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Wharton's jelly derived-mesenchymal stem cells (WJ-MSCs) have a same developmental origin with primordial germ cells. WJ-MSCs perhaps differentiate into oocyte and germ like-cells (OLCs/GLCs) in the presence of appropriate inducers. Human follicular fluid (FF) and cumulus cells conditioned medium (CCM) are naturally rich sources for oocyte development. The aim of this study was to evaluate WJ-MSCs potential for differentiating into OLCs and GLCs exposed to FF and CCM. WJ-MSCs were cultured in two different induction media (10% FF, 10% CCM) for 21 days. Morphological changes and expression of developmental genes were evaluated on days 0, 7, 14 and 21 of culture. Also, on 21st day of culture, the expression of oocyte and germ cell proteins investigated using immunofluorescence staining. Appearance of round shaped cells from 7th day onwards indicated that WJ-MSCs can differentiate into OLCs when exposed to FF and CCM. The size of produced OLCs and expression of oocyte specific genes and proteins were increased more positively in FF group rather than CCM group. Although, WJ-MSCs could differentiate into OLCs by FF and CCM, however, the induction potential of FF for producing OLCs was better than CCM.

To differentiate stem cells into endothelial cells, vascular endothelia growth factors (VEGF) serve as the major signal for stimulating the cells. However, there are other cytokines or growth factors associated with endothelial cell development and differentiation. Human platelet lysate (hPL) has been a promising reagent in cell-based therapy since it is considered as a source of bioactive molecules and growth factors. The aim of this study was to investigate the in vitro differentiation of human amniotic fluid mesenchymal stem cells (hAF-MSCs) into endothelial-like cells under hPL together with VEGF or endothelial cell growth medium 2 (EGM-2), a commercially induced medium. In this study, hAF-MSCs were isolated from human amniotic fluid cells (hAFCs) using the direct adherence method. The cells expressed CD44, CD73, CD90, and HLA-ABC at high levels and expressed Oct-4 (octamer-binding transcription factor 4) at low levels. The cells were negative for CD31, CD34, CD45, CD105 and HLA-DR. This study found that hAF-MSCs induced with hPL and VEGF had the ability to differentiate into endothelial-like cells by presenting endothelial specific markers (vWF, VEGFR2 and eNOS), forming a network-like structure on Matrigel, and producing nitric oxide (NO). This outcome was similar to those of experiments involving EGM-2 induced cells. The present findings indicate that hPL + VEGF can induce hAF-MSCs to express endothelial cell characteristics. Our findings represent an important step forward in the development of a clinically compliant process for the production of endothelial cell-derived hAF-MSCs, and their subsequent testing in future clinical trials.

Objectives: The objective of this study was to investigate iron sucrose labeling in mesenchymal stem cell (MSCs) tracking. Background: Adipose-derived mesenchymal stem cell-based therapy is a promising strategy for promoting musculoskeletal repair. Methods: Iron sucrose-labeled adipose-derived mesenchymal stem cells (IS-labeled ASCs) were tracked using T2-and T2∗-weighted sequences by 1.5 and 3 T MRI in an in vitro model. ASCs were isolated from cosmetic liposuction specimens. ASCs from passages 4–6 were labeled with iron sucrose (Venofer®) which was added to the cell culture medium. Pre- and post-iron sucrose labeled ASCs were evaluated for cell surface immunophenotypes. Cell viability as well as chondrogenic, adipogenic and osteogenic differentiation of IS-labeled-ASCs were evaluated. The IS-labeled ASCs were titrated into microtubes at 1 × 103, 1 × 104, 1 × 105 and 1 × 106 cells/ml/microtube and their intensities were determined by 1.5 and 3T MRI using T2-and T2∗-weighted sequences. Results: The expression markers of IS-labeled ASCs from flow cytometry were equivalent to control. The mean cell viability was 97.73 ± 2.06%. Cell differentiations of IS-labeled ASCs were confirmed in each lineage using specific staining solutions. T2∗-weighted sequences (T2∗) were able to detect iron sucrose labeled-ASCs at a minimum of 1 × 105 cells/ml/microtube using 1.5 and 3T MRI, but the detection sensitivity was lower with T2-weighted sequences (T2). Conclusions: Iron sucrose incubation is a safe alternative method for ASCs labeling and tracking using MRI following treatment. Clinicians and researchers should be able to visualize the location of ASCs engraftment without secondary surgical investigation involving tissue sampling.

