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Design: The research used an in vitro cell exposure model and multi-omics integration of transcriptome and epigenome profiling to compare the molecular effects of e-cigarettes and tobacco smoke on dental stem cells., Aim: The study aimed to compare the effects of e-cigarette and tobacco smoke on periodontal stem cells using a multi-omics approach to understand gene regulation., Methods: This research studied primary human gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs) obtained from healthy donors. The cells were subjected to tobacco smoke, e-cigarette aerosol (both tobacco and menthol flavors), e-cigarette liquid (both tobacco and menthol flavors), or untreated conditions using an in vitro exposure system. RNA sequencing and bioinformatics analysis were used to profile the transcriptome and identify differential gene expression. Additionally, chromatin immunoprecipitation sequencing (ChIP-seq) was used to conduct genome-wide histone modification mapping for H3K27me3. Transcriptome profiling was combined with histone modification characterization to understand gene regulatory mechanisms. The study compared the effects of smoke versus e-cigarette, aerosol versus liquid exposure, and tobacco versus menthol flavor on gene expression and epigenetic landscapes in the two oral stem cell populations., Results: The use of tobacco smoke caused damage to the DNA and nucleus in GMSCs, as well as mitochondrial dysfunction in PDLSCs. Regarding e-cigarettes, the aerosol and liquid affected non-coding RNA expression differently. The chemokine CXCL2 was found to be downregulated by aerosol but upregulated by liquid in GMSCs. An integrative analysis revealed that the upregulation of CXCL2 caused by e-liquid involved reduced H3K27me3 and activation of distal enhancers. On the other hand, aerosol exposure maintained H3K27me3 levels, while direct e-liquid exposure resulted in genome-wide reductions in H3K27me3, particularly in enhancer regions. Overall, the specific delivery methods and components of e-cigarettes caused unique changes in the transcriptome and epigenome of oral stem cells., Conclusions: E-cigarettes affect oral stem cells differently than tobacco smoke. Their aerosol and liquid have varying impacts on gene expression and regulatory landscapes in oral cells. Multi-omics approaches are important to understanding the molecular changes caused by e-cigarette components. This can help with toxicological assessments and determine their impact on periodontal health. Transcriptome and epigenome profiling are powerful tools to examine the unique molecular mechanisms involved in cellular responses to e-cigarettes., (© 2023. The Author(s), under exclusive licence to British Dental Association.)

Context: Clinicians can use stem cells to repair kidney injury. The kidneys' exosome secretions hold the secret to this therapeutic impact. Exosomes from urine-derived stem cells can prevent and treat glomerular damage that diabetes can cause, but the underlying process has remained a mystery., Objective: The study aimed to investigate the protective impact of exosomes from urine-derived stem cells (USCs) against diabetic nephropathy (DN) and to determine the mechanisms involved., Design: The research team performed an animal study., Setting: The study took place at the Affiliated Hospital of Jiujiang University in Jiujiang, Jiangxi, China., Animals: The animals were rats, SD male rats, weighing 200-220g, 40 animals, purchased from Weitong Lihua Experimental Animal Technology Co., Ltd. (certificate number: SCXK (Beijing) 2021-0006)., Intervention: Except for a control group, the rats in the groups had induced DN. The five groups, with 10 rats each, were: (1) the negative control group, which received 0.2 ml of PBS solution; (2) the DN group, a second negative control group, which received 0.2 ml of PBS solution, (3) the inhibitor group, an intervention group that received 20 mg/kg of autophagy inhibitor; (4) the exosomes group, an intervention group that received 100 ug/kg of exosomes; and (5) the exosomes + inhibitor group, an intervention group that received 100 ug/kg of exosomes + 20 mg/kg of autophagy inhibitor. From week 8, for four weeks the team injected the inhibitor, exosomes, and exosomes + inhibitor groups with the appropriate treatments using the rats' tail veins., Outcome Measures: The research team: (1) examined the USCs in the exosomes of stem cells; (2) assessed the rats' weights and fasting blood glucose (FBG), using a blood glucose meter; (3) used Coomassie brilliant blue (CBB) staining to determine the amount of protein in the rats' urine and assessed their biochemical indexes; and (4) used Western blot (WB) and a quantitative polymerase chain reaction (Q-PCR) to detect autophagy and the signal transduction pathway., Results: Human exosomes from USCs alleviated injury in the rats that DN caused by reducing urinary-protein levels, serum creatinine (SCR), blood urea nitrogen (BUN), glomerular cell accumulation, and kidney weights. In rats with induced DN, the exosomes + inhibitor significantly reduced the activation of the mTOR signaling pathway, reduced the autophagy of their kidney cells, increased the protein expression of Bcl-2 in the kidney tissues, and lessened the damage to glomerular cells., Conclusions: Human urine-derived stem cell exosomes can significantly reduce the activation of the mTOR signaling pathway, reduce the autophagy of rats' kidney cells, increase the protein expression of LC3B in kidney tissues, and reduce the damage to glomerular cells. By blocking the mTOR signaling pathway, human urogenic exosomes can alleviate the signs and symptoms of DN.

Stem cell differentiation towards various somatic cells and body organs has proven to be an effective technique in the understanding and progression of regenerative medicine. Despite the advances made, concerns regarding the low efficiency of differentiation and the remaining differences between stem cell products and their in vivo counterparts must be addressed. Biomaterials that mimic endogenous growth conditions represent one recent method used to improve the quality and efficiency of stem cell differentiation, though the mechanisms of this improvement remain to be completely understood. The effectiveness of various biomaterials can be analyzed through a multidisciplinary approach involving bioinformatics and systems biology tools. Here, we aim to use bioinformatics to accomplish two aims: 1) determine the effect of different biomaterials on stem cell growth and differentiation, and 2) understand the effect of cell of origin on the differentiation potential of multipotent stem cells. First, we demonstrate that the dimensionality (2D versus 3D) and the degradability of biomaterials affects the way that the cells are able to grow and differentiate at the transcriptional level. Additionally, according to transcriptional state of the cells, the particular cell of origin is an important factor in determining the response of stem cells to same biomaterial. Our data demonstrates the ability of bioinformatics to understand novel molecular mechanisms and context by which stem cells are most efficiently able to differentiate. These results and strategies can be used to suggest proper combinations of biomaterials and stem cells to achieve high differentiation efficiency and functionality of desired cell types., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study experimentally substantiates that the micromechanical compatibility between cell and substrate is essential for cells to achieve energetically favorable mechanotransduction that directs phenotypic transitions. The argument for this compatibility is based on a thermodynamic model that suggests that the response of cells to their substrate mechanical environment is a consequence of the interchange between forms of energy governing the cell-substrate interaction. Experimental validation for the model has been carried out by investigating the osteogenic differentiation of dental follicle stem cells (DFSCs) seeded on electrospun fibrous scaffolds. Electrospinning of blends containing polycaprolactone (PCL) and silk fibroin (SF) with varying composition of cellulose nanocrystals (CNCs) resulted in three-dimensional (3D) fibrous scaffolds with bimodal distribution of fiber diameter, which provides both macroscopically stiff and microscopically compliant scaffolds for cells without affecting the surface chemical functionality of scaffolds. Atomic force microscopy (AFM) with a colloidal probe and single-cell force spectroscopy were used to characterize cell stiffness and scaffold