The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells

Induction of a rat model of premature ovarian insufficiency using D-galactose feeding during the critical periods of development: A pilot study

Background: Premature ovarian insufficiency (POI) affects about 1% of women of reproductive ages (15-45 yr), with no curative treatment. Objective: We aimed to present a rat model of POI using a D-galactose enriched diet. Materials and Methods: In a pilot study, 4 pregnant Wistar rats were divided into 4 groups; 3 groups were fed galactose-enriched diets at days 3-15 of pregnancy (G1); on the 3rd day of pregnancy to parturition (G2), and the 3rd day of pregnancy until the end of the weaning period (G3). Also, group 4, as the control group (G0), was fed standard pellets during the study. After confirming the lack of adverse effects of dieting with galactose in terms of offsprings’ birth weight, we performed our study designed the same as the pilot study. A total of 40 pregnant Wistar rats were randomly divided into 4 groups. Ovarian histology, reproductive hormones, and immunological characteristics of the female offspring were examined in all experimental groups and compared. Results: The pilot study revealed no significant differences in the birth weight of the offspring of the 4 study groups (p = 0.96). The ovarian index in the female offspring of those with a gal-exposed diet was significantly lower than that of the control group offspring (p < 0.01). Conclusion: As the birth weights of the offspring of our experimental and control groups were similar, it can be concluded that the reduction of ovarian follicles after prenatal exposure to D-galactose is due to the ovotoxicity of galactose. The results of our final study will provide more information about the rat POI model induced by prenatal exposure to D-galactose. Key words: Premature ovarian insufficiency, Animal model, D-galactose

Peripheral axotomy-induced changes of motor function and histological structure of spinal anterior horn

The aim of this study was to evaluate changes of both peripheral motor function and histology of spinal anterior horn in adult rats after unilateral sciatectomy. Ten adult healthy rats served as control group, while in the ten rat experimental group the right sciatic nerve was severed. We followed-up nerve motor function using a sciatic function index and electromyography activity of the gastrocnemious muscle. The rats of the experimental group presented the expected gross locomotor deficit and leg muscle atrophy. At 12 weeks post sciatectomy, L4 and L5 spinal cord segments were removed from the twenty rats and were analysed by istological stereological methods. In the axotomized animals volume of the anterior horn and its content of motor neurons decreased, while the content of astrocytes increased (p<0.05). Thus, in adult rats, beside the obvious peripheral nerve disfuction, the sciatic nerve axotomy have severe consequences on the soma of the injured motor neurons in the spinal anterior horn. All these quantitative analyses may be usefull to quantify changes occurring in adult animals after axotomy and eventual management to modify the final outcomes in peripheral nerve disorders

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells

The structure of Human Mesenchymal Stem Cells Differentiated into Cartilage in Micromass Culture System

Introduction: The aim of this study was to differentiate humanmesenchymal stem cells (hMSCs) into cartilage in a micromass culturesystem and study of their structure by light and electron microscopy.Material and Methods: Human bone marrow cells obtained from volunteerpatients were plated in 75-cm2 flasks and their MSCs were expandedthrough several sub-cultures. The passage 4 cells were used to establishmicromass culture system for chondrogenic differentiation. For this purpose,200,000 fibroblastic cells were placed in centrifuge tubes and pelleted at 250g for 5 minutes. About 0.5 ml chondrogenic induction medium was thenadded to the pellet and the culture incubated in 5% CO2 at 37°C for 21 days.Then, some pellets were utilized to evaluate chondrogenic differentiation byeither RT-PCR analysis of some cartilage marker molecules or specificstaining for detecting cartilage matrix, and other pellets were used for lightand electron microscopic study of differentiated tissue.Results: Primary culture of the bone marrow cells were initially composed ofthe spindle- and round shaped cells, from which the spindle cells remainedand expanded through several passages. At the end of differentiation period,RT-PCR analysis showed high production of collagen II and X and aggrecanmRNA inside the differentiated cells, and toluidine blue staining indicatedintermediate accumulation of the metachromatic matrix among the inducedcells. In general, light micrograph indicated a rather cellular state of thedifferentiated tissue in which the peripheral part had more metachromaticmatrix than central zone. More detailed study of the sections revealed thatinduced aggregates of the cells were composed externally of very thin layerof elongated cells reminiscent of perichondrium and internally a mass of ovalcells comprising the main part of the pellet. Ultra-thin sections showed thatthe cells in perichondrium-like layer were very similar to fibroblastic cells andthose located centrally had a set of well-developed organelles, characteristicof highly active cells. Some fat cells were seen in central zone.Conclusion: Cartilage tissue differentiated from MSCs in micromass culturesystem seemed to be structurally very similar to developing cartilage not toadult mature cartilage

Hepatic stellate cell activation by TGFβ induces hedgehog signaling and endoplasmic reticulum stress simultaneously.

