Mizrahi O, Sheyn D, Tawackoli W, Kallai I, Oh A, Su S, Da X, Zarrini P, Cook-Wiens G, Gazit D, Gazit Z
. BMP-6 is more efficient in bone formation than BMP-2 when overexpressed in mesenchymal stem cells
[Internet]. Gene Ther 2013;20(4):370-7. Publisher's VersionAbstract
Bone regeneration achieved using mesenchymal stem cells (MSCs) and nonviral gene therapy holds great promise for patients with fractures seemingly unable to heal. Previously, MSCs overexpressing bone morphogenetic proteins (BMPs) were shown to differentiate into the osteogenic lineage and induce bone formation. In the present study, we evaluated the potential of osteogenic differentiation in porcine adipose tissue- and bone marrow-derived MSCs (ASCs and BMSCs, respectively) in vitro and in vivo when induced by nucleofection with rhBMP-2 or rhBMP-6. Our assessment of the in vivo efficiency of this procedure was made using quantitative micro-computed tomography (micro-CT). Nucleofection efficiency and cell viability were similar in both cell types; however, the micro-CT analyses demonstrated that in both ASCs and BMSCs, nucleofection with rhBMP-6 generated bone tissue faster and of higher volumes than nucleofection with rhBMP-2. RhBMP-6 induced more efficient osteogenic differentiation in vitro in BMSCs, and in fact, greater osteogenic potential was identified in BMSCs both in vitro and in vivo than in ASCs. On the basis of our findings, we conclude that BMSCs nucleofected with rhBMP-6 are superior at inducing bone formation in vivo than all other groups studied.
Mizrahi O, Sheyn D, Tawackoli W, Ben-David S, Su S, Li N, Oh A, Bae H, Gazit D, Gazit Z
. Nucleus pulposus degeneration alters properties of resident progenitor cells
[Internet]. Spine J 2013;13(7):803-14. Publisher's VersionAbstract
BACKGROUND CONTEXT: The intervertebral disc (IVD) possesses a minimal capability for self-repair and regeneration. Changes in the differentiation of resident progenitor cells can represent diminished tissue regeneration and a loss of homeostasis. We previously showed that progenitor cells reside in the nucleus pulposus (NP). The effect of the degenerative process on these cells remains unclear. PURPOSE: We sought to explore the effect of IVD degeneration on the abundance of resident progenitor cells in the NP, their differentiation potential, and their ability to give rise to NP-like cells. We hypothesize that disc degeneration affects those properties. STUDY DESIGN: Nucleus pulposus cells derived from healthy and degenerated discs were methodically compared for proliferation, differentiation potential, and ability to generate NP-like cells. METHODS: Intervertebral disc degeneration was induced in 10 skeletally, mature mini pigs using annular injury approach. Degeneration was induced in three target discs, whereas intact adjacent discs served as controls. The disc degeneration was monitored using magnetic resonance imaging for 6 to 8 weeks. After there was a clear evidence of degeneration, we isolated and compared cells from degenerated discs (D-NP cells [NP-derived cells from porcine degenerated discs]) with cells isolated from healthy discs (H-NP cells) obtained from the same animal. RESULTS: The comparison showed that D-NP cells had a significantly higher colony-forming unit rate and a higher proliferation rate in vitro. Our data also indicate that although both cell types are able to differentiate into mesenchymal lineages, H-NP cells exhibit significantly greater differentiation toward the chondrogenic lineage and NP-like cells than D-NP cells, displaying greater production of glycosaminoglycans and higher gene expression of aggrecan and collagen IIa. CONCLUSIONS: Based on these findings, we conclude that IVD degeneration has a meaningful effect on the cells in the NP. D-NP cells clearly go through the regenerative process; however, this process is not powerful enough to facilitate full regeneration of the disc and reverse the degenerative course. These findings facilitate deeper understanding of the IVD degeneration process and trigger further studies that will contribute to development of novel therapies for IVD degeneration.
Benjamin S, Sheyn D, Ben-David S, Oh A, Kallai I, Li N, Gazit D, Gazit Z
. Oxygenated environment enhances both stem cell survival and osteogenic differentiation
[Internet]. Tissue Eng Part A 2013;19(5-6):748-58. Publisher's VersionAbstract
Osteogenesis of mesenchymal stem cells (MSCs) is highly dependent on oxygen supply. We have shown that perfluorotributylamine (PFTBA), a synthetic oxygen carrier, enhances MSC-based bone formation in vivo. Exploring this phenomenon's mechanism, we hypothesize that a transient increase in oxygen levels using PFTBA will affect MSC survival, proliferation, and differentiation, thus increasing bone formation. To test this hypothesis, MSCs overexpressing bone morphogenetic protein 2 were encapsulated in alginate beads that had been supplemented with an emulsion of PFTBA or phosphate-buffered saline. Oxygen measurements showed that supplementation of PFTBA significantly increased the available oxygen level during a 96-h period. PFTBA-containing beads displayed an elevation in cell viability, which was preserved throughout 2 weeks, and a significantly lower ratio of dead cells throughout the experiment. Furthermore, the cells from the control group expressed significantly more hypoxia-related genes such as VEGF, DDIT3, and PKG1. Additionally, PFTBA supplementation led to an increase in the osteogenic differentiation and to a decrease in chondrogenic differentiation of MSCs. In conclusion, PFTBA increases the oxygen availability in the vicinity of the MSCs, which suffer oxygen exhaustion shortly after encapsulation in alginate beads. Consequently, cell survival is increased, and hypoxia-related genes are downregulated. In addition, PFTBA promotes osteogenic differentiation over chondrogeneic differentiation, and thereby can accelerate MSC-based bone regeneration.
