Matilda H.-C. Sheng

Ex vivo gene therapy with stromal cells transduced with a retroviral vector containing the BMP4 gene completely heals critical size calvarial defect in rats

In order to develop a successful gene therapy system for the healing of bone defects, we developed a murine leukemia virus (MLV)-based retroviral system expressing the human bone morphogenetic protein (BMP) 4 transgene with high transduction efficiency. The bone formation potential of BMP4 transduced cells was tested by embedding 2.5 × 106 transduced stromal cells in a gelatin matrix that was then placed in a critical size defect in calvariae of syngenic rats. Gelatin matrix without cells or with untransduced stromal cells were the two control groups. The defect area was completely filled with new bone in experimental rats after 4 weeks, while limited bone formation occurred in either control group. Bone mineral density (BMD) of the defect in the gene therapy group was 67.8 ± 5.7 mg/cm2 (mean ± s.d., n = 4), which was 119 ± 10% of the control BMD of bone surrounding the defect (57.2 ± 1.5 mg/cm2). In contrast, BMD of rats implanted with untransduced stromal cells was five-fold lower (13.8 ± 7.4 mg/cm2, P < 0.001). Time course studies revealed that there was a linear increase in BMD between 2–4 weeks after inoculation of the critical size defect with 2.5 × 106 implanted BMP4 cells. In conclusion, the retroviral-based BMP4 gene therapy system that we have developed has the potential for regeneration of large skeletal defects.

Histomorphometric studies show that bone formation and bone mineral apposition rates are greater in C3H/HeJ (high-density) than C57BL/6J (low-density) mice during growth

High-density C3H/HeJ (C3H) and low-density C57BL/6J (B6) mice, with femoral bone density differing by 50%, were chosen as a model to investigate the mechanisms controlling peak bone density and to map peak bone density genes. The present longitudinal study was undertaken to further establish the bone biologic phenotypes of these two inbred strains of mice. To evaluate phenotypic differences in bone formation parameters in C3H and B6 mice between the ages of 6 and 26 weeks, undecalcified ground sections from the diaphyses of the tibia and femur were prepared from mice receiving two injections of tetracycline. Histomorphometric analyses revealed that the cortical bone area was significantly greater (16%-56%, p < 0.001) in both the femur and tibia of the C3H mice than in the B6 mice at all timepoints. This difference in cortical bone area was due to significantly smaller medullary areas in the C3H mice than in the B6 mice. The bone formation rates (BFR) at the endosteum in both the femur and tibia were significantly greater (28%-117%,p < 0.001) in the young C3H mice (6-12 weeks old) than in B6 mice. The higher bone formation in C3H mice was associated with higher values of the bone mineral apposition rate (25%-94%, p < 0.001), and was not associated with higher values of the forming surface length as measured by tetracycline label length. Similar interstrain differences in mineral apposition and bone formation rates were observed in the periosteum of the femur and tibia. In conclusion, the greater bone area in the high-density C3H mice vs. the low-density B6 mice was, in part, due to the greater periosteal and endosteal bone formation rates during growth in the C3H mice. Because the C3H and B6 mice were maintained under identical environmental conditions (diet, lighting, etc.), the observed interstrain differences in bone parameters were the result of the action of genetic factors. Consequently, these two inbred strains of mice are suitable as a model to identify genetic factors responsible for high bone formation rates.

Effects of liver-derived insulin-like growth factor I on bone metabolism in mice

Insulin‐like growth factor (IGF) I is an important regulator of both skeletal growth and adult bone metabolism. To better understand the relative importance of systemic IGF‐I versus locally expressed IGF‐I we have developed a transgenic mouse model with inducible specific IGF‐I gene inactivation in the liver (LI‐IGF‐I−/−). These mice are growing normally up to 12 weeks of age but have a disturbed carbohydrate and lipid metabolism. In this study, the long‐term effects of liver‐specific IGF‐I inactivation on skeletal growth and adult bone metabolism were investigated. The adult (week 8–55) axial skeletal growth was decreased by 24% in the LI‐IGF‐I−/− mice whereas no major reduction of the adult appendicular skeletal growth was seen. The cortical cross‐sectional bone area, as measured in the middiaphyseal region of the long bones, was decreased in old LI‐IGF‐I−/− mice. This reduction in the amount of cortical bone was caused mainly by decreased periosteal circumference and was associated with a weaker bone determined by a decrease in ultimate load. In contrast, the amount of trabecular bone was not decreased in the LI‐IGF‐I−/− mice. DNA microarray analysis of 30‐week‐old LI‐IGF‐I−/− and control mice indicated that only four genes were regulated in bone whereas ∼40 genes were regulated in the liver, supporting the hypothesis that liver‐derived IGF‐I is of minor importance for adult bone metabolism. In summary, liver‐derived IGF‐I exerts a small but significant effect on cortical periosteal bone growth and on adult axial skeletal growth while it is not required for the maintenance of the trabecular bone in adult mice.