Background: Evaluations for the tumorigenicity of transplantation of stem cell products is mandatory for clinical application. It is of importance to establish a system to accurately quantify contaminated tumorigenic cells regardless of the format of stem cell product. In the present report, we aimed to examine the accuracy of the quantification of tumorigenic cell numbers with commonly used 2 methods, quantitative polymerase chain reaction (qPCR) and flow cytometry (FCM) using experimental models of stem cell products spiked with tumorigenic cells. Methods: Human mesenchymal stem cells (hMSCs) and melanoma Mewo-Luc cells constitutively expressing luciferase were used. We stained Mewo-Luc cells with a cell linker then spiked onto hMSC suspensions and hMSC sheets. We validated the accuracy of 10-fold serial dilution technique for Mewo-Luc cell suspension using a Coulter counter. The samples spiked with Mewo-Luc cells were subjected to qPCR and FCM analyses, respectively for the quantification of Mewo-Luc cells. Results: Ten-fold serial dilutions of Mewo-Luc cells were performed accurately with small deviation. In samples spiked with or less than 100 cells in hMSC suspensions, and samples spiked with or less than 1,000 cells in hMSC sheets showed significantly higher cell numbers in calculations by FCM, respectively (suspensions; qPCR vs FCM: 100 cells: 59 ± 25 vs 232 ± 35 cells, p = 0.022/10 cells: 21 ± 7 vs 114 ± 27 cells, p = 0.030, sheets; qPCR vs FCM: 1,000 cells: 1723 ± 258 vs 5810 ± 878 cells, p = 0.012/100 cells: 110 ± 18 vs 973 ± 232 cells, p = 0.012/10 cells: 20 ± 6 vs 141 ± 36 cells, p = 0.030). Conclusion: Differences in accuracy between quantification methods should be considered in designing a tumorigenicity study model.

Matrix Gla protein (MGP), a modulator of the BMP-SMAD signals, inhibits arterial calcification in a Glu γ-carboxylation dependent manner but the role of MGP highly expressed in a subset of bone marrow (BM) mesenchymal stem/stromal cells is unknown. Here we provide evidence that MGP might be a niche factor for both normal and malignant myelopoiesis. When mouse BM hematopoietic cells were cocultured with mitomycin C-treated BM stromal cells in the presence of anti-MGP antibody, growth of hematopoietic cells was reduced by half, and maintenance of long-term culture-initiating cells (LTC-ICs) was profoundly attenuated. Antibody-mediated blockage of MGP also inhibited growth (by a fifth) and cobblestone formation (by half) of stroma-dependent MB-1 myeloblastoma cells. MGP was undetectable in normal hematopoietic cells but was expressed in various mesenchymal cells and was aberrantly high in MB-1 cells. MGP and bone morphogenetic protein (BMP)-4 were co-induced in stromal cells cocultured with both normal hematopoietic cells and MB-1 myeloblastoma cells in an oscillating several days-periodic manner. BMP-2 was also induced in stromal cells cocultured with normal hematopoietic cells but was barely expressed when cocultured with MB-1 cells. GST-pulldown and luciferase reporter assays showed that uncarboxylated MGP interacted with BMP-4 and that anti-MGP antibody abolished this interaction. LDN-193189, a selective BMP signaling inhibitor, inhibited growth and cobblestone formation of MB-1 cells. The addition of warfarin, a selective inhibitor of vitamin K-dependent Glu γ-carboxylation, did not affect MB-1 cell growth, suggesting that uncarboxylated MGP has a biological effect in niche. These results indicate that MGP may maintain normal and malignant hematopoietic progenitor cells, possibly by modulating BMP signals independently of Glu γ-carboxylation. Aberrant MGP by leukemic cells and selective induction of BMP-4 relative to BMP-2 in stromal cells might specify malignant niche.

Advances in prenatal molecular testing have made it possible to diagnose most genetic disorders early in gestation. In utero mesenchymal stem cell (MSC) therapy can be a powerful tool to cure the incurable. With this in mind, this method could ameliorate potential physical and functional damage. However, the presence of maternal T cells trafficking in the fetus during pregnancy is thought to be the major barrier to achieving the engraftment into the fetus. We investigated the possibility of using maternal adipose-derived stem cells (ADSCs) for in utero transplantation to improve engraftment, thus lowering the risk of graft rejection. Herein, fetal brain engraftment using congenic and maternal ADSC grafts was examined via in utero stem cell transplantation in a mouse model. ADSCs were purified using the mesenchymal stem cell markers, PDGFRα, and Sca-1 via fluorescence-activated cell sorting. The PDGFRα+Sca-1+ ADSCs were transplanted into the fetal intracerebroventricular (ICV) at E14.5. The transplanted grafts grew for at least 28 days after in utero transplantation with PDGFRα+Sca-1+ ADSC, and mature neuronal markers were also detected in the grafts. Furthermore, using the maternal sorted ADSCs suppressed the innate immune response, preventing the infiltration of CD8 T cells into the graft. Thus, in utero transplantation into the fetal ICV with the maternal PDGFRα+Sca-1+ ADSCs may be beneficial for the treatment of congenital neurological diseases because of the ability to reduce the responses after in utero stem cell transplantation and differentiate into neuronal lineages.

