Comparing the osteogenic potential of schneiderian membrane and dental pulp mesenchymal stem cells: an in vitro study

Cell and Tissue Banking - Mesenchymal stem cells, being characterized by high self-renewal capacity and multi-lineage differentiation potential, are widely used in regenerative medicine especially...     

Integration of neuronally predifferentiated human dental pulp stem cells into rat brain in vivo

Pluripotency and their neural crest origin make dental pulp stem cells (DPSCs) an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the integration of transplanted DPSC derived neuronal pecursors into the central nervous system. To address this issue, neuronally predifferentiated DPSCs, labeled with a vital cell dye Vybrant DiD were introduced into postnatal rat brain. DPSCs were transplanted into the cerebrospinal fluid of 3-day-old male Wistar rats. Cortical lesion was induced by touching a cold (−60 °C) metal stamp to the calvaria over the forelimb motor cortex. Four weeks later cell localization was detected by fluorescent microscopy and neuronal cell markers were studied by immunohistochemistry. To investigate electrophysiological properties of engrafted, fluorescently labeled DPSCs, 300 μm-thick horizontal brain slices were prepared and the presence of voltage-dependent sodium and potassium channels were recorded by patch clamping.Predifferentiated donor DPSCs injected into the cerebrospinal fluid of newborn rats migrated as single cells into a variety of brain regions. Most of the cells were localized in the normal neural progenitor zones of the brain, the subventricular zone (SVZ), subgranular zone (SGZ) and subcallosal zone (SCZ). Immunohistochemical analysis revealed that transplanted DPSCs expressed the early neuronal marker N-tubulin, the neuronal specific intermediate filament protein NF-M, the postmitotic neuronal marker NeuN, and glial GFAP. Moreover, the cells displayed TTX sensitive voltage dependent (VD) sodium currents (I_Na) and TEA sensitive delayed rectifier potassium currents (K_DR). Four weeks after injury, fluorescently labeled cells were detected in the lesioned cortex. Neurospecific marker expression was increased in DPSCs found in the area of the cortical lesions compared to that in fluorescent cells of uninjured brain. TTX sensitive VD sodium currents and TEA sensitive K_DR significantly increased in labeled cells of the cortically injured area. In conclusion, our data demonstrate that engrafted DPSC-derived cells integrate into the host brain and show neuronal properties not only by expressing neuron-specific markers but also by exhibiting voltage dependent sodium and potassium channels. This proof of concept study reveals that predifferentiated hDPSCs may serve as useful sources of neuro- and gliogenesis in vivo, especially when the brain is injured.     

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。     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

Treatment of stress urinary incontinence with adipose tissue-derived stem cells

Background aimsEffective treatment for stress urinary incontinence (SUI) is lacking. This study investigated whether transplantation of adipose tissue-derived stem cells (ADSC) can treat SUI in a rat model.MethodsRats were induced to develop SUI by postpartum vaginal balloon dilation and bilateral ovariectomy. ADSC were isolated from the peri-ovary fat, examined for stem cell properties, and labeled with thymidine analog BrdU or EdU. Ten rats received urethral injection of saline as a control. Twelve rats received urethral injection of EdU-labeled ADSC and six rats received intravenous injection of BrdU-labeled ADSC through the tail vein. Four weeks later, urinary voiding function was assessed by conscious cystometry. The rats were then killed and their urethras harvested for tracking of ADSC and quantification of elastin, collagen and smooth muscle contents.ResultsCystometric analysis showed that eight out 10 rats in the control group had abnormal voiding, whereas four of 12 (33.3%) and two of six (33.3%) rats in the urethra-ADSC and tail vein-ADSC groups, respectively, had abnormal voiding. Histologic analysis showed that the ADSC-treated groups had significantly higher elastin content than the control group and, within the ADSC-treated groups, rats with normal voiding pattern also had significantly higher elastin content than rats with voiding dysfunction. ADSC-treated normal-voiding rats had significantly higher smooth muscle content than control or ADSC-treated rats with voiding dysfunction.ConclusionsTransplantation of ADSC via urethral or intravenous injection is effective in the treatment and/or prevention of SUI in a pre-clinical setting.     

In vitro differentiation and mineralization of human dental pulp cells induced by dentin extract

Summary In this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], β-glycerophosphate [β-GP]) and MS+DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS+DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, β-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.     

Increased strength in the Col-Tgel induces apoptosis in the human dental pulp stem cells: 3D culturing of human dental pulp stem cells at different strengths of collagen

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9–1.5 kPa and 14–20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.     

Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells

Dental pulp stem cells (DPSCs) are an attractive alternative mesenchymal stem cell (MSC) source because of their isolation simplicity compared with the more invasive methods associated with harvesting other MSC sources. However, the isolation method to be favored for obtaining DPSC cultures remains under discussion. This study compares the stem cell properties and multilineage differentiation potential of DPSCs obtained by the two most widely adapted isolation procedures. DPSCs were isolated either by enzymatic digestion of the pulp tissue (DPSC-EZ) or by the explant method (DPSC-OG), while keeping the culture media constant throughout all experiments and in both isolation methods. Assessment of the stem cell properties of DPSC-EZ and DPSC-OG showed no significant differences between the two groups with regard to proliferation rate and colony formation. Phenotype analysis indicated that DPSC-EZ and DPSC-OG were positive for CD29, CD44, CD90, CD105, CD117 and CD146 expression without any significant differences. The multilineage differentiation potential of both stem cell types was confirmed by using standard immuno(histo/cyto)chemical staining together with an in-depth ultrastructural analysis by means of transmission electron microscopy. Our results indicate that both DPSC-EZ and DPSC-OG could be successfully differentiated into adipogenic, chrondrogenic and osteogenic cell types, although the adipogenic differentiation of both stem cell populations was incomplete. The data suggest that both the enzymatic digestion and outgrowth method can be applied to obtain a suitable autologous DPSC resource for tissue replacement therapies of both bone and cartilage.     

In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells

Cardiomyocyte loss in the ischemically injured human heart often leads to irreversible defects in cardiac function. Recently, cellular cardiomyoplasty with mesenchymal stem cells, which are multipotent cells with the ability to differentiate into specialized cells under appropriate stimuli, has emerged as a new approach for repairing damaged myocardium. In the present study, the potential of human umbilical cord-derived mesenchymal stem cells to differentiate into cells with characteristics of cardiomyocyte was investigated. Mesenchymal stem cells were isolated from endothelial/subendothelial layers of the human umbilical cords using a method similar to that of human umbilical vein endothelial cell isolation. Isolated cells were characterized by transdifferentiation ability to adipocytes and osteoblasts, and also with flow cytometry analysis. After treatment with 5-azacytidine, the human umbilical cord-derived mesenchymal stem cells were morphologically transformed into cardiomyocyte-like cells and expressed cardiac differentiation markers. During the differentiation, cells were monitored by a phase contrast microscope and their morphological changes were demonstrated. Immunostaining of the differentiated cells for sarcomeric myosin (MF20), desmin, cardiac troponin I, and sarcomeric alpha-actinin was positive. RT-PCR analysis showed that these differentiated cells express cardiac-specific genes. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure and typical sarcomers. These observations confirm that human umbilical cord-derived mesenchymal stem cells can be chemically transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty.     

Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.     

In vitro endothelial potential of human UC blood-derived mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (MSC) possess powerful ex vivo expansion and versatile differentiation potential, placing themselves at the forefront of the field of stem cell-based therapy and transplantation. Of high clinical relevance is the endothelial differentiation potential of MSC, which can be used to treat various forms of ischemic vascular disease. METHODS: We investigated whether human umbilical cord blood (UCB)-derived MSC are able to differentiate in vitro along an endothelial lineage, by using flow cytometry, RT-PCR and immunofluorescence analyzes, as well as an Ab array method. RESULTS: When the cells were incubated for up to 3 weeks in the presence of VEGF, EGF and hydrocortisone, they began to express a variety of endothelial lineage surface markers, such as Flk-1, Flt-1, VE-Cadherin, vWF, VCAM-1, Tie-1 and Tie-2, and to secrete a specific set of cytokines. Differentiated cells were also found to be able to uptake low-density lipoprotein and form a tubular network structure. DISCUSSION: These observations have led us to conclude that UCB-derived MSC retain endothelial potential that is suitable for basic and clinical studies aimed at the development of vasculature-directed regenerative medicine.     

