Ultrastructural characteristics of ovine bone marrow‐derived mesenchymal stromal cells cultured with a silicon stabilized tricalcium phosphate bioceramic

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM‐MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM‐MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM‐MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM‐MSCs were isolated from the iliac crest, cultured until they reached near‐confluence and incubated with SiTCP. After 48 hr the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT‐PCR analysis. RT‐PCR displayed that oBM‐MSCs express typical surface marker for MSCs. TEM revealed the presence of electron‐lucent cells and electron‐dense cells, both expressing the CD90 surface antigen. The prominent feature of electron‐lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM‐MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM‐MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic. Skelite cultured ovine BM‐MSCs display electron‐dense and electron‐lucent cells which are differently affected by this bioceramic. This suggests that they could play a different role in bioceramic based therapy.     

The effects of human menstrual blood stem cells‐derived granulosa cells on ovarian follicle formation in a rat model of premature ovarian failure

Many studies have reported that human endometrial mesenchymal stem cells (HuMenSCs) are capable of repairing damaged tissues. The aim of the present study was to investigate the effects of HuMenSCs transplantation as a treatment modality in premature ovarian failure (POF) associated with chemotherapy‐induced ovarian damage. HuMenSCs were isolated from menstrual blood samples of five women. After the in vitro culture of HuMenSCs, purity of the cells was assessed by cytometry using CD44, CD90, CD34, and CD45 FITC conjugate antibody. Twenty‐four female Wistar rats were randomly divided into four groups: negative control, positive control, sham, and treatment groups. The rat models of POF used in our study were established by injecting busulfan intraperitoneally into the rats during the first estrus cycle. HuMenSCs were transplanted by injection via the tail vein into the POF‐induced rats. Four weeks after POF induction, ovaries were collected and the levels of Amh, Fst, and Fshr expression in the granulosa cell (GC) layer, as well as plasma estradiol (E2) and progesterone (P4) levels were evaluated. Moreover, migration and localization of DiI‐labeled HuMenSCs were detected, and the labeled cells were found to be localized in GCs layer of immature follicles. In addition to DiI‐labelled HuMenSCs tracking, increased levels of expression of Amh and Fshr and Fst, and the high plasma levels of E2 and P4 confirmed that HuMenSC transplantation had a significant effect on follicle formation and ovulation in the treatment group compared with the negative control (POF) group.     

Effects of human umbilical cord mesenchymal stem cells on co‐cultured ovarian carcinoma cells

Ovarian carcinoma is mainly treated by surgery aided by chemotherapy. If supplemented by stem cells treatment, its recurrence rate and mortality rate will be decreased. This is a new therapy. In this study, ovarian cancer cells were cultured together with umbilical cord mesenchymal stem cells (UCMSCs), and the interactions between them were observed. The results showed that the survival rates of UCMSCs increased to 83.8 ± 2.2% from 56.5 ± 5.5%, and the survival rates of ovarian cancer cells decreased to 16.2 ± 2.2% from 43.5 ± 5.5% with the progression of the cultural time from 24 to 96 hr. There was a significant difference between them (p < .05). It revealed that UCMSCs could inhibit the proliferation of ovarian cancer cells.     

Transplantation of BMP-7 gene-transfected bone marrow mesenchymal stem cells for the treatment of spinal cord injury in rats

Background: Spinal cord injury (SCI) is a serious traumatic disease of the central nervous system, and there is currently no effective treatment for SCI because of its complicated pathophysiology. Bone marrow mesenchymal stem cells (BMSCs) have multidirectional differentiation abilities. Our study aims to explore the effects of bone morphogenetic protein 7 (BMP-7)-modified BMSCs transplantation on the repair of SCI in rats. Methods: In this study, a rat spinal cord injury model was established with the modified Allen method. Then, BMSCs transfected with the BMP7 gene were transplanted to treat the spinal cord injury in rats. Forty Sprague-Dawley rats were randomly divided into the sham operation group (sham group), spinal cord injury group (model group), BMSC treatment group (BMSC group) and LV-BMP7-BMSC treatment group (LV-BMP7-BMSC group). The Basso, Beattie, and Bresnahan (BBB) score was used to evaluate the recovery of hindlimb function in the rats. The levels of neurofilament protein NF-200 (NF-200) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence, RT-PCR and Western blotting. Results: At 14 d, 21 d, and 28 d after treatment, the BBB score of the rats in the LV-BMP7-BMSC group was higher than that of the rats in the model group and BMSC group. The results showed that NF-200 was expressed at the local spinal cord injury site. Compared with that of the sham group, the NF-200 expression level of the BMSC group and LV-BMP7-BMSC group was increased (P < 0.05). The results showed that the mRNA expression levels of NF-200 in the spinal cord tissue of the BMSC group and LV-BMP7-BMSC group were increased compared with those of the sham group (P < 0.05). The western blotting results further confirmed the PCR results. Conclusion: BMP-7 gene-modified BMSC transplantation can promote the repair of spinal cord functions after SCI in rats.     

