- Simulated microgravity culture enhances the neuroprotective effects of human cranial bone-derived mesenchymal stem cells in traumatic brain injury. [Journal Article]
- SCStem Cells Dev 2018 May 23
- Fundamental cures of central nervous system (CNS) diseases are rarely achieved due to the low regenerative ability of the CNS. Recently, cell-based therapy using mesenchymal stem cells (MSCs) has bee...
Fundamental cures of central nervous system (CNS) diseases are rarely achieved due to the low regenerative ability of the CNS. Recently, cell-based therapy using mesenchymal stem cells (MSCs) has been explored as effective treatments for CNS diseases. Among the various tissue-derived MSCs, we have isolated human cranial bone-derived MSCs (cMSCs) in our laboratory. In addition, we have focused on simulated microgravity (MG) as a valuable culture environment of MSCs. However, the detailed mechanisms underlying functional recovery from the transplantation of MSCs cultured under MG conditions remain unclear. In this study, we investigated the therapeutic mechanisms of cMSCs transplantation cultured under MG conditions in traumatic brain injury (TBI) model mice. Human cMSCs were cultured under 1G and MG conditions, and cMSCs cultured under MG conditions expressed significantly higher mRNA levels of hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-β). In TBI model mice, the transplantation of cMSCs cultured under MG conditions (group MG) showed greater motor functional improvement compared to the only phosphate buffered saline administration (group PBS). Moreover, the protein expressions of tumor necrosis factor alpha (TNF-α) and the Bax/Bcl-2 ratio were significantly lower at brain injury sites in mice of group MG than those of group PBS. In addition, an in vitro study showed that the conditioned medium of the cMSCs cultured under MG conditions significantly suppressed the cell death of NG108-15 cells exposed to oxidative or inflammatory stress through anti-inflammatory and anti-apoptosis effects. These findings demonstrate that culturing cMSCs under simulated microgravity increases the neuroprotective effects, suggesting that simulated microgravity cultures may be a useful method for cell-based therapy strategies for CNS diseases.
- Long-term Effects of Simulated Microgravity and Vibration Exposure on Skeletal Development in Zebrafish. [Journal Article]
- SCStem Cells Dev 2018 May 23
- Most studies utilising fish to study the effects of simulated microgravity (SMG) only observe the effects during the first week of development. They also do not take into account the potential impact...
Most studies utilising fish to study the effects of simulated microgravity (SMG) only observe the effects during the first week of development. They also do not take into account the potential impact on development of vibrations caused by the equipment. Here we analyse the effects of both SMG and vibration on development of the skeleton. We analyse three different exposure durations and starting points that coincide with cranial neural crest cell migration. We use a combination of bone staining and morphometrics to analyse the effects. Our data show that both vibration and SMG affect vertebrae number and body size, however not all vertebrae are equally affected by each treatment. We also show that delayed ossification manifests during development, particularly after SMG exposure, and this translates into buckled and bent bones in adults. This study highlights the large impact of even very short exposure periods when they coincide with critical time points of development.
- The fate of autologous endometrial mesenchymal stromal cells after application in the healthy equine uterus. [Journal Article]
- SCStem Cells Dev 2018 May 23
- Because of their distinct differentiation, immunomodulatory and migratory capacities, endometrial mesenchymal stromal cells (MSCs) may provide an optimum source of therapeutic cells not only in relat...
Because of their distinct differentiation, immunomodulatory and migratory capacities, endometrial mesenchymal stromal cells (MSCs) may provide an optimum source of therapeutic cells not only in relation to the uterus but also for regeneration of other tissues. This study reports the fate of endometrial MSCs following intrauterine application in mares. Stromal cell fractions were isolated from endometrial biopsies taken from seven reproductively healthy mares, expanded and fluorescence-labeled in culture. MSCs (15 x 106) or PBS were autologously infused into each uterine horn during early diestrus and subsequently tracked by fluorescence microscopy and flow cytometry of endometrial biopsies and blood samples taken periodically after infusion. The inflammatory response to cell infusion was monitored in endometrial cytology samples. MSCs were detected in endometrial sections at 6, 12 and 24 hours but not later (7 or 14 days) after cell infusion. Cells were in all cases located in the uterine lumen, never within endometrial tissue. No fluorescence signal was detected in blood samples at any time point after infusion. Cytology analyses showed an increase in %PMN between 1 and 3 hours after uterine infusion with either MSCs or PBS, and a further increase by 6 hours only in mares infused with PBS. In summary, endometrial MSCs were detected in the uterine lumen for up to 24 h after infusion but did not migrate into healthy endometrium. Moreover, MSCs effectively attenuated the inflammatory response to uterine infusion. We conclude that endometrial MSCs obtained from routine uterine biopsies could provide a safe and effective cell source for treatment of inflammatory conditions of the uterus and potentially other tissues.