Human dental pulp stem cells (hDPSCs) are promising source of cells for numerous and varied regenerative medicine applications as those possess high proliferation potential with multilineage differentiation capacity compare to other sources of adult stem cells; therefore, hDPSCs could be the good source for autologous transplantation in tissue engineering and regenerative medicine. In this study stem cells were isolated from dental pulp and were characterised by flowcytometry and immunocytochemistry. The controlled cells as well as, 7-day cultured cells were positive for transcription factors, OCT 4 and SOX 2 thatconfirmed isolated cellsasmesenchymal stem cells (MSCs). These cells showed positive expression for CD 19, CD 73, CD 90, CD 105 and are negative for CD 34, CD 45. Viability of hDPSCS were studied by trypan blue (TB) staining and fluorescent microscopic study. After 7 days of passaging by using several growth factors, cells express neural cell markers oligodendrocyte and glial fibrillary acidic protein. Specifically, osteocytes were grown from dental pulp MSCSsin vitro with the help of growth factors, dexamethasone, ascorbic acid-2- phosphate and β-glycerophosphate whereas, adipocytes were grown with indomethacin, 3-isobutyl-1-methylxanthine and insulin. Osteocytes and adipocytes were characterized by von Kossa and Oil red O staining, respectively. Chromosomal analysis of dental pulp-MSCs was done for qualitative assessment of MSCs. Karyotyping indicated diploid chromosome number in dental pulp derived MSCs. In vitro grown osteocytes could be used for bone fracture reunion cases, and adipocytes could be used for further research purposes.

Background: Human multipotent adult progenitor cells (MAPC®) are an emerging therapy for traumatic brain injury (TBI); however, clinically translating a therapy involves overcoming many factors in vivo which are not present in pre-clinical testing. In this study we examined clinical parameters in vitro that may impact cell therapy efficacy. Methods: MAPC were infused through varying gauged needles and catheters with and without chlorhexidine, and their viability tested with trypan blue exclusion. MAPC were co-cultured with phenytoin and celecoxib at relevant clinical concentrations for 1 h and 24 h. Anti-inflammatory potency was tested using a stimulated rat splenocyte co-culture and ELISA for TNF-α production. MAPC were cultured under different osmolar concentrations and stained with propidium iodide for viability. Anti-inflammatory potency was tested by co-culture of MAPC with naïve lymphocytes activated by CD3/CD28 beads, and Click-iT® Plus EdU was used to quantify proliferation by flow cytometry. Results: The mean viability of the MAPC infused via needles was 95 ± 1%; no difference was seen with varying flow rate, but viability was notably reduced by chlorhexidine. MAPC function was not impaired by co-culture with phenytoin, celecoxib, or combination with both. Co-culture with phenytoin showed a decrease in TNF-α production as compared to the MAPC control. MAPC cultured at varying osmolar concentrations all had viabilities greater than 90% with no statistical difference between them. Co-culture of MAPC with CD3/CD28 activated PBMCs showed a significant reduction in proliferation as measured by EdU uptake. Discussion: Needle diameter, phenytoin, celecoxib, and a relevant range of osmolarities do not impair MAPC viability or anti-inflammatory potency in vitro.

Aims: The aim of this study was to present a new method for removing Sodium dodecyl sulfate (SDS) detergent from decellularized bovine pericardium using vacuum. Materials and Methods: The cows’ pericardia were collected and decellularized. The samples were incubated with SDS1% for 48 h at 40 °C. To perform vacuum washing (VW: negative pressure was used to wash and remove detergents), every decellularized tissue was cut in 75mm diameter and fixed via a stainless-steel ring with 60mm diameter in the center of filtration Buchner Funnel which was connected to glass filtration flask The system was connected to a vacuum pump by a hose, and a negative pressure of -100 mmHg was applied for 15 min. Then, the samples were shaken and washed at 40-rpm in 100 ml of distilled water for 45 min. This process was repeated for samples of each group (6 times for sample VW6h, 12 times for sample VW12h, and 24 times for sample VW24h). At the end of every cycle, the effluent was collected to take a sample for SDS measurement. The normal washing (NW) group containing distilled water (NWd) and PBS (Phosphate buffered saline) (NWp) were used to wash and remove detergents. SDS measurements, MTT Assay, histological and tensile test, to compare two methods were used. Results: The highest SDS in the effluent was in groups VW12h and VW24h (P ≤ 0.001) and the lowest residual SDS in scaffold was in two groups of VW12h and VW24h (P ≤ 0.001). MTT assay showed that cell survival in the VW12h and VW24h groups was higher than other groups and there' was no significant difference between cell survival in the VW12h and VW24h groups. Histological study showed destruction of tissue in the VW24h group. The results of the tensile test were shown that the native group had the highest module and the lowest amount was the VW24h sample which was reported with P ≤ 0.001 significance for all groups. Conclusion: VW12h can be used as an effective method for SDS removal from decellularized pericardium which morphologically demonstrated a good structure in ECM.