Cell-surface ultrastructural changes during the in vitro neuron-like differentiation of rat bone marrow-derived mesenchymal stem cells

The neuron-like differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) has been extensively studied. However, the alternations of the cell-surface ultrastructures and the membrane tension/reservoir of the cells during this differentiation process are poorly understood. Therefore, atomic force microscopy (AFM) was utilized in this study to observe the cell-surface ultrastructural changes among rat bone marrow-derived mesenchymal stem cells (rBMMSCs), partially differentiated cells, and fully differentiated neuron-like cells. By analyzing the stiffness of plasma membranes, lamellipodial extensions, average heights of small membrane protrusions and relatively larger uplifted structures, and peak-peak spacing among protrusions and/or uplifted structures, we found that the membrane reservoir may potentially decrease upon the differentiation from rBMMSCs to partially differentiated cells and to fully differentiated neuron-like cells. The results may help to better understanding the membrane tension of various types of cells and related biological processes, such as membrane traffic, cell adhesion, motility, differentiation, among others. The data also implies that AFM may be a useful tool for evaluating membrane reservoir by imaging cell-surface ultrastructures.     

The osteogenic effects of swimming on bone mass,strength, and microarchitecture in rats with unloading‐induced bone loss

The effect of nonweight‐bearing exercise on osteoporotic bones remains controversial and inconclusive. The purpose of this study was to evaluate the effects of swimming on osteoporotic tibias of rats submitted to hindlimb suspension. Initially, 20 Wistar rats were used to confirm a significant bone loss following 21 days of unloading. Thirty rats were then divided into 3 groups and followed during 51 days: CON (nonsuspended rats), S + WB (suspended rats for 21 days and then released for regular weight‐bearing) and, S + Swim (suspended rats for 21 days and then released from suspension and submitted to swimming exercise). We observed that swimming exercise was effective at fully recovering the bone deterioration caused by suspension, with significant increments in BMD, bone strength and bone volume. On the other hand, regular weight‐bearing failed at fully restoring the bone loss induced by unloading. These results indicate that swimming exercise may be a potential tool to improve bone density, strength, and trabecular volume in tibias with bone loss induced by mechanical unloading in suspended rats. We conclude that this modality of activity could be beneficial in improving bone mass, strength, and architecture in osteoporotic individuals induced by disuse, such as bed rest or those exposed to microgravity, who may not be able to perform weight‐bearing exercises. Microsc. Res. Tech. 78:784–791, 2015. © 2015 Wiley Periodicals, Inc.     

The antioxidant trolox inhibits aging and enhances prostaglandin E-2 secretion in mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been widely used in regenerative medicine and clinical therapy due to their capabilities of proliferation, differentiation, and immune regulation. However, during in vitro expansion, MSCs are prone to aging, which largely limits their application. Prostaglandin E-2 (PGE-2) is a key effector secreted by MSCs to exert immunomodulatory effects. By screening the compound library for PGE-2 secretion, the antioxidant trolox was verified as a stimulator of MSCs to secrete PGE-2. The effect of antioxidant trolox on biological characteristics of MSCS, including aging, proliferation, and gene expression, was examined. The results demonstrated that trolox can resist aging, promote proliferation, and enhance PGE-2 secretion of MSCs without affecting their surface marker expression. Furthermore, trolox treatment up-regulates miR-17-92 clusters in MSCs and may contribute to its anti-aging effects. Thus, trolox addition might be beneficial for MSCs expansion and their application.     

Role of spermatogonial stem cells extract in transdifferentiation of 5‐Aza‐2′‐deoxycytidine‐treated bone marrow mesenchymal stem cells into germ‐like cells

As one of the induced pluripotent stem cells (iPSCs) methods, spermatogonial stem cells (SSC_S) extract is considered as new approach in stem cell therapy of infertility. 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) inhibits methyltransferase enzyme, and induces gene reprogramming; herein, the effects of SSC_S extract incubation in 5‐aza‐dC‐treated bone marrow mesenchymal stem cells (BMMSCs) has been surveyed. BMMSCs were isolated from femurs of three to four weeks old male NMRI mice, and the cells at passage three were treated with 2 µM 5‐aza‐dC for 72 hours. SSCs were isolated, cultured, and harvested at passage three to collect SSC_S extract; BMMSCs were then incubated with SSC_S extract in the three time periods: 72 hours, one week and two weeks. There were five groups: control, sham, extract, 5‐aza‐dC and extract‐5‐aza‐dC. After one week of incubation, flow cytometry and real‐time polymerase chain reaction (PCR) exhibited high levels of expression for β1‐ and α6‐integrins and promyelocytic leukaemia zinc finger (PLZF) in extract and extract‐5‐aza‐dC groups (P < 0.05 vs. control and 5‐aza‐dC), and cells in these two groups had two forms of morphology, round and fusiform, similar to germ‐like cells. 5‐aza‐dC had no significant effects during the three time periods of evaluation. These data disclose the effectiveness of SSCs extract incubation in transdifferentiation of BMMSCs into germ‐like cells; this strategy could introduce a new approach for treatment of male infertility in clinic. Microsc. Res. Tech. 79:365–373, 2016. © 2016 Wiley Periodicals, Inc.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.     

Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

Nowadays, infertility is no longer considered as an unsolvable disorder due to progresses in germ cells derived from stem lineage with diverse origins. Technical and ethical challenges push researchers to investigate various tissue sources to approach more efficient gametes. The purpose of the current study is to investigate the efficacy of a combined medium, retinoic acid (RA) together with Bone Morphogenic Protein‐4 (BMP4), on differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and adipose‐derived mesenchymal stem cells (ADMSCs) into germ cells. Murine MSCs were obtained from both Bone Marrow (BM) and Adipose Tissue (AT) samples and were analyzed for surface markers to get further verification of their nature. BMMSCs and ADMSCs were induced into osteogenic and adipogenic lineage cells respectively, to examine their multipotency. They were finally differentiated into germ cells using media enriched with BMP4 for 4 days followed by addition of RA for 7 days (11 days in total). Analyzing of differentiation potential of BMMSCs‐ and ADMSCs were performed via Immunofluorescence, Flowcytometry and Real time‐PCR techniques for germ cell‐specific markers (Mvh, Dazl, Stra8 and Scp3). Mesenchymal surface markers (CD90 and CD44) were expressed on both BMMSCs and ADMSCs, while endothelial and hematopoietic cell markers (CD31 and CD45) had no expression. Finally, all germ‐specific markers were expressed in both BM and AT. Although germ cells differentiated from ADMSCs showed faster growth and proliferation as well as easy collection, they significantly expressed germ‐specific markers lower than BMMSCs. This suggests stronger differentiation potential of murine BMMSCs than ADMSCs.     

Ultrastructural evaluation of mesenchymal stem cells from inflamed periodontium in different in vitro conditions

This research aimed to observe the behavior of mesenchymal stem cells (MSCs) isolated from periodontal granulation tissue (gt) when manipulated ex vivo to induce three‐dimensional (3D) spheroid (aggregates) formation as well as when seeded on two bone scaffolds of animal origin. Periodontal gt was chosen as a MSC source because of its availability, considering that it is eliminated as a waste material during conventional surgical therapies. 3D aggregates of cells were generated; they were grown for 3 and 7 days, respectively, and then prepared for transmission electron microscopic analysis. The two biomaterials were seeded for 72 h with gtMSCs and prepared for scanning electronic microscopic observation. The ultrastructural analysis of 3D spheroids remarked some differences between the inner and the outer cell layers, with a certain commitment observed at the inner cells. Both scaffolds showed a relatively smooth surface at low magnification. Macro‐ and micropores having a scarce distribution were observed on both bone substitutes. gtMSCs grew with relative difficulty on the biomaterials. After 72 h of proliferation, gtMSCs scarcely covered the surface of bovine bone scaffolds, demonstrating fibroblast‐like or star‐like shapes with elongated filiform extensions. Our results add other data on the possible usefulness of gtMSC and could question the current paradigm regarding the complete removal of chronically inflamed gts from the defects during periodontal surgeries. Until optimal protocols for ex vivo manipulation of MSCs are available for clinical settings, it is advisable to use biocompatible bone substitutes that allow the development of progenitor cells. Microsc. Res. Tech. 78:792–800, 2015. © 2015 Wiley Periodicals, Inc.     

Fate of donor bone marrow cells in medial collateral ligament after simulated autologous transplantation

A potential strategy to enhance ligament healing by transplantation of mesenchymal stem cells (MSCs), which are demonstrated to differentiate into fibroblast-like cells in vitro, is presented. The objective of this study was to follow transplanted nucleated cells from bone marrow, which contain MSCs, in the healing medial collateral ligament (MCL) over time, and to examine their phenotype and survivability. It was hypothesized that MSCs in nucleated cells from bone marrow would differentiate into fibroblast-like cells in the healing ligament following adaptation to the environment. The transplantation model employed in this study eliminates the immune response to a donor by the recipient using a transgenic rat (donor), which does not produce foreign protein from transgenes, and its wild-type rat (recipient) in order to simulate autologous transplantation. The MCL of the wild-type rat was ruptured, where 1 x 10(6) nucleated cells of bone marrow from the transgenic rat were injected. The transgenes in transplanted nucleated cells were detected throughout the healing MCL for 28 days by in situ hybridization. At 3 days, many donor cells were evident in the injury site and fascial pocket, and some were found in the midsubstance. Morphologically, transplanted cells with elongated nuclei were found at the ruptured edge of the midsubstance and surface of the unruptured site after 3 days. At 28 days, these cells continued to survive in the healing MCL. Their shapes were similar to those of surrounding recipient MCL fibroblasts. Thus, transplanted cells might differentiate into fibroblasts. Therefore, it was demonstrated that there is a potential for nucleated cells from bone marrow to serve as a vehicle for therapeutic molecules as well as to be a source in enhancing healing of ligaments.     