Cortical tibial bone volume in two strains of mice: effects of sciatic neurectomy and genetic regulation of bone response to mechanical loading

Although C3H/HeJ (C3H) and C57BL/6J (B6) mice are similar in body size (and adult weight), and have bones of similar external size, C3H mice have higher peak bone densities than B6 mice (e.g., 53% higher peak bone density in the femora). The current studies were intended to assess the role of mechanical loading/unloading as a possible determinant of the bone density difference between these inbred strains of mice and, specifically, to assess the effect of sciatic neurectomy on histomorphometric indices of bone formation and resorption in the tibiae of female C3H and B6 mice. Groups of 10 mice of each strain were subjected to left-side sciatic neurectomy (left hindlimb immobilization) or a sham procedure. The contralateral (right) legs of each mouse were used as controls. Four weeks of immobilization produced no systemic changes in bone formation indices in either strain of mice (i.e., no change in serum alkaline phosphatase or serum osteocalcin). However, histomorphometric assessments at the tibiofibular junction showed that 4 weeks of immobilization caused a time-dependent decrease in the length of the endosteal bone forming perimeter (e.g., 14% of control single-labeled, noneroded surface at 4 weeks, p < 0.005) with a concomitant increase in the length of the endosteal bone resorbing perimeter (i.e., 424% of control eroded surface at 4 weeks, p < 0.005), in the B6 mice. These effects were associated with an increase in medullary area (132% of control, p < 0.05) at this site, in the B6 mice. The pattern of response was different in the tibiae of the C3 mice-a much smaller decrease in bone forming perimeter (88% of control at 4 weeks, p < 0.05), with no associated increase in bone resorbing perimeter, and no change in medullary area. Similar effects were seen at a second cross-sectional sampling site, in the proximal tibia. Together, these findings indicate that B6 mice are more sensitive to endosteal bone loss from hindlimb immobilization than C3H mice.

In vivo bone formation in fracture repair induced by direct retroviral-based gene therapy with bone morphogenetic protein-4

This study sought to develop an in vivo gene therapy to accelerate the repair of bone fractures. In vivo administration of an engineered viral vector to promote fracture healing represents a potential high-efficacy, low-risk procedure. We selected a murine leukemia virus (MLV)-based retroviral vector, because this vector would be expected to target transgene expression to the proliferating periosteal cells arising shortly after bone fracture. This vector transduced a hybrid gene that consisted of a bone morphogenetic protein (BMP)-4 transgene with the BMP-2 secretory signal to enhance the secretion of mature BMP-4. The MLV vector expressing this BMP-2/4 hybrid gene or beta-galactosidase control gene was administered at the lateral side of the fracture periosteum at 1 day after fracture in the rat femoral fracture model. X-ray examination by radiograph and peripheral quantitative computed tomography at 7, 14, and 28 days after fracture revealed a highly significant enhancement of fracture tissue size in the MLV-BMP-2/4-treated fractures compared to the control fractures. The tissue was extensively ossified at 14 and 28 days, and the newly formed bone exhibited normal bone histology. This tissue also exhibited strong immunohistochemical staining of BMP-4. Additional control and MLV-BMP-2/4-treated animals each were monitored for 70 days to determine the fate of the markedly enhanced fracture callus. Radiographs showed that the hard callus had been remodeled and substantial healing at the fracture site had occurred, suggesting that the union of the bone at the fracture site was at least as high in the BMP-4-treated bone as in the control bone. There was no evidence of viral vector infection of extraskeletal tissues, suggesting that this in vivo gene therapy for fracture repair is safe. In summary, we have demonstrated for the first time that a MLV-based retroviral vector is a safe and effective means of introducing a transgene to a fracture site and that this procedure caused an enormous augmentation of fracture bone formation.

Osteoclast formation in bone marrow cultures from two inbred strains of mice with different bone densities.

