Photobiomodulation:a novel approach to promote trans-differentiation of adipose-derived stem cells into neuronal-like cells

Photobiomodulation,originally used red and near-infrared lasers,can alter cellular metabolism.It has been demonstrated that the visible spectrum at 451-540 nm does not necessarily increase cell proliferation,near-infrared light promotes adipose stem cell proliferation and affects adipose stem cell migration,which is necessary for the cells homing to the site of injury.In this in vitro study,we explored the potential of adipose-derived stem cells to differentiate into neurons for future translational regenerative treatments in neurodegenerative disorders and brain injuries.We investigated the effects of various biological and chemical inducers on trans-differentiation and evaluated the impact of photobiomodulation using 825 nm near-infrared and 525 nm green laser light at 5 J/cm2.As adipose-derived stem cells can be used in autologous grafting and photobiomodulation has been shown to have biostimulatory effects.Our findings reveal that adipose-derived stem cells can indeed trans-differentiate into neuronal cells when exposed to inducers,with pre-induced cells exhibiting higher rates of proliferation and trans-differentiation compared with the control group.Interestingly,green laser light stimulation led to notable morphological changes indicative of enhanced trans-differentiation,while near-infrared photobiomodulation notably increased the expression of neuronal markers.Through biochemical analysis and enzyme-linked immunosorbent assays,we observed marked improvements in viability,proliferation,membrane permeability,and mitochondrial membrane potential,as well as increased protein levels of neuron-specific enolase and ciliary neurotrophic factor.Overall,our results demonstrate the efficacy of photobiomodulation in enhancing the trans-differentiation ability of adipose-derived stem cells,offering promising prospects for their use in regenerative medicine for neurodegenerative disorders and brain injuries.

Reversal of hyperglycemia in diabetic rats by portal vein transplantation of islet-like cells generated from bone marrow mesenchymal stem cells

AIM: To study the capacity of bone marrow mesenchymal stem cells (BM-MSCs) trans-differentiating into islet-like cells and to observe the effect of portal vein transplantation of islet-like cells in the treatment of streptozotocin-induced diabetic rat. METHODS: BM-MSCs were isolated from SD rats and induced to differentiate into islet-like cells under defined conditions. Differentiation was evaluated with electron microscopy, RT-PCR, immunofluorescence and flow cytometry. insulin release after glucose challenge was tested with ELiSA. Then allogeneic islet-like cells were transplanted into diabetic rats via portal vein. Blood glucose levels were monitored and islet hormones were detected in the liver and pancreas of the recipient by immunohistochemistry. RESULTS: BM-MSCs were spheroid adherent monolayers with high CD90, CD29 and very low CD45 expression. Typical islet-like cells clusters were formed after induction. Electron microscopy revealed that secretory granules were densely packed within the cytoplasm of the differentiated cells. The spheroid cells expressed islet related genes and hormones. The insulin-positive cells accounted for 19.8% and mean fluorescence intensity increased by 2.6 fold after induction. The cells secreted a small amount of insulin that was increased 1.5 fold after glucose challenge. After transplantation, islet-like cells could locate in the liver expressing islet hormones and lower the glucose levels of diabetic rats during d 6 to d 20.CONCLUSION: Rat BM-MSCs could be transdifferentiated into islet-like cells in vitro . Portal vein transplantation of islet-like cells could alleviate the hyperglycemia of diabetic rats.

Skeletal Muscle-derived Stem Cells Exhibit Cardiocyte Competences

Adult stem cells from skeletal muscle cells were induced to differentiate into cardiocytes to see if stem cells from another different but histologically-comparable tissues can differentiate to the target cells. Skeletal muscles-derived stem cells （MDSCs） were isolated from adult skeleton muscle tissues by differential adhesion, and immunocytochemically identified by using Sca-1. In order to induce the proliferation but not differentiation of MDSCs, the cells were cultured in Dulbecco’s modified Eagle’s medium/F12 （DMEM/F12） supplemented with 1：50 B27, 20 ng/mL basic fibroblast growth factor （bFGF）, 20 ng/mL epidermal growth factor （EGF） in a suspension for 6 days. Then these stem cells were treated with 5 μmol/L 5-azacytidine for 24 h in an adherence culture. The characteristics of induced cells were examined by immunocytochemistry, quantitative real time RT-PCR and morphological observation of cell phenotype. Our results showed that the appearance of some cells gradually changed from spindle-shape into polygonal or short-column-shape. Some of these post-treated cells could contract spontaneously and rhythmically. The expression of GATA-4 and cTnT was increased 1 and 2 week（s） after the treatment. And about 16.6% of post-treated cells were cTnT-positive. Therefore, we are led to conclude that skeletal muscle-derived stem cells could differentiate into cardiocyte-like cells, which exhibited some characteristics of cardiocytes.