- Autologous induced Pluripotent Stem Cell-derived Neurons to treat Parkinson's disease. [Journal Article]
- SCStem Cells Dev 2018 May 23
- In 2012, we planned a program to develop a neuron replacement therapy for Parkinson's disease (PD) that would have the greatest promise to help the patients. PD is a movement disorder caused by the p...
In 2012, we planned a program to develop a neuron replacement therapy for Parkinson's disease (PD) that would have the greatest promise to help the patients. PD is a movement disorder caused by the progressive, inevitable loss of a specific type of dopamine neuron in the brain. The only viable treatment to reverse the progress of the disease is to replace those neurons; we decided to make dopamine neurons that matched the patients, by differentiating induced pluripotent stem cells (iPSCs) that we generated from individuals with PD. This autologous cell therapy is entering the regulatory approval process this year with the US FDA to begin to transplant the cells in the following one to two years.
- Rat cranial bone-derived mesenchymal stem cell transplantation promotes functional recovery in ischemic stroke model rats. [Journal Article]
- SCStem Cells Dev 2018 May 22
- The functional disorders caused by central nervous system (CNS) diseases, such as ischemic stroke, are clinically incurable and current treatments have limited effects. Previous studies suggested tha...
The functional disorders caused by central nervous system (CNS) diseases, such as ischemic stroke, are clinically incurable and current treatments have limited effects. Previous studies suggested that cell-based therapy using mesenchymal stem cells (MSCs) exerts therapeutic effects for ischemic stroke. In addition, the characteristics of MSCs may depend on their sources. Among the derived tissues of MSCs, we have focused on cranial bones originating from the neural crest. We previously demonstrated that the neurogenic potential of human cranial bone-derived MSCs (cMSCs) was higher than that of human iliac bone-derived MSCs. Therefore, we presumed that cMSCs have a higher therapeutic potential for CNS diseases. However, the therapeutic effects of cMSCs have not yet been elucidated in detail. In the present study, we aimed to demonstrate the therapeutic effects of transplantation with rat cranial bone-derived MSCs (rcMSCs) in ischemic stroke model rats. The mRNA expression of brain-derived neurotrophic factor and nerve growth factor was significantly stronger in rcMSCs than in rat bone marrow-derived MSCs (rbMSCs). Ischemic stroke model rats in the rcMSC transplantation group showed better functional recovery than those in the no transplantation and rbMSC transplantation groups. Furthermore, in the in vitro study, the conditioned medium of rcMSCs significantly suppressed the death of neuroblastoma × glioma hybrid cells (NG108-15) exposed to oxidative and inflammatory stresses. These results suggest that cMSCs have potential as a candidate cell-based therapy for CNS diseases.
- Calcium, sodium and TRP channel expression in human fetal midbrain-derived neural progenitor cells. [Journal Article]
- SCStem Cells Dev 2018 May 19
- Voltage-gated sodium and calcium channels as well as transient receptor potential (TRP) channels are expressed during the differentiation of human neural progenitor cells (hNPCs) and are likely to be...