Activation of hepatic stellates (HSCs) is known as the major cause of initiation and progression of liver fibrosis. A wide array of events occurs during HSC activation including induction of hedgehog (Hh) signaling and endoplasmic reticulum (ER) stress. Targeting HSC activation may provide promising insights into liver fibrosis treatment. In this regard, establishing in vitro models which can mimic the molecular pathways of interest is very important. We aimed to activate HSC in which Hh signaling and ER stress are stimulated simultaneously. We used 5 ng/ml TGFβ to activate LX-2 cells, HSC cell line. Gene expression analysis using qRT-PCR, immunostaining and immunoblotting were performed to show HSC activation associated markers. Furthermore, the migration capacity of the TGFβ treated cells is evaluated. The results demonstrated that major fibrogenic markers including collagen1a, lysyl oxidase, and tissue inhibitor of matrix metalloproteinase 1 genes are up-regulated significantly. In addition, our immunofluorescence and immunoblotting results showed that protein levels of GLI-2 and XBP1, were enhanced. Moreover, we found that TGFβ treatment reduced the migration of LX-2 cells. Our results are compatible with high throughput data analysis with respect to differentially expressed genes of activated HSC compared to the quiescent ones. Moreover, our findings suggest that quercetin can reduce fibrogenic markers of activated HSCs as well as osteopontin expression, a target gene of hedgehog signaling

Athletes’ Mesenchymal Stem Cells Could Be the Best Choice for Cell Therapy in Omicron-Infected Patients

New severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, Omicron, contains 32 mutations that have caused a high incidence of breakthrough infections or re-infections. These mutations have reduced vaccine protection against Omicron and other new emerging variants. This highlights the need to find effective treatment, which is suggested to be stem cell-based therapy. Stem cells could support respiratory epithelial cells and they could restore alveolar bioenergetics. In addition, they can increase the secretion of immunomodulatory cytokines. However, after transplantation, cell survival and growth rate are low because of an inappropriate microenvironment, and stem cells face ischemia, inflammation, and oxidative stress in the transplantation niche which reduces the cells’ survival and growth. Exercise-training can upregulate antioxidant, anti-inflammatory, and anti-apoptotic defense mechanisms and increase growth signaling, thereby improving transplanted cells’ survival and growth. Hence, using athletes’ stem cells may increase stem-cell therapy outcomes in Omicron-affected patients

Neural differentiation of choroid plexus epithelial cells: role of human traumatic cerebrospinal fluid

As the key producer of cerebrospinal fluid (CSF), the choroid plexus (CP) provides a unique protective system in the central nervous system. CSF components are not invariable and they can change based on the pathological conditions of the central nervous system. The purpose of the present study was to assess the effects of non-traumatic and traumatic CSF on the differentiation of multipotent stem-like cells of CP into the neural and/or glial cells. CP epithelial cells were isolated from adult male rats and treated with human non-traumatic and traumatic CSF. Alterations in mRNA expression of Nestin and microtubule-associated protein (MAP2), as the specific markers of neurogenesis, and astrocyte marker glial fibrillary acidic protein (GFAP) in cultured CP epithelial cells were evaluated using quantitative real-time PCR. The data revealed that treatment with CSF (non-traumatic and traumatic) led to increase in mRNA expression levels of MAP2 and GFAP. Moreover, the expression of Nestin decreased in CP epithelial cells treated with non-traumatic CSF, while treatment with traumatic CSF significantly increased its mRNA level compared to the cells cultured only in DMEM/F12 as control. It seems that CP epithelial cells contain multipotent stem-like cells which are inducible under pathological conditions including exposure to traumatic CSF because of its compositions

Peripheral axotomy-induced changes of motor function and histological structure of spinal anterior horn

The aim of this study was to evaluate changes of both peripheral motor function and histology of spinal anterior horn in adult rats after unilateral sciatectomy. Ten adult healthy rats served as control group, while in the ten rat experimental group the right sciatic nerve was severed. We followed-up nerve motor function using a sciatic function index and electromyography activity of the gastrocnemious muscle. The rats of the experimental group presented the expected gross locomotor deficit and leg muscle atrophy. At 12 weeks post sciatectomy, L4 and L5 spinal cord segments were removed from the twenty rats and were analysed by istological stereological methods. In the axotomized animals volume of the anterior horn and its content of motor neurons decreased, while the content of astrocytes increased (p<0.05). Thus, in adult rats, beside the obvious peripheral nerve disfuction, the sciatic nerve axotomy have severe consequences on the soma of the injured motor neurons in the spinal anterior horn. All these quantitative analyses may be usefull to quantify changes occurring in adult animals after axotomy and eventual management to modify the final outcomes in peripheral nerve disorders