Origin of insulin secreted from islet-like cell clusters derived from murine embryonic stem cells

Islet-like cell clusters (ILCCs) were derived from murine embryonic stem cells using a slightly modified version of the protocol originally described by Lumelsky et al. in 2001. Analysis with enzyme-linked immunosorbent assays (ELISAs) that distinguish human from murine insulin demonstrated that insulin released from these ILCCs, upon initial in vitro glucose challenge, was of non-murine origin and in fact corresponded to the species of insulin, human or bovine, that had been added to the culture media used to derive ILCCs. This finding convincingly supports the hypothesis that ILCCs are not synthesizing insulin de novo, but rather simply regurgitating insulin taken up during tissue culture. In further experiments, ILCCs were derived in media in which insulin had been replaced by IGF-I with which it shares a common signaling pathway. These ILCCs failed to release any detectable insulin. In contrast, ILCCs produced by various protocols stained positive (dithizone and immunoselective antibodies) for intracellular insulin and, in some cases, C-peptide. Despite the presence of at least some level of de novo, synthesized insulin in ILCCs, the majority of insulin released by ILCCs was sequestered from the exogenous medium.     

Differentiation of mesenchymal stem cells derived from human bone marrow and subcutaneous adipose tissue into pancreatic islet-like clusters in vitro

Although stem cells are present in various adult tissues and body fluids, bone marrow has been the most popular source of stem cells for treatment of a wide range of diseases. Recent results for stem cells from adipose tissue have put it in a position to compete for being the leading therapeutic source. The major advantage of these stem cells over their counterparts is their amazing proliferative and differentiation potency. However, their pancreatic lineage transdifferentiation competence was not compared to that for bone marrow-derived stem cells. This study aims to identify an efficient source for transdifferentiation into pancreatic islet-like clusters, which would increase potential application in curative diabetic therapy. The results reveal that mesenchymal stem cells (MSC) derived from bone marrow and subcutaneous adipose tissue can differentiate into pancreatic islet-like clusters, as evidenced by their islet-like morphology, positive dithizone staining and expression of genes such as Nestin, PDX1, Isl 1, Ngn 3, Pax 4 and Insulin. The pancreatic lineage differentiation was further corroborated by positive results in the glucose challenge assay. However, the results indicate that bone marrow-derived MSCs are superior to those from subcutaneous adipose tissue in terms of differentiation into pancreatic islet-like clusters. In conclusion, bone marrow-derived MSC might serve as a better alternative in the treatment of diabetes mellitus than those from adipose tissue.     

Neural crest stem cell property of apical pulp cells derived from human developing tooth

Recent reports have described that NCSCs (neural crest-derived stem cells) are not only present in the embryonic neural crest but also in the adult tissues. Dental pulp is one of mesenchymal soft tissues origin from cranial neural crest cells, and thought to be a source of adult stem cells. Here, we investigated the existence of NCSC-like cells in apical pulp of human developing tooth. Human impacted third molars with immature apex freshly extracted were obtained. The cells derived from the apical pulp tissue not framed by dentin or the coronal pulp tissues were cultured by primary explant culture. APDCs (apical pulp-derived cells) and CPCs (coronal pulp cells) formed spheres under neurosphere culture condition. The number of spheres from APDCs was larger than that from CPCs. The sphere-forming cells derived from APDCs had self-renewal capacity, and expressed neural crest-associated markers (p75, Snail and Slug) and NSC (neural stem cell) markers (Nestin and Musashi1). The expression pattern of mesenchymal stem cell markers, CD105 and CD166, on the surface of sphere-forming cells derived APDCs was different from that of APDCs. These sphere-forming cells could differentiate into multiple mesenchymal lineages (osteoblasts, adipocytes, chondrocytes and smooth muscle cells) and neural lineage (neurons) in vitro, and generated ectopic bone tissues on the border of HA (hydroxyapatite) scaffold in vivo. The results of this study suggest that APDCs contain cells with characteristics of NCSCs reported previously in mice. Humans developing tooth with immature apex is an effective source of cells for neural crest lineage tissue regeneration.     

Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee

Background  

Chimpanzee dental pulp stem/stromal cells (ChDPSCs) are very similar to human bone marrow derived mesenchymal stem/stromal cells (hBMSCs) as demonstrated by the expression pattern of cell surface markers and their multipotent differentiation capability.     

Isolation of pluripotent stem cells from human third molar dental pulp

Potent stem/progenitor cells have been isolated from normal human dental pulps, termed dental pulp stem cells (DPSCs). However, no study has described the presence of stem cell populations in human dental pulp from the third molar with embryonic phenotypes. The dental pulp tissue was cultured in media with the presence of LIF, EGF, and PDGF. In the present study, we describe a new population of pluripotent stem cells that were isolated from dental pulp (DPPSC). These cells are SSEA-4(+), Oct4(+), Nanog(+), FLK-1(+), HNF3beta(+), Nestin(+), Sox2(+), Lin28(+), c-Myc(+), CD13(+), CD105(+), CD3(-), CD45(-), CD90(low), CD29(+), CD73(low), STRO-1(low) and CD146(-). We have investigated by SEM analysis and q-RT-PCR the capacity of DPPSCs to 3D differentiate in vitro using the Cell Carrier 3D glass scaffold into tissues that have similar characteristics to embryonic mesoderm and endoderm layers. These data would support the use of these cells, which are derived from an easily accessible source and can be used in future regeneration protocols for many tissue types that differentiate from the three embryonic layers.     