For the purpose of identifying genes that affect bone volume, we previously identified two inbred mouse strains (C57BL/6J and C3H/HeJ) with large differences in femoral bone density and medullary cavity volume. The lower density and larger medullary cavity volume in C57BL/6J mice could result from either decreased formation or increased resorption or both. We recently reported evidence suggesting that bone formation was increased in vivo and that osteoblast progenitor cells are more numerous in the bone marrow of C3H/HeJ compared with C57BL/6J mice. In the present study, we determined whether osteoclast numbers in vivo and osteoclast formation from bone marrow cells in vitro might also differ between the two mouse strains. We have found that the number of osteoclasts on bone surfaces of distal humerus secondary spongiosa was 2‐fold higher in 5.5‐week‐old C57BL/6J mice than in C3H/HeJ mice of the same age (p < 0.001). Bone marrow cells of C57BL/6J mice cocultured with Swiss/Webster mouse osteoblasts consistently produced more osteoclasts than did C3H/HeJ bone marrow cells at all ages tested from 3.5–14 weeks of age (p < 0.001). Osteoclast formation was also greater from spleen cells of 3.5‐week‐old C57BL/6J mice than C3H/HeJ mice. The distribution of nuclei per osteoclast and the 1,25‐dihydroxyvitamin D3 dose dependence of osteoclast production from bone marrow cells were similar. Osteoclasts that developed from both C57BL/6J and C3H/HeJ marrow cells formed pits in dentin slices. Cultures from C57BL/6J marrow cells formed 2.5‐fold more pits than cultures from C3H/HeJ marrow cells (p < 0.02). We compared the abilities of C57BL/6J and C3H/HeJ osteoblasts to support osteoclast formation. When bone marrow cells from either C57BL/6J or C3H/HeJ mice were cocultured with osteoblasts from either C57BL/6J or C3H/HeJ newborn calvaria, the strain from which osteoblasts were derived did not affect the number of osteoclasts formed from marrow cells of either strain. Together, these observations suggest that genes affecting the bone marrow osteoclast precursor population may contribute to the relative differences in bone density that occur between C3H/HeJ and C57BL/6J mouse strains.

Efficient Reprogramming of Human Cord Blood CD34+ Cells Into Induced Pluripotent Stem Cells With OCT4 and SOX2 Alone

The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34+ cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34+ cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34(+) cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34(+) cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.

Osteoactivin is a novel osteoclastic protein and plays a key role in osteoclast differentiation and activity

This study presents gene expression, protein expression, and in situ immunohistochemical evidence that osteoclasts express high levels of osteoactivin (OA), which had previously been reported to be an osteoblast‐specific protein in bone. OA expression in osteoclasts was up‐regulated upon receptor activator of NFκB ligand‐induced differentiation. Suppression of functional activity of OA with neutralizing antibody reduced cell size, number of nuclei, fusion, and bone resorption activity of osteoclasts. OA was co‐immunoprecipitated with integrin β3 and β1, indicating that OA co‐localizes with integrin β3 and/or β1 in a hetero‐polymeric complex in osteoclasts. These findings indicate that OA is a novel osteoclastic protein and plays a role in osteoclast differentiation and/or activity.

Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in mice.

Bone loss in osteoporosis is caused by an imbalance between resorption and formation on endosteal surfaces of trabecular and cortical bone. We investigated the feasibility of increasing endosteal bone formation in mice by ex vivo gene therapy with bone marrow stromal cells (MSCs) transduced with a MLV‐based retroviral vector to express human bone morphogenetic protein 4 (BMP4).

Disruption of the insulin-like growth factor-1 gene in osteocytes impairs developmental bone growth in mice

This study evaluated the role of osteocyte-derived insulin-like growth factor 1 (IGF-1) in developmental bone growth by assessing the bone phenotype of osteocyte Igf1 conditional knockout (KO) mice, generated by crossing the Dmp1-driven Cre-expressing transgenic mice with Igf1 floxed mice containing loxP sites that flank exon 4 of the Igf1 gene. The periosteal diameter of femurs of homozygous conditional KO mutants was 8-12% smaller than wild-type (WT) littermates. The conditional mutants had 14-20%, 10-21%, and 15-31% reduction in total, trabecular, and cortical bone mineral contents, respectively. However, there were no differences in the total, trabecular, or cortical bone mineral densities, or in trabecular bone volume, thickness, number, and separation at secondary spongiosa between the mutants and WT littermates. The conditional KO mutants showed reduction in dynamic bone formation parameters at both periosteal and endosteal surfaces at the mid-diaphysis and in trabecular bone formation rate and resorption parameters at secondary spongiosa. The lower plasma levels of PINP and CTx in conditional KO mice support a regulatory role of osteocyte-derived IGF-1 in the bone turnover. The femur length of conditional KO mutants was 4-7% shorter due to significant reduction in the length of growth plate and hypertropic zone. The effect on periosteal expansion appeared to be bigger than that on longitudinal bone growth. The conditional KO mice had 14% thinner calvaria than WT littermates, suggesting that deficient osteocyte IGF-1 production also impairs developmental growth of intramembraneous bone. Conditional disruption of Igf1 in osteocytes did not alter plasma levels of IGF-1, calcium, or phosphorus. In summary, this study shows for the first time that osteocyte-derived IGF-1 plays an essential role in regulating bone turnover during developmental bone growth.