Complement-mediated perivascular inflammation contributes to hypertensive vascular injury

Adipose tissue plays pivotal roles in the development of hypertension,including white and brown adipocytes.Immunity and inflammation provide a bridge between adipose dysfunction and hypertensive target organ damage.We firstly found that perivascular adipose tissue(PVAT)expressed abundant C3,which stimulated adventitial fibroblast migration and phenotype trans-differentiation.Subsequently,we showed that C5a regulated M1/M2 macrophage polarization and inhibited adiponectin production in the PVAT,which contributed to vascular inflammation in hypertension.

Resolving the lineage relationship between malignant cells and vascular cells in glioblastomas

Glioblastoma multiforme(GBM),a highly malignant and heterogeneous brain tumor,contains various types of tumor and non-tumor cells.Whether GBM cells can trans-differentiate into non-neural cell types,including mural cells or endothelial cells(ECs),to support tumor growth and invasion remains controversial.Here we generated two genetic GBM models de novo in immunocompetent mouse brains,mimicking essential pathological and molecular features of human GBMs.Lineage-tracing and transplantation studies demonstrated that,although blood vessels in GBM brains underwent drastic remodeling,evidence of trans-differentiation of GBM cells into vascular cells was barely detected.Intriguingly,GBM cells could promiscuously express markers for mural cells during gliomagenesis.Furthermore,single-cell RNA sequencing showed that patterns of copy number variations(CNVs)of mural cells and ECs were distinct from those of GBM cells,indicating discrete origins of GBM cells and vascular components.Importantly,single-cell CNV analysis of human GBM specimens also suggested that GBM cells and vascular cells are likely separate lineages.Rather than expansion owing to trans-differentiation,vascular cell expanded by proliferation during tumorigenesis.Therefore,cross-lineage trans-differentiation of GBM cells is very unlikely to occur during gliomagenesis.Our findings advance understanding of cell lineage dynamics during gliomagenesis,and have implications for targeted treatment of GBMs.

The overexpression of Rps14 in Lgr5+progenitor cells promotes hair cell regeneration in the postnatal mouse cochlea

Sensory hair cells(HCs)in the cochlea cannot regenerate spontaneously in adult mammals after being damaged by external or genetic factors.However,several genes and signaling pathways are reported to induce cochlear HC regeneration at the early neonatal stage.Rps14 encodes a ribosomal protein that is involved in the regulation of cell differentiation and proliferation in mammals.However,its roles in the cochlea have not been reported in vivo.Here,we specifically overexpressed Rps14 in Lgr5+progenitor cells in the newborn mouse cochlea and found that Rps14 conditional overexpression(cOE)mice had significantly increased the ectopic HCs,including inner and outer HCs.We further explored the source of these ectopic HCs and found no EdU+supporting cells observed in the Rps14 cOE mice.The lineage tracing results,on the other hand,revealed that Rps14 cOE mice had significantly more tdTomato+HCs in their cochleae than control mice.These results indicated that regenerated HCs by cOE of Rps14 are most likely derived from inducing the direct trans-differentiation of Lgr5+progenitor cells into HCs.Moreover,real-time qPCR results suggested that the transcription factor genes Atoh1 and Gfi1,which are important in regulating HC differentiation,were upregulated in the cochlear basilar membrane of Rps14 cOE mice.In summary,this study provides in vivo evidence that,in the postnatal mouse cochlea,Rps14 is a potential gene that can promote the spontaneous trans-differentiation of Lgr5+progenitor cells into HCs.This gene may one day be exploited as a therapeutic target for treating hearing loss.

Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epi-dermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immuno-histology and RT-PCR were conducted to identify the expression of specific markers (β1, α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epi-dermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being co-cultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β 1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.