Whilst the potential of neural stem cell (NSC)-based treatment is recognized worldwide and seems to offer a promising therapeutic option for stroke treatments, there is currently no full understanding regarding the effects of hypoxic and baicalein-enriched fraction (BEF) preconditioning approaches on the therapeutic potential of these cells for stroke. The potential of preconditioned NSC can be determined based on the expression of several key neuroprotective genes using qRT-PCR technique. However, prior to that, it is imperative and extremely important to carefully select reference gene(s) for accurate qRT-PCR data normalization to avoid error in data interpretation. This study aimed to evaluate the stability of ten candidate reference genes via comprehensive analysis using three algorithms software: geNorm, NormFinder and BestKeeper. Our results revealed that HPRT1 and RPL13A were the most reliable reference genes for BEF-preconditioned NSCs, but ironically, HPRT1 was ranked as the least stable reference gene for hypoxic-preconditioned NSCs. On the other hand, RPLP1 and RPL13A were selected as the most stably expressed pair of reference genes for hypoxic-preconditioned NSCs. In conclusion, this study has pointed out the importance of identifying valid reference genes and has presented the first significant validation on best reference genes recommended for qRT-PCR study involves NSC preconditioned with hypoxia or with BEF extracted from Oroxylum indicum medicinal plant.

Demineralized bone allografts and mesenchymal stromal cells have been used to promote bone regeneration. However, the degree to which cortical bone should be demineralized for use in combination with adipose-derived mesenchymal stromal cells (Ad-MSCs) remains to be clarified. In this study, the in vitro osteogenic ability of Ad-MSCs on allografts was investigated in relation to the extent of demineralization. Three treatment groups were established by varying exposure time to 0.6 N HCL: partially demineralized (PDB; 12 h), fully demineralized (FDB; 48 h), and non-demineralized bone (NDB; 0 h, as a control). Allografts were prepared as discs 6 mm in diameter for in vitro evaluation, and their demineralization and structure were evaluated by micro-computed tomography and scanning electron microscopy. Ad-MSC adhesion and proliferation were measured by MTS assay, and osteogenesis-related gene expression was assessed by quantitative reverse transcription polymerase chain reaction. PDB and FDB demineralization rates were 57.13 and 92.30%, respectively. Moreover, Ad-MSC adhesion rates on NDB, PDB, and FDB were 53.41, 60.65, and 61.32%, respectively. Proliferation of these cells on FDB increased significantly after 2 days of culture compared to the other groups (P < 0.05). Furthermore, expression of the osteogenic genes ALP, BMP-7, and TGF-β in the FDB group on culture day 3 was significantly elevated in comparison to the other treatments. Given its biocompatibility and promotion of the osteogenic differentiation of Ad-MSCs, our results suggest that FDB may be a suitable scaffold for use in the repair of bone defects.

The development of central nervous system is a highly coordinated and complex process. Any alteration of this process can lead to disturbances in the structure and function of the brain, which can cause deficits in neurological development, resulting in neurodevelopmental disorders, including, for example, autism or attention-deficit hyperactivity disorder. Exposure to certain chemicals during the fetal period and childhood is known to cause developmental neurotoxicity and has serious consequences that persist into adult life. For regulatory purposes, determination of the potential for developmental neurotoxicity is performed according the OECD Guideline 426, in which the test substance is administered to animals during gestation and lactation. However, these animal models are expensive, long-time consuming and may not reflect the physiology in humans; that makes it an unsustainable model to test the large amount of existing chemical products, hence alternative models to the use of animals are needed. One of the most promising methods is based on the use of stem cell technology. Stem cells are undifferentiated cells with the ability to self-renew and differentiate into more specialized cell types. Because of these properties, these cells have gained increased attention as possible therapeutic agents or as disease models. Here, we provide an overview of the current models both animal and cellular, available to study developmental neurotoxicity and review in more detail the usefulness of human stem cells, their properties and how they are becoming an alternative to evaluate and study the mechanisms of action of different environmental toxicants., Competing Interests: The authors declare no conflict of interest., (© 2020 Published by Elsevier Ltd.)

Wharton's jelly derived-mesenchymal stem cells (WJ-MSCs) have a same developmental origin with primordial germ cells. WJ-MSCs perhaps differentiate into oocyte and germ like-cells (OLCs/GLCs) in the presence of appropriate inducers. Human follicular fluid (FF) and cumulus cells conditioned medium (CCM) are naturally rich sources for oocyte development. The aim of this study was to evaluate WJ-MSCs potential for differentiating into OLCs and GLCs exposed to FF and CCM. WJ-MSCs were cultured in two different induction media (10% FF, 10% CCM) for 21 days. Morphological changes and expression of developmental genes were evaluated on days 0, 7, 14 and 21 of culture. Also, on 21 st day of culture, the expression of oocyte and germ cell proteins investigated using immunofluorescence staining. Appearance of round shaped cells from 7 th day onwards indicated that WJ-MSCs can differentiate into OLCs when exposed to FF and CCM. The size of produced OLCs and expression of oocyte specific genes and proteins were increased more positively in FF group rather than CCM group. Although, WJ-MSCs could differentiate into OLCs by FF and CCM, however, the induction potential of FF for producing OLCs was better than CCM., (© 2020 Published by Elsevier Ltd.)