Sheyn D, Cohn Yakubovich D, Kallai I, Su S, Da X, Pelled G, Tawackoli W, Cook-Weins G, Schwarz EM, Gazit D, Gazit Z
. PTH promotes allograft integration in a calvarial bone defect
[Internet]. Mol Pharm 2013;10(12):4462-71. Publisher's VersionAbstract
Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones. Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and microcomputed tomography (muCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes. Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The muCT scans revealed a significant increase in bone formation in allograft + PTH treated mice comparing to allograft + PBS treated mice. The osteogenic genes osteocalcin (Oc/Bglap) and integrin binding sialoprotein (Ibsp) were upregulated in the allograft + PTH treated animals. In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.
Kimelman NB, Kallai I, Sheyn D, Tawackoli W, Gazit Z, Pelled G, Gazit D
. Real-time bioluminescence functional imaging for monitoring tissue formation and regeneration
[Internet]. Methods Mol Biol 2013;1048:181-93. Publisher's VersionAbstract
Real-time bioluminescence functional imaging holds great promise for regenerative medicine because it improves the researcher's ability to analyze and understand the healing process. Using transgenic mice coupled with gene-modified cells, one can employ this method to monitor host and graft activity in various models of tissue regeneration. We implemented real-time bioluminescence functional imaging to analyze bone formation by following a unique protocol in which the luciferase reporter gene, driven by an osteocalcin promoter, is used to visualize host and graft activity during bone formation. Real-time bioluminescence functional imaging can be used to assess the "host reaction" in transgenic mice models; it can also be used to assess "graft activity" in other animals in which genetically labeled stem cells have been implanted or direct gene delivery has been applied. The suggested imaging protocol requires 25 min per sample. However, special attention must be given to the layout of the experimental design, which determines the specific activity that will be analyzed.
Liu Q, Jin N, Fan Z, Natsuaki Y, Tawackoli W, Pelled G, Bae H, Gazit D, Li D
. Reliable chemical exchange saturation transfer imaging of human lumbar intervertebral discs using reduced-field-of-view turbo spin echo at 3.0 T
[Internet]. NMR Biomed 2013;26(12):1672-9. Publisher's VersionAbstract
The reduced field-of-view (rFOV) turbo-spin-echo (TSE) technique, which effectively suppresses bowel movement artifacts, is developed for the purpose of chemical exchange saturation transfer (CEST) imaging of the intervertebral disc (IVD) in vivo. Attempts to quantify IVD CEST signals in a clinical setting require high reliability and accuracy, which is often compromised in the conventionally used technique. The proposed rFOV TSE CEST method demonstrated significantly superior reproducibility when compared with the conventional technique on healthy volunteers, implying it is a more reliable measurement. Phantom study revealed a linear relation between CEST signal and glycosaminoglycan (GAG) concentration. The feasibility of detecting IVD degeneration was demonstrated on a healthy volunteer, indicating that the proposed method is a promising tool to quantify disc degeneration.
Sheyn D, Pelled G, Tawackoli W, Su S, Ben-David S, Gazit D, Gazit Z
. Transient overexpression of Ppargamma2 and C/ebpalpha in mesenchymal stem cells induces brown adipose tissue formation
[Internet]. Regen Med 2013;8(3):295-308. Publisher's VersionAbstract
BACKGROUND: Brown adipose tissue plays a pivotal role in mammal metabolism and thermogenesis. It has a great therapeutic potential in several metabolic disorders such as obesity and diabetes. Mesenchymal stem cells (MSCs) are suitable candidates for brown adipose tissue formation de novo. Ppargamma2 and C/ebpalpha are nucleic receptors known to mediate adipogenic differentiation. We hypothesized that overexpression of the Ppargamma2 and C/ebpalpha genes in MSCs would lead to the formation of adipose tissue. MATERIALS & METHODS: MSCs bearing the Luc reporter gene were transfected to overexpress Ppargamma2 and C/ebpalpha. Differentiation of nucleofected cells was evaluated in vitro and in vivo following ectopic implantation of the cells in C3H/HeN mice. RESULTS: After implantation, the engineered cells survived for 5 weeks and brown adipose-like tissue was observed in histological samples. Immunostaining and bioluminescent imaging showed new adipocytes expressing Luc and the brown adipose tissue marker, UCP1, in vitro and in vivo. CONCLUSION: We show that gene delivery of transcription factors into MSCs generates brown adipose tissue in vitro and in vivo.