Transitions between epithelial and mesenchymal states during cell fate conversions

Cell fate conversion is considered as the changing of one type of cells to another type including somatic cell reprogramming （de-differentiation）, differentiation, and trans-differentiation, Epithelial and mesenchymal cells are two major types of cells and the transitions between these two cell states as epithelial-mesenchymal transi- tion （EMT） and mesenchymal-epithelial transition （MET） have been observed during multiple cell fate conversions including embryonic development, tumor progression and somatic cell reprogramming. In addition, MET and sequential EMT-MET during the generation of induced pluripotent stem cells （iPSC） from fibroblasts have been reported recently. Such observation is consistent with multiple rounds of sequential EMT-MET during embryonic development which could be considered as a reversed process of reprogramming at least partially. Therefore in current review, we briefly discussed the potential roles played by EMT, MET, or even sequential EMT-MET during different kinds of cell fate conversions. We also provided some preliminary hypotheses on the mechanisms that connect cell state transitions and cell fate conversions based on results collected from cell cycle, epigenetic regulation, and sternness acquisition.

The role of dendritic cells derived osteoclasts in bone destruction diseases

The bone is previously considered as a dominant organ involved in the processes of locomotion.However,in the past two decades,a large number of studies have suggested that the skeletal system closely coordinated with the immune system so as to result in the emerging area of'osteoimmunology'.In the evolution of many kinds of bone destruction-related dis-eases,osteoclasts could differentiate from dendritic cells,which contributed to increased expression of osteoclast-related membrane receptors and relatively higher activity of bone destruction,inducing sewere bone destruction under inflammatory conditions.Numerous fac-tors could influence the interaction between osteoclasts and dendritic cells,contributing to the pathogenesis of several bone diseases in the context of inflammation,including both im-munocytes and a large number of cytokines.In addition,the products of osteoclasts released from bone destruction area serve as important signals for the differentiation and activation of immature dendritic cells.Therefore,the border between the dendritic cell-rela ted immune response and osteoclast-related bone destruction has gradually unravelled.Dendritic cells and osteoclasts cooperate with each other to mediate bone destruction and bone remodelling under inflammatory conditions.In this review,we will pay attention to the interactions be-tween dendritic cells and osteoclasts in physiological and pathological conditions to further understand the skeletal system and identifty potential new therapeutic targets for the future by summarizing their significant roles and molecular mechanisms in bone destruction.

Reprogramming somatic cells to cells with neuronal characteristics by defined medium both in vitro and in vivo

Background:Currently,direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds.Delivery of transcriptional factors may affect genome stability,while small-molecule compounds may require more evaluations when applied in vivo.Thus,a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation.Results:Here,we report that a defined medium(5C)consisting of basic fibroblast growth factor(bFGF),N2 supplement,leukemia inhibitory factor,vitamin C(Vc),andβ-mercaptoethanol(βMe)induces the direct conversion of somatic cells to cells with neuronal characteristics.Application of 5C medium converted mouse embryonic fibroblasts(MEFs)into TuJ+neuronal-like cells,which were capable of survival after being transplanted into the mouse brain.The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury,possibly by inducing similar conversions,when infused into the mouse brain in vivo.Crucially,5C medium could also induce neuronal characteristics in several human cell types.Conclusions:In summary,this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.

Regeneration of β cells from cell phenotype conversion among the pancreatic endocrine cells

The prevalence of diabetes has increased dramatically, with over 537 million adults affected worldwide.1 In both type 1 and type 2 diabetes, pancreatic islet β cell dysfunction is common pathogenesis.

Transitions between mesenchymal and epithelial states and the concomitant gene expression changes

Epithelial-mesenchymal transition(EMT),mesenchymal-epithelial transition(MET),and even the sequential EMT-MET can be observed during multiple cell fate conversions including cancer progression and embryonic development.In the current review,we first focused on the existence and beneficial roles of the sequential EMT-MET during three typical cell fate conversions,differentiation from pluripotent stem cells(PSCs)to neurons,de-differentiation from mouse embryonic fibroblasts(MEFs)to PSCs,and trans-differentiation from MEFs to neurons.We tried to provide some preliminary hypotheses to connect EMT-MET and cell fate conversions,like the possible contributions of the intermediate mesenchymal state to iPSCs generation and neurontrans-differentiation.The intermediate mesenchymal state during sequential EMT-MET was further discussed by exploring the conserved signatures on gene expression during a variety of EMT.Discussion on the interaction among vitamin C,DNA methylation,and EMT/MET was also provided.