To differentiate stem cells into endothelial cells, vascular endothelia growth factors (VEGF) serve as the major signal for stimulating the cells. However, there are other cytokines or growth factors associated with endothelial cell development and differentiation. Human platelet lysate (hPL) has been a promising reagent in cell-based therapy since it is considered as a source of bioactive molecules and growth factors. The aim of this study was to investigate the in vitro differentiation of human amniotic fluid mesenchymal stem cells (hAF-MSCs) into endothelial-like cells under hPL together with VEGF or endothelial cell growth medium 2 (EGM-2), a commercially induced medium. In this study, hAF-MSCs were isolated from human amniotic fluid cells (hAFCs) using the direct adherence method. The cells expressed CD44, CD73, CD90, and HLA-ABC at high levels and expressed Oct-4 (octamer-binding transcription factor 4) at low levels. The cells were negative for CD31, CD34, CD45, CD105 and HLA-DR. This study found that hAF-MSCs induced with hPL and VEGF had the ability to differentiate into endothelial-like cells by presenting endothelial specific markers (vWF, VEGFR2 and eNOS), forming a network-like structure on Matrigel, and producing nitric oxide (NO). This outcome was similar to those of experiments involving EGM-2 induced cells. The present findings indicate that hPL + VEGF can induce hAF-MSCs to express endothelial cell characteristics. Our findings represent an important step forward in the development of a clinically compliant process for the production of endothelial cell-derived hAF-MSCs, and their subsequent testing in future clinical trials., (© 2020 The Author(s).)

Objectives: The objective of this study was to investigate iron sucrose labeling in mesenchymal stem cell (MSCs) tracking., Background: Adipose-derived mesenchymal stem cell-based therapy is a promising strategy for promoting musculoskeletal repair., Methods: Iron sucrose-labeled adipose-derived mesenchymal stem cells (IS-labeled ASCs) were tracked using T2-and T2∗-weighted sequences by 1.5 and 3 T MRI in an in vitro model. ASCs were isolated from cosmetic liposuction specimens. ASCs from passages 4-6 were labeled with iron sucrose (Venofer®) which was added to the cell culture medium. Pre- and post-iron sucrose labeled ASCs were evaluated for cell surface immunophenotypes. Cell viability as well as chondrogenic, adipogenic and osteogenic differentiation of IS-labeled-ASCs were evaluated. The IS-labeled ASCs were titrated into microtubes at 1 × 10 3 , 1 × 10 4 , 1 × 10 5 and 1 × 10 6 cells/ml/microtube and their intensities were determined by 1.5 and 3T MRI using T2-and T2∗-weighted sequences., Results: The expression markers of IS-labeled ASCs from flow cytometry were equivalent to control. The mean cell viability was 97.73 ± 2.06%. Cell differentiations of IS-labeled ASCs were confirmed in each lineage using specific staining solutions. T2∗-weighted sequences (T2∗) were able to detect iron sucrose labeled-ASCs at a minimum of 1 × 10 5 cells/ml/microtube using 1.5 and 3T MRI, but the detection sensitivity was lower with T2-weighted sequences (T2)., Conclusions: Iron sucrose incubation is a safe alternative method for ASCs labeling and tracking using MRI following treatment. Clinicians and researchers should be able to visualize the location of ASCs engraftment without secondary surgical investigation involving tissue sampling., (© 2020 The Author(s).)

Background: Evaluations for the tumorigenicity of transplantation of stem cell products is mandatory for clinical application. It is of importance to establish a system to accurately quantify contaminated tumorigenic cells regardless of the format of stem cell product. In the present report, we aimed to examine the accuracy of the quantification of tumorigenic cell numbers with commonly used 2 methods, quantitative polymerase chain reaction (qPCR) and flow cytometry (FCM) using experimental models of stem cell products spiked with tumorigenic cells., Methods: Human mesenchymal stem cells (hMSCs) and melanoma Mewo-Luc cells constitutively expressing luciferase were used. We stained Mewo-Luc cells with a cell linker then spiked onto hMSC suspensions and hMSC sheets. We validated the accuracy of 10-fold serial dilution technique for Mewo-Luc cell suspension using a Coulter counter. The samples spiked with Mewo-Luc cells were subjected to qPCR and FCM analyses, respectively for the quantification of Mewo-Luc cells., Results: Ten-fold serial dilutions of Mewo-Luc cells were performed accurately with small deviation. In samples spiked with or less than 100 cells in hMSC suspensions, and samples spiked with or less than 1,000 cells in hMSC sheets showed significantly higher cell numbers in calculations by FCM, respectively (suspensions; qPCR vs FCM: 100 cells: 59 ± 25 vs 232 ± 35 cells, p = 0.022/10 cells: 21 ± 7 vs 114 ± 27 cells, p = 0.030, sheets; qPCR vs FCM: 1,000 cells: 1723 ± 258 vs 5810 ± 878 cells, p = 0.012/100 cells: 110 ± 18 vs 973 ± 232 cells, p = 0.012/10 cells: 20 ± 6 vs 141 ± 36 cells, p = 0.030)., Conclusion: Differences in accuracy between quantification methods should be considered in designing a tumorigenicity study model., (© 2020 The Author(s).)