Voltage-gated sodium and calcium channels as well as transient receptor potential (TRP) channels are expressed during the differentiation of human neural progenitor cells (hNPCs) and are likely to be involved in regulating neurogenesis. However, the molecular composition of these ion channels in proliferating and differentiating hNPCs is largely unknown. Here, we investigated fetal mesencephalic hNPCs in respect to their sodium, calcium, and TRP channel subunit expression and function. Quantitative real-time PCR indicated a significant upregulation of voltage-gated sodium and calcium channel subunits in hNPCs after differentiation for 3 weeks in vitro. In contrast the TRP channel expression did not increase significantly during hNPC maturation. Intracellular Ca2+ measurements showed the marked reduction of KCl-induced Ca2+ transients through inhibition of voltage-gated Ca2+ channels by verapamil and mibefradil in differentiated hNPCs. Application of TRP channel agonists induced intracellular Ca2+ peaks already in proliferating hNPCs without affecting their cell division. The co-incubation of hNPCs with TRP-channel agonists pregnenolone sulfate or RN1747 did not have any significant effect on their proliferation and differentiation. These data indicate that hNPCs derived from fetal midbrain tissue acquire essential voltage-gated sodium and calcium channel properties during neuronal maturation in vitro. An early role of TRP channels in neurogenesis which may be important for regenerative clinical applications or cellular models could not be elucidated using hNPCs.
- Development of postmeiotic cells in vitro from spermatogonial cells of prepubertal cancer patients. [Journal Article]
- SCStem Cells Dev 2018 May 19
- Aggressive chemotherapy in childhood often results in testicular damage and consequently jeopardizes future fertility. The presence of spermatogonial cells (SPGCs) in the testes of prepubertal cancer...
Aggressive chemotherapy in childhood often results in testicular damage and consequently jeopardizes future fertility. The presence of spermatogonial cells (SPGCs) in the testes of prepubertal cancer patient boys (PCPBs) can be used to develop future strategies for male fertility preservation. In the present study, we examined the presence of SPGCs in testes of chemotherapy-treated PCPBs and their ability to develop spermatogenesis in vitro using a three-dimensional (3D) culture system. Seven testicular biopsies were obtained from chemotherapy-treated PCPBs and one from a patient with β-thalassemia major. Isolated testicular cells were cultured in a methylcellulose culture system (MCS)-containing StemPro enriched with growth factors for 5-15 weeks. The presence of pre-meiotic, meiotic and post-meiotic cells was examined by immunofluorescence staining (IF) and/or RT-PCR analysis. We observed SPGCs in the examined testicular biopsies. Isolated testicular cells cultured in MCS developed into colonies and contained pre-meiotic, meiotic and post-meiotic cells. Furthermore, we identified sperm-like cells that had developed from testicular cells of a PCPB. Our results demonstrate for the first time, the presence of biologically active SPGCs in testicular biopsies of chemotherapy-treated PCPBs, and their capacity to develop in vitro to different stages of spermatogenesis including the generation of sperm-like cells. This study may open the way for new therapeutic strategies for fertility preservation of PCPBs and for azoospermic patients. Key words: Chemotherapy; Radiotherapy; Leukemia, Male infertility; Pre-pubertal cancer patient boys; Spermatogenesis; Testis; In vitro culture; spermatogonial cells.
- Efficient generation of cynomolgus monkey induced pluripotent stem cell-derived intestinal organoids with pharmacokinetic functions. [Journal Article]
- SCStem Cells Dev 2018 May 10
- In preclinical studies, the cynomolgus monkey (CM) model is frequently used to predict the pharmacokinetics of drugs in the human small intestine, because of its evolutionary closeness to humans. Int...
In preclinical studies, the cynomolgus monkey (CM) model is frequently used to predict the pharmacokinetics of drugs in the human small intestine, because of its evolutionary closeness to humans. Intestinal organoids that mimic the intestinal tissue have attracted attention in regenerative medicine and drug development. In this study, we generated intestinal organoids from CM induced pluripotent stem (CMiPS) cells and analysed their pharmacokinetic functions. CMiPS cells were induced into the hindgut; then, the cells were seeded on microfabricated culture vessel plates to form spheroids. The resulting floating spheroids were differentiated into intestinal organoids in a medium containing small-molecule compounds. The mRNA expression of intestinal markers and pharmacokinetic-related genes was markedly increased in the presence of small-molecule compounds. The organoids possessed a polarised epithelium and contained various cells constituting small intestinal tissues. The intestinal organoids formed functional tight junctions and expressed drug transporters proteins. In addition, in the organoids generated, cytochrome P450 3A8 (CYP3A8) activity was inhibited by the specific inhibitor ketoconazole and was induced by rifampicin. Therefore, in the present work, we successfully generated intestinal organoids, with pharmacokinetic functions, from CMiPS cells using small-molecule compounds.
