did not affect growth or migration of the primary tumor, it elicited several changes in the tumor gene expression profile resulting in a less metastatic phenotype. In conclusion, PTH treatment in mice alters the bone microenvironment, resulting in decreased cancer cell engraftment, reduced incidence of metastases, preservation of bone microarchitecture and prolonged survival.
View details for PubMedID 28878134
In vivo rescue of the hematopoietic niche by pluripotent stem cell complementation of defective osteoblast compartments.
Stem cells (Dayton, Ohio)
Bone-forming osteoblasts play critical roles in supporting bone marrow hematopoiesis. Pluripotent stem cells (PSCs), including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, 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 employed 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 GFP(+) ES and YFP(+) iPS cells 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% wild-type 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. This article is protected by copyright. All rights reserved.
View details for PubMedID 28741855
Loss of Gsa in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy.
journal of biological chemistry
2016; 291 (4): 1631-1642
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 (PTHR1) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTHR1 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-GsαOsxKO 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-GsαOsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-GsαOsxKO mice. In mice that express a mutated PTHR1 that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C (PLC) via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that PLC activation is not required for increased bone turnover in response to PTH. Therefore while 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.
View details for DOI 10.1074/jbc.M115.679753
View details for PubMedID 26598522