Bone defect healing is markedly impaired in osteoporotic patient due to poor bone regeneration ability. Stromal cell derived factor-1α (SDF-1α) plays a pivotal role in the repair of various injured tissues including bone. Here, we definite that SDF-1α hydrogels potentiates in vivo osteogenesis of bone marrow-derived stromal stem cells (BMSCs) in osteoporosis. The characteristics of rat primary BMSCs including superficial markers by flow cytometry and multi-lineage differentiation by induction were determined. At different time intervals, the release media from the SDF-1α-releasing hydrogels were collected to identificate SDF-1α exhibited a sustained release profile and maintained its bioactivity after release from the hydrogels to stimulate chemotaxis of BMSCs in a time dependent manner. Bilateral alveolar defects were operated in ovariectomized (OVX) rats and repaired with systemic BMSCs transplantation with or without the hydrogels. Local administration of SDF-1α significantly enhanced BMSCs recruitment and promoted more bone regeneration as well as the expression of OCN and Runx2 compared with the effect of BMSCs transplantation alone. Moreover, after BMSCs transplantation with SDF-1α delivery, macrophage polarization was promoted toward the M2 phenotype, that is identified as an important symbol in tissue regeneration process. Taken together, local SDF-1α application enhances the efficacy of BMSCs transplantation therapy in osteoporotic bone healing, suggesting clinical potential of SDF-1α to serve as a therapeutic drug target for osteoporosis treatment., (© 2020 The Authors. Published by Elsevier Ltd.)

Organs and tissues, such as cartilage and limbs, are formed during development through an orchestration of growth factors that function as morphogens. Examples of growth factors include growth differentiation factor 5 (GDF5) and transforming growth factors beta 1 and 3 (TGFβ-1 and TGFβ-3) which can specify creation of more than one cell type after forming a concentration gradient in vivo . Here, we studied the impact of morphogen gradients during differentiation of induced pluripotent stem cells (iPSCs) into the chondrocyte lineage. Cell budding zones, consisting of condensed cell aggregates, were observed only in gradients of GDF5. T-box transcription factor 3 (TBX3) was detected specifically in the budding zones (ranging from 500-1,500 particles/μm 2 ) of nuclei and cell vesicles. A homogenous density of GDF5 of 900 particles/μm 2 on a surface induced budding and expression of TBX3 after five days in iPSCs. Therefore, we conclude that a gradient of GDF5, as well as the specific homogenous density of GDF5, support the induction of TBX3 in iPCSs. Moreover, differentiation of iPSCs first on GDF5 gradient or homogenous surfaces for five days and then in a three-dimensional structure for five weeks resulted in pellets that expressed TBX3., (© 2020 The Authors.)

Matrix Gla protein (MGP), a modulator of the BMP-SMAD signals, inhibits arterial calcification in a Glu γ-carboxylation dependent manner but the role of MGP highly expressed in a subset of bone marrow (BM) mesenchymal stem/stromal cells is unknown. Here we provide evidence that MGP might be a niche factor for both normal and malignant myelopoiesis. When mouse BM hematopoietic cells were cocultured with mitomycin C-treated BM stromal cells in the presence of anti-MGP antibody, growth of hematopoietic cells was reduced by half, and maintenance of long-term culture-initiating cells (LTC-ICs) was profoundly attenuated. Antibody-mediated blockage of MGP also inhibited growth (by a fifth) and cobblestone formation (by half) of stroma-dependent MB-1 myeloblastoma cells. MGP was undetectable in normal hematopoietic cells but was expressed in various mesenchymal cells and was aberrantly high in MB-1 cells. MGP and bone morphogenetic protein (BMP)-4 were co-induced in stromal cells cocultured with both normal hematopoietic cells and MB-1 myeloblastoma cells in an oscillating several days-periodic manner. BMP-2 was also induced in stromal cells cocultured with normal hematopoietic cells but was barely expressed when cocultured with MB-1 cells. GST-pulldown and luciferase reporter assays showed that uncarboxylated MGP interacted with BMP-4 and that anti-MGP antibody abolished this interaction. LDN-193189, a selective BMP signaling inhibitor, inhibited growth and cobblestone formation of MB-1 cells. The addition of warfarin, a selective inhibitor of vitamin K-dependent Glu γ-carboxylation, did not affect MB-1 cell growth, suggesting that uncarboxylated MGP has a biological effect in niche. These results indicate that MGP may maintain normal and malignant hematopoietic progenitor cells, possibly by modulating BMP signals independently of Glu γ-carboxylation. Aberrant MGP by leukemic cells and selective induction of BMP-4 relative to BMP-2 in stromal cells might specify malignant niche., (© 2020 The Author(s).)