Transplantation of male germ line stem cells restores fertility in infertile mice

Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.     

人体牙髓干细胞的分离与鉴定

目的探讨牙髓干细胞（DPSC）对牙周病,外伤及肿瘤等造成下颌骨缺损、口腔软组织与神经损伤的修复治疗作用。方法本研究利用组织块培养法分离出人体DPSC,用流式细胞仪进行了鉴定,并进行DPSC成骨、成脂、成神经的分化研究。结果分离出3株DPSC,流式细胞分析表明DPSC表达CD73和CD90标志物,但不表达生血干细胞标志物CD34。用茜素红染色表明DPSC能分化成骨细胞,油红O染色表明DPSC能分化成脂肪细胞,免疫免疫荧光染色表明DPSC分化的细胞表达神经细胞特异标志物TUJ1。结论组织块培养能够高效快速分离表达CD73和CD90的DPSC,在体外诱导条件下DPSC能分化为成骨细胞、脂肪细胞和神经细胞,此研究为DPSC在治疗和修复骨组织缺损和神经损伤中的临床应用提供了实验依据。     

Folic acid-mediated mitochondrial activation for protection against oxidative stress in human dental pulp stem cells derived from deciduous teeth

Enzymatic antioxidant systems, mainly involving mitochondria, are critical for minimizing the harmful effects of reactive oxygen species, and these systems are enhanced by interactions with nonenzymatic antioxidant nutrients. Because fetal growth requires extensive mitochondrial respiration, pregnant women and fetuses are at high risk of exposure to excessive reactive oxygen species. The enhancement of the antioxidant system, e.g., by nutritional management, is therefore critical for both the mother and fetus. Folic acid supplementation prevents homocysteine accumulation and epigenetic dysregulation associated with one-carbon metabolism. However, few studies have examined the antioxidant effects of folic acid for healthy pregnancy outcomes. The purpose of this study was to elucidate the association between the antioxidant effect of folic acid and mitochondria in undifferentiated cells during fetal growth. Neural crest-derived dental pulp stem cells of human exfoliated deciduous teeth were used as a model of undifferentiated cells in the fetus. Pyocyanin induced excessive reactive oxygen species, resulting in a decrease in cell growth and migration accompanied by mitochondrial fragmentation and inactivation in dental pulp stem cells. This damage was significantly improved by folic acid, along with decreased mitochondrial reactive oxygen species, PGC-1α upregulation, DRP1 downregulation, mitochondrial elongation, and increased ATP production. Folic acid may protect undifferentiated cells from oxidative damage by targeting mitochondrial activation. These results provide evidence for a new benefit of folic acid in pregnant women and fetuses.     

Differentiation of mesenchymal stem cells derived from pancreatic islets and bone marrow into islet-like cell phenotype

Background

Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself.

Methodology/Principal Findings

In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin.

Conclusions/Significance

Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.     

Isolation and characterization of neural crest-derived stem cells from dental pulp of neonatal mice

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues.     

Stage-specific embryonic antigen-4 identifies human dental pulp stem cells

Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4- cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells.     

Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

Background  

We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells.     

Combination stem cell therapy using dental pulp stem cells and human umbilical vein endothelial cells for critical hindlimb ischemia

Narrowing of arteries supplying blood to the limbs provokes critical hindlimb ischemia (CLI). Although CLI results in irreversible sequelae, such as amputation, few therapeutic options induce the formation of new functional blood vessels. Based on the proangiogenic potentials of stem cells, in this study, it was examined whether a combination of dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) could result in enhanced therapeutic effects of stem cells for CLI compared with those of DPSCs or HUVECs alone. The DPSCs+ HUVECs combination therapy resulted in significantly higher blood flow and lower ischemia damage than DPSCs or HUVECs alone. The improved therapeutic effects in the DPSCs+ HUVECs group were accompanied by a significantly higher number of microvessels in the ischemic tissue than in the other groups. In vitro proliferation and tube formation assay showed that VEGF in the conditioned media of DPSCs induced proliferation and vessel-like tube formation of HUVECs. Altogether, our results demonstrated that the combination of DPSCs and HUVECs had significantly better therapeutic effects on CLI via VEGF-mediated crosstalk. This combinational strategy could be used to develop novel clinical protocols for CLI proangiogenic regenerative treatments.     

The role of asporin in mineralization of human dental pulp stem cells

Human adult dental pulp stem cells (hDPSCs) are a unique precursor population isolated from postnatal dental pulp and have the ability to regenerate a reparative dentin-like complex. In this study, we investigated the role of Asporin in hDPSCs, which was identified as a matrix protein in our previous dentin proteomic analysis. We isolated a clonogenic, highly proliferative population of cells from adult human dental pulp. These isolated hDPSCs were confirmed by fluorescence activated cell sorting (FACS) using stem cell-specific markers and have shown multilineage differentiation potential. The localization of Asporin was identified by immunohistochemistry in the globular calcification region in the junction of predentin and dentin. The gene and protein expression levels of Asporin were enhanced at the early stage of and then reduced during the late stage of differentiation of hDPSCs in mineralization media. ASPN knock-down using a lentiviral system suppressed the mineralization of hDPSCs. These results suggest that ASPN plays positive roles in the mineralization of hDPSCs and predentin to dentin.