- BMS 493 Modulates Retinoic Acid Induced Differentiation During Expansion of Human Hematopoietic Progenitor Cells for Islet Regeneration. [Journal Article]
- SCStem Cells Dev 2018 May 08
- Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cor...
Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cord blood (UCB)-derived hematopoietic progenitor cells (HPC) isolated by high aldehyde dehydrogenase activity (ALDH<sup>hi</sup>) have previously been shown to reduce hyperglycemia after intrapancreatic (iPan) transplantation into streptozotocin (STZ)-treated non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice. However, these cells are rare and require ex vivo expansion to reach clinically applicable numbers for human therapy. Therefore, we investigated whether BMS 493, an inverse retinoic acid receptor agonist, could prevent retinoic acid-induced differentiation and preserve islet regenerative functions during expansion. After 6-day expansion, BMS 493-treated cells showed a two-fold increase in the number of ALDH<sup>hi </sup>cells available for transplantation compared to untreated controls. Newly expanded ALDH<sup>hi</sup> cells showed increased numbers of CD34 and CD133 positive cells, as well as a reduction in CD38 expression, a marker of hematopoietic cell differentiation. BMS 493-treated cells showed similar hematopoietic colony forming capacity compared to untreated cells, with ALDH<sup>hi</sup> subpopulations producing more colonies than ALDH<sup>lo</sup> subpopulations for expanded cells. To determine if the secreted proteins of these cells could augment the survival and/or proliferation of β-cells in vitro, conditioned media (CM) from cells expanded with or without BMS 493 was added to human islet cultures. The total number of proliferating β-cells was increased after 3 or 7-day culture with CM generated from BMS 493-treated cells. In contrast to freshly isolated ALDH<sup>hi</sup> cells, 6-day expansion with or without BMS 493 generated progeny that were unable to reduce hyperglycemia after iPan transplantation into STZ-treated NOD/SCID mice. Further strategies to reduce retinoic acid differentiation during HPC expansion is required to expand ALDH<sup>hi</sup> cells without the loss of islet regenerative functions.
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- Human mesenchymal stromal cell sheets induce macrophages predominantly to an anti-inflammatory phenotype. [Journal Article]
- SCStem Cells Dev 2018 May 08
- Tissue healing is a highly complex process involving a cascade of biochemical and cellular events. Excessive inflammation can impair the healing response. Previous in vitro studies have shown that me...
Tissue healing is a highly complex process involving a cascade of biochemical and cellular events. Excessive inflammation can impair the healing response. Previous in vitro studies have shown that mesenchymal stromal cells can modulate macrophage-induced inflammation and therefore are promising candidates for cell-based therapies aimed at promoting tissue repair. Recently, cell sheets were introduced as a new method of delivering stromal cells to the repair-site. The goal of the current study was to compare the effect of different types of stromal cell sheets on the inflammatory state of macrophages <i>in vitro</i>. We compared the effects of adipose tissue-derived stromal cell (ASC) sheets, bone marrow derived stromal cell (BMSC) sheets and fibroblast sheets on macrophage functional phenotype using flow cytometric analysis, gene expression as well as cell sheet protein secretion. This was evaluated with and without inflammatory stimulation. Viability and senescence for the different types of sheet were also evaluated. Macrophages cultured in ASC sheet conditioned medium (CM) displayed a higher fluorescence intensity of the anti-inflammatory CD206 surface marker than when cultured in BMSC sheet CM, and expressed more <i>CCL18</i> and <i>IL1RA</i> than when cultured in fibroblast sheet CM. Moreover, ASC sheets had higher cell viability and less senescent cells than BMSC sheets and fibroblast sheets. Taken together, ASC and BMSC can stimulate the anti-inflammatory macrophage (M2) phenotype to a better extent than fibroblasts. It is suggested, that ASC sheets might outperform BMSC sheets in an inflammatory situation since ASC sheet CM induced-macrophages have more M2 characteristics and ASC in the sheet were more viable.