Phenotypic, trophic, and regenerative properties of mesenchymal stem cells from different osseous tissues.

Mesenchymal stem cells (MSCs) have broad-based therapeutic potential in regenerative medicine. However, a major barrier to their clinical utility is that MSCs from different tissues are highly variable in their regenerative properties. In this study, we defined the molecular and phenotypic identities of different MSC populations from different osseous tissue sites of different patients and, additionally, determined their respective regenerative properties. MSCs from 6 patients were isolated from either bone marrow of the iliac crest (BMSCs) or alveolar bone tissue (aBMSCs), and flow cytometry revealed that regardless of the tissue source, MSC immunotypes had the same expression of MSC markers CD73, CD90, and CD105. However, transcriptomic analyses revealed 589 genes differentially expressed (DE) between BMSCs and aBMSCs, including eightfold higher levels of bone morphogenetic protein 4 (BMP-4) in aBMSCs. In striking contrast, gene expression of MSCs derived from the same tissue, but between different patients (i.e., BMSCs to BMSCs, aBMSCs to aBMSCs), showed only 38 DE BMSC genes and 51 DE aBMSC genes. A protein array showed that aBMSC and BMSC produced equivalent levels of angiogenic cytokines; however, when placed in angiogenesis model systems, aBMSCs induced significantly more capillaries in vitro and in vivo. Finally, cell transplantation of MSCS into osseous defects showed that the bone regenerative capacity of aBMSCs was significantly greater than that of BMSCs. This study is the first to link the molecular, phenotypic, and regenerative properties of different MSCs from different patients and provides novel insights toward MSC differences based on the osseous tissue origin.

Dental Pulp Tissue Regeneration Using Dental Pulp Stem Cells Isolated and Expanded in Human Serum.

INTRODUCTION: Dental pulp-derived stem cells (DPSCs) have the potential to regenerate dentin and dental pulp tissue because of their differentiation capacity and angiogenic properties. However, for regenerative approaches to gain regulatory and clinical acceptance, protocols are needed to determine more feasible ways to cultivate DPSCs, namely, without the use of xenogeneic-derived components (animal sera) and exogenous growth factors. METHODS: In this study, human DPSCs were isolated from third molars and expanded in standard culture conditions containing fetal bovine serum (DPSCs-FBS) or conditions containing human serum (DPSCs-HS). After cell characterization and evaluation of their angiogenic secretome, DPSCs were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. After 30 days, tooth slices were retrieved and evaluated for dental pulp tissue regeneration. Immunohistochemistry and confocal microscopy were used to quantify blood vessel formation and evaluate predentin and dentin formation. RESULTS: After culture, DPSCs-HS produced concentrations of angiogenic growth factors equivalent to DPSCs-FBS. Additionally, in DPSCs-HS, several angiogenic factors were produced in at least 1-fold higher concentrations than in DPSCs-FBS. In vivo, it was determined that DPSCs-HS produced a robust angiogenic response and regeneration of dentin equivalent to DPSCs-FBS. CONCLUSIONS: These findings show that DPSCs can be isolated and expanded to clinical scale numbers in media devoid of animal serum or exogenous growth factors and still maintain their pulp regenerative properties. The implications of these findings are significant for further development of clinical protocols using DPSCs in cell therapies.

Biochemical Basis for Dominant Inheritance, Variable Penetrance, and Maternal Effects in RBP4 Congenital Eye Disease.

Gestational vitamin A (retinol) deficiency poses a risk for ocular birth defects and blindness. We identified missense mutations in RBP4, encoding serum retinol binding protein, in three families with eye malformations of differing severity, including bilateral anophthalmia. The mutant phenotypes exhibit dominant inheritance, but incomplete penetrance. Maternal transmission significantly increases the probability of phenotypic expression. RBP normally delivers retinol from hepatic stores to peripheral tissues, including the placenta and fetal eye. The disease mutations greatly reduce retinol binding to RBP, yet paradoxically increase the affinity of RBP for its cell surface receptor, STRA6. By occupying STRA6 nonproductively, the dominant-negative proteins disrupt vitamin A delivery from wild-type proteins within the fetus, but also, in the case of maternal transmission, at the placenta. These findings establish a previously uncharacterized mode of maternal inheritance, distinct from imprinting and oocyte-derived mRNA, and define a group of hereditary disorders plausibly modulated by dietary vitamin A.

Tooth storage, dental pulp stem cell isolation, and clinical scale expansion without animal serum.