Ultrastructural study of mouse adipose‐derived stromal cells induced towards osteogenic direction

We investigated the ultrastructural characteristics of mouse adipose‐derived stem/stromal cells (ASCs) induced towards osteogenic lineage. ASCs were isolated from adipose tissue of FVB‐Cg‐Tg(GFPU)5Nagy/J mice and expanded in monolayer culture. Flow cytometry, histochemical staining, and electron microscopy techniques were used to characterize the ASCs with respect to their ability for osteogenic differentiation capacity. Immunophenotypically, ASCs were characterized by high expression of the CD44 and CD90 markers, while the relative content of cells expressing CD45, CD34 and CD117 markers was <2%. In assays of differentiation, the positive response to osteogenic differentiation factors was observed and characterized by deposition of calcium in the extracellular matrix and alkaline phosphatase production. Electron microscopy analysis revealed that undifferentiated ASCs had a rough endoplasmic reticulum with dilated cisterns and elongated mitochondria. At the end of the osteogenic differentiation, the ASCs transformed from their original fibroblast‐like appearance to having a polygonal osteoblast‐like morphology. Ultrastructurally, these cells were characterized by large euchromatic nucleus and numerous cytoplasm containing elongated mitochondria, a very prominent rough endoplasmic reticulum, Golgi apparatus and intermediate filament bundles. Extracellular matrix vesicles of variable size similar to the calcification nodules were observed among collagen fibrils. Our data provide the ultrastructural basis for further studies on the cellular mechanisms involved in osteogenic differentiation of mouse adipose‐derived stem/stromal cells. Microsc. Res. Tech. 79:557–564, 2016. © 2016 Wiley Periodicals, Inc.     

Ultrastructural analysis of human bone marrow mesenchymal stem cells during in vitro osteogenesis and chondrogenesis

The main purpose of this article was to describe the morphology of mesenchymal stem cells (MSCs) differentiated in vitro towards osteogenic and chondrogenic lineages and to focus on the ultrastructural features associated with these processes. Human mononuclear cells (hMNC) were isolated, expanded, and analyzed for the expression of specific cell surface markers to demonstrate their stem cell characteristics. Human mononuclear cells were differentiated in vitro in an osteogenic and in a chondrogenic sense for 7, 14, 21, and 28 days. Subsequently, they were processed using electron microscopic analysis (FEISEM). Alizarin red and alcian blue staining were carried out to demonstrate the deposition of mineral salts and proteoglycans in the extracellular matrix. Undifferentiated MSCs showed a cell surface covered by filopodia and ondulopodia. During differentiation, the MSCs changed their shape from a round to a fibroblastic-like shape. At the end of the differentiation, several filaments with a parallel orientation in the osteogenic samples as well as a network organization in the chondrogenic samples were detected in the extracellular spaces. This study demonstrated that there are morphological features associated with the undifferentiated and differentiated states of the MSCs, which could be utilized as new parameters for identifying and classifying these cells.     

<i>miR-103-3p</i> regulates the differentiation of bone marrow mesenchymal stem cells in myelodysplastic syndrome

The pathogenesis of myelodysplastic syndrome (MDS) may be related to the abnormal expression of microRNAs(miRNAs), which could influence the differentiation capacity of mesenchymal stem cells (MSCs) towards adipogenic andosteogenic lineages. In this study, exosomes from bone marrow plasma were successfully extracted and identified.Assessment of miR-103-3p expression in exosomes isolated from BM in 34 MDS patients and 10 controls revealed its0.52-fold downregulation in patients with MDS compared with controls (NOR) and was downregulated 0.55-fold inMDS-MSCs compared with NOR-MSCs. Transfection of MDS-MSCs with the miR-103-3p mimic improved osteogenicdifferentiation and decreased adipogenic differentiation in vitro, while inhibition of miR-103-3p showed the oppositeresults in NOR-MSCs. Thus, the expression of miR-103-3p decreases in MDS BM plasma and MDS-MSCs, significantlyimpacting MDS-MSCs differentiation. The miR-103-3p mimics may boost MDS-MSCs osteogenic differentiation whileweakening lipid differentiation, thereby providing possible target for the treatment of MDS pathogenesis.