Effects of doxorubicin on human dental pulp cells in vitro

There is substantial information concerning the effects of continuous exposure to supratherapeutic or therapeutic concentrations of doxorubicin on human molar pulpal cells; the effects of continuous exposure to subtherapeutic concentrations of this agent are undetermined. To this end, we studied the proliferation of human fibroblasts and pulpal cells and their pattern of mineralized nodule deposition in vitro. Cell proliferation was assessed at 1, 3, 5, and 7 days from populations with either no exposure (control) or exposure to 10⁻⁶–10⁻⁹ mol/L doxorubicin. Mineralized nodule deposition and calcium-45 incorporation were assessed at 7 and 21 days of culture. Data were compared by factorial ANOVA and a post-hoc Tukey test. 10⁻⁶ and 10⁻⁷ mol/L doxorubicin significantly reduced the total number of viable pulpal cells in cultures from days 1 to 3 (p < 0.05); doxorubicin 10⁻⁶–10⁻⁹ mol/L significantly inhibited cell proliferation (p < 0.05) and DNA synthesis 5 days after plating (p < 0.001). After 21 days, doxorubicin 10⁻⁶–10⁻⁸ mol/L significantly decreased calcium-45 incorporation into pulpal cultures (p < 0.001); all dilutions significantly reduced the number of mineralized nodules within the 21-day pulpal cultures (p < 0.05). In addition, all dilutions of doxorubicin significantly inhibited fibroblast cell proliferation and incorporation of [³H]thymidine. In contrast, the fibroblast cultures did not produce mineralized nodules, suggesting that the mineralized nodules within the pulpal cell cultures did not result from dystrophic calcification. Thus, exposure to subtheraputic doxorubicin concentrations has potential adverse effects on mineralized tissue formation within the pulp, which could affect the rates of reparative dentin deposition within the tooth pulps of patients receiving this chemotherapeutic agent.     

Vascular endothelial growth factor enhances in vitro proliferation and osteogenic differentiation of human dental pulp stem cells

Mesenchymal stem cells (MSC), isolated from dental tissues, are largely studied for future application in regenerative dentistry. In this study, we used MSC obtained from human dental pulp (DPSC) of normal impacted third molars that, when cultured in lineage-specific inducing media, differentiate into osteoblasts and adipocytes (evaluated by Alizarin Red S and Red Oil O stainings, respectively), thus showing a multipotency. We confirmed that DPSC, grown under undifferentiating conditions, are negative for hematopoietic (CD45, CD31, CD34, CD144) and positive for mesenchymal (CD29, CD90, CD105, CD166, CD146, STRO-1) markers, that underwent down-regulation when cells were grown in osteogenic medium for 3 weeks. In this condition, they also exhibit an increase in the expression of osteogenic markers (RUNX-2, alkaline phosphatase) and extracellular calcium deposition, whereas the expression of receptors (VEGFR-1 and -2) for vascular endothelial growth factors (VEGF) and related VEGF binding proteins was similar to that found in undifferentiated DPSC. Exposure of DPSC growing under undifferentiating or osteogenic conditions to VEGF-A165 peptide (10-40 ng/ml) for 8 days dose- and time-dependently increased the number of proliferating cells without inducing differentiation towards endothelial lineage, as evaluated by the lack of expression of specific markers (CD31, CD34, CD144). Additionally, exposure of DPSC cultured in osteogenic medium to VEGF-A165 for a similar period enhanced cell differentiation towards osteoblasts as evaluated after 14 and 21 days by Alizarin Red S staining and alkaline phosphatase activity quantification. These findings may have clinical implications possibly facilitating tissue repair and remodeling.     

In vitro and in vivo neurogenic potential of mesenchymal stem cells isolated from different sources

Regenerative medicine is an evolving interdisciplinary topic of research involving numerous technological methods that utilize stem cells to repair damaged tissues. Particularly, mesenchymal stem cells (MSCs) are a great tool in regenerative medicine because of their lack of tumorogenicity, immunogenicity and ability to perform immunomodulatory as well as anti-inflammatory functions. Numerous studies have investigated the role of MSCs in tissue repair and modulation of allogeneic immune responses. MSCs derived from different sources hold unique regenerative potential as they are self-renewing and can differentiate into chondrocytes, osteoblasts, adipocytes, cardiomyocytes, hepatocytes, endothelial and neuronal cells, among which neuronal-like cells have gained special interest. MSCs also have the ability to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation. In this review we focus on neural differentiation potential of MSCs isolated from different sources and how certain growth factors/small molecules can be used to derive neuronal phenotypes from MSCs. We also discuss the efficacy of MSCs when transplanted in vivo and how they can generate certain neurons and lead to relief or recovery of the diseased condition. Furthermore, we have tried to evaluate the appropriate merits of different sources of MSCs with respect to their propensity towards neurological differentiation as well as their effectiveness in preclinical studies.     

In vitro and in vivo characterization of neural stem cells

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.