INTRODUCTION: Dental pulp stem cells (DPSCs) have therapeutic potential for dentin and dental pulp regeneration. For regenerative approaches to gain clinical acceptance, protocols are needed to determine feasible ways to store teeth, isolate DPSCs, and expand them to clinical scale numbers. METHODS: In this study, 32 third molars were obtained from patients and immediately placed in saline or tissue culture medium followed by overnight storage at 4°C or immediate isolation of DPSCs. Upon isolation, cells were expanded in medium containing either fetal bovine serum (FBS) or human serum (HS). Cell proliferation (population doubling time [PDT]), cell surface marker expression, and multipotency were compared between DPSCs in FBS and DPSCs in HS. RESULTS: The time frame of storage and storage medium did not affect the ability to isolate DPSCs. However, using HS instead of FBS in the initial isolation of DPSCs significantly decreased (P < .01) the isolation success rate from 89% (FBS) to 23% (HS). Yet, incorporating fibronectin in the DPSC initial isolation (using HS) significantly (P < .01) increased the isolation success rate to 83%. Interestingly, it was found that the proliferation rate was significantly (P < .05) higher for DPSCs in HS (PDT = 1.59 ± 0.46) than that for DPSCs in FBS (PDT = 2.84 ± 2.5). Finally, there was no difference in the expression of CD73, CD90, CD105, or multipotency (as measured by osteogenic, adipogenic, and chondrogenic differentiation) between DPSCs in FBS and DPSCs in HS. CONCLUSIONS: These findings show a clinically feasible method of storing third molars for the isolation of DPSCs. Additionally, DPSCs can be isolated and expanded to clinical scale numbers in media devoid of FBS and still maintain their phenotypic properties.

Stem cells derived from tooth periodontal ligament enhance functional angiogenesis by endothelial cells.

In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential, which could make them a very attractive cell population for utilization in regenerative cell therapies.

Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial.

Stem cell therapy offers potential in the regeneration of craniofacial bone defects; however, it has not been studied clinically. Tissue repair cells (TRCs) isolated from bone marrow represent a mixed stem and progenitor population enriched in CD90- and CD14-positive cells. In this phase I/II, randomized, controlled feasibility trial, we investigated TRC cell therapy to reconstruct localized craniofacial bone defects. Twenty-four patients requiring localized reconstruction of jawbone defects participated in this longitudinal trial. For regenerative therapy, patients were randomized to receive either guided bone regeneration (GBR) or TRC transplantation. At 6 or 12 weeks following treatment, clinical and radiographic assessments of bone repair were performed. Bone biopsies were harvested and underwent quantitative micro-computed tomographic (µCT) and bone histomorphometric analyses. Oral implants were installed, subsequently restored, and functionally loaded with tooth restorations. Reconstructed sites were assessed for 1 year following therapy. No study-related, serious adverse events were reported. Following therapy, clinical, radiographic, tomographic, and histological measures demonstrated that TRC therapy accelerated alveolar bone regeneration compared to GBR therapy. Additionally, TRC treatment significantly reduced the need for secondary bone grafting at the time of oral implant placement with a five fold decrease in implant bony dehiscence exposure (residual bone defects) as compared to GBR-treated sites(p < 0.01). Transplantation of TRCs for treatment of alveolar bone defects appears safe and accelerates bone regeneration, enabling jawbone reconstruction with oral implants. The results from this trial support expanded studies of TRC therapy in the treatment of craniofacial deformities (ClinicalTrials.gov number CT00755911).

Angiogenic and osteogenic potential of bone repair cells for craniofacial regeneration.

There has been increased interest in the therapeutic potential of bone marrow derived cells for tissue engineering applications. Bone repair cells (BRCs) represent a unique cell population generated via an ex vivo, closed-system, automated cell expansion process, to drive the propagation of highly osteogenic and angiogenic cells for bone engineering applications. The aims of this study were (1) to evaluate the in vitro osteogenic and angiogenic potential of BRCs, and (2) to evaluate the bone and vascular regenerative potential of BRCs in a craniofacial clinical application. BRCs were produced from bone marrow aspirates and their phenotypes and multipotent potential characterized. Flow cytometry demonstrated that BRCs were enriched for mesenchymal and vascular phenotypes. Alkaline phosphatase and von Kossa staining were performed to assess osteogenic differentiation, and reverse transcriptase-polymerase chain reaction was used to determine the expression levels of bone specific factors. Angiogenic differentiation was determined through in vitro formation of tube-like structures and fluorescent labeling of endothelial cells. Finally, 6 weeks after BRC transplantation into a human jawbone defect, a biopsy of the regenerated site revealed highly vascularized, mineralized bone tissue formation. Taken together, these data provide evidence for the multilineage and clinical potential of BRCs for craniofacial regeneration.

Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute.

Ribosomal protein mutations, termed Minutes, have been instrumental in studying the coordination of cell and tissue growth in Drosophila. Although abundant in flies, equivalent defects in mammals are relatively unknown. Belly spot and tail (Bst) is a semidominant mouse mutation that disrupts pigmentation, somitogenesis and retinal cell fate determination. Here, we identify Bst as a deletion within the Rpl24 riboprotein gene. Bst significantly impairs Rpl24 splicing and ribosome biogenesis. Bst/+ cells have decreased rates of protein synthesis and proliferation, and are outcompeted by wild-type cells in C57BLKS<-->ROSA26 chimeras. Bacterial artificial chromosome (BAC) and cDNA transgenes correct the mutant phenotypes. Our findings establish Bst as a mouse Minute and provide the first detailed characterization of a mammalian ribosomal protein mutation.