Advances in prenatal molecular testing have made it possible to diagnose most genetic disorders early in gestation. In utero mesenchymal stem cell (MSC) therapy can be a powerful tool to cure the incurable. With this in mind, this method could ameliorate potential physical and functional damage. However, the presence of maternal T cells trafficking in the fetus during pregnancy is thought to be the major barrier to achieving the engraftment into the fetus. We investigated the possibility of using maternal adipose-derived stem cells (ADSCs) for in utero transplantation to improve engraftment, thus lowering the risk of graft rejection. Herein, fetal brain engraftment using congenic and maternal ADSC grafts was examined via in utero stem cell transplantation in a mouse model. ADSCs were purified using the mesenchymal stem cell markers, PDGFRα, and Sca-1 via fluorescence-activated cell sorting. The PDGFRα + Sca-1 + ADSCs were transplanted into the fetal intracerebroventricular (ICV) at E14.5. The transplanted grafts grew for at least 28 days after in utero transplantation with PDGFRα + Sca-1 + ADSC, and mature neuronal markers were also detected in the grafts. Furthermore, using the maternal sorted ADSCs suppressed the innate immune response, preventing the infiltration of CD8 T cells into the graft. Thus, in utero transplantation into the fetal ICV with the maternal PDGFRα + Sca-1 + ADSCs may be beneficial for the treatment of congenital neurological diseases because of the ability to reduce the responses after in utero stem cell transplantation and differentiate into neuronal lineages., (© 2020 The Author(s).)

Human dental pulp stem cells (hDPSCs) are promising source of cells for numerous and varied regenerative medicine applications as those possess high proliferation potential with multilineage differentiation capacity compare to other sources of adult stem cells; therefore, hDPSCs could be the good source for autologous transplantation in tissue engineering and regenerative medicine. In this study stem cells were isolated from dental pulp and were characterised by flowcytometry and immunocytochemistry. The controlled cells as well as, 7-day cultured cells were positive for transcription factors, OCT 4 and SOX 2 thatconfirmed isolated cellsasmesenchymal stem cells (MSCs). These cells showed positive expression for CD 19, CD 73, CD 90, CD 105 and are negative for CD 34, CD 45. Viability of hDPSCS were studied by trypan blue (TB) staining and fluorescent microscopic study. After 7 days of passaging by using several growth factors, cells express neural cell markers oligodendrocyte and glial fibrillary acidic protein. Specifically, osteocytes were grown from dental pulp MSCSs in vitro with the help of growth factors, dexamethasone, ascorbic acid-2- phosphate and β-glycerophosphate whereas, adipocytes were grown with indomethacin, 3-isobutyl-1-methylxanthine and insulin. Osteocytes and adipocytes were characterized by von Kossa and Oil red O staining, respectively. Chromosomal analysis of dental pulp-MSCs was done for qualitative assessment of MSCs. Karyotyping indicated diploid chromosome number in dental pulp derived MSCs. In vitro grown osteocytes could be used for bone fracture reunion cases, and adipocytes could be used for further research purposes., (© 2019 The Authors.)

Background: Human multipotent adult progenitor cells (MAPC®) are an emerging therapy for traumatic brain injury (TBI); however, clinically translating a therapy involves overcoming many factors in vivo which are not present in pre-clinical testing. In this study we examined clinical parameters in vitro that may impact cell therapy efficacy., Methods: MAPC were infused through varying gauged needles and catheters with and without chlorhexidine, and their viability tested with trypan blue exclusion. MAPC were co-cultured with phenytoin and celecoxib at relevant clinical concentrations for 1 h and 24 h. Anti-inflammatory potency was tested using a stimulated rat splenocyte co-culture and ELISA for TNF-α production. MAPC were cultured under different osmolar concentrations and stained with propidium iodide for viability. Anti-inflammatory potency was tested by co-culture of MAPC with naïve lymphocytes activated by CD3/CD28 beads, and Click-iT® Plus EdU was used to quantify proliferation by flow cytometry., Results: The mean viability of the MAPC infused via needles was 95 ± 1%; no difference was seen with varying flow rate, but viability was notably reduced by chlorhexidine. MAPC function was not impaired by co-culture with phenytoin, celecoxib, or combination with both. Co-culture with phenytoin showed a decrease in TNF-α production as compared to the MAPC control. MAPC cultured at varying osmolar concentrations all had viabilities greater than 90% with no statistical difference between them. Co-culture of MAPC with CD3/CD28 activated PBMCs showed a significant reduction in proliferation as measured by EdU uptake., Discussion: Needle diameter, phenytoin, celecoxib, and a relevant range of osmolarities do not impair MAPC viability or anti-inflammatory potency in vitro ., (© 2019 The Author(s).)

Aims: The aim of this study was to present a new method for removing Sodium dodecyl sulfate (SDS) detergent from decellularized bovine pericardium using vacuum., Materials and Methods: The cows' pericardia were collected and decellularized. The samples were incubated with SDS1% for 48 h at 40 °C. To perform vacuum washing (VW: negative pressure was used to wash and remove detergents), every decellularized tissue was cut in 75mm diameter and fixed via a stainless-steel ring with 60mm diameter in the center of filtration Buchner Funnel which was connected to glass filtration flask The system was connected to a vacuum pump by a hose, and a negative pressure of -100 mmHg was applied for 15 min. Then, the samples were shaken and washed at 40-rpm in 100 ml of distilled water for 45 min. This process was repeated for samples of each group (6 times for sample VW6h, 12 times for sample VW12h, and 24 times for sample VW24h). At the end of every cycle, the effluent was collected to take a sample for SDS measurement. The normal washing (NW) group containing distilled water (NWd) and PBS (Phosphate buffered saline) (NWp) were used to wash and remove detergents. SDS measurements, MTT Assay, histological and tensile test, to compare two methods were used., Results: The highest SDS in the effluent was in groups VW12h and VW24h (P ≤ 0.001) and the lowest residual SDS in scaffold was in two groups of VW12h and VW24h (P ≤ 0.001). MTT assay showed that cell survival in the VW12h and VW24h groups was higher than other groups and there' was no significant difference between cell survival in the VW12h and VW24h groups. Histological study showed destruction of tissue in the VW24h group. The results of the tensile test were shown that the native group had the highest module and the lowest amount was the VW24h sample which was reported with P ≤ 0.001 significance for all groups., Conclusion: VW12h can be used as an effective method for SDS removal from decellularized pericardium which morphologically demonstrated a good structure in ECM.

Whilst the potential of neural stem cell (NSC)-based treatment is recognized worldwide and seems to offer a promising therapeutic option for stroke treatments, there is currently no full understanding regarding the effects of hypoxic and baicalein-enriched fraction (BEF) preconditioning approaches on the therapeutic potential of these cells for stroke. The potential of preconditioned NSC can be determined based on the expression of several key neuroprotective genes using qRT-PCR technique. However, prior to that, it is imperative and extremely important to carefully select reference gene(s) for accurate qRT-PCR data normalization to avoid error in data interpretation. This study aimed to evaluate the stability of ten candidate reference genes via comprehensive analysis using three algorithms software: geNorm, NormFinder and BestKeeper. Our results revealed that HPRT1 and RPL13A were the most reliable reference genes for BEF-preconditioned NSCs, but ironically, HPRT1 was ranked as the least stable reference gene for hypoxic-preconditioned NSCs. On the other hand, RPLP1 and RPL13A were selected as the most stably expressed pair of reference genes for hypoxic-preconditioned NSCs. In conclusion, this study has pointed out the importance of identifying valid reference genes and has presented the first significant validation on best reference genes recommended for qRT-PCR study involves NSC preconditioned with hypoxia or with BEF extracted from Oroxylum indicum medicinal plant.

Demineralized bone allografts and mesenchymal stromal cells have been used to promote bone regeneration. However, the degree to which cortical bone should be demineralized for use in combination with adipose-derived mesenchymal stromal cells (Ad-MSCs) remains to be clarified. In this study, the in vitro osteogenic ability of Ad-MSCs on allografts was investigated in relation to the extent of demineralization. Three treatment groups were established by varying exposure time to 0.6 N HCL: partially demineralized (PDB; 12 h), fully demineralized (FDB; 48 h), and non-demineralized bone (NDB; 0 h, as a control). Allografts were prepared as discs 6 mm in diameter for in vitro evaluation, and their demineralization and structure were evaluated by micro-computed tomography and scanning electron microscopy. Ad-MSC adhesion and proliferation were measured by MTS assay, and osteogenesis-related gene expression was assessed by quantitative reverse transcription polymerase chain reaction. PDB and FDB demineralization rates were 57.13 and 92.30%, respectively. Moreover, Ad-MSC adhesion rates on NDB, PDB, and FDB were 53.41, 60.65, and 61.32%, respectively. Proliferation of these cells on FDB increased significantly after 2 days of culture compared to the other groups ( P < 0.05). Furthermore, expression of the osteogenic genes ALP , BMP-7 , and TGF-β in the FDB group on culture day 3 was significantly elevated in comparison to the other treatments. Given its biocompatibility and promotion of the osteogenic differentiation of Ad-MSCs, our results suggest that FDB may be a suitable scaffold for use in the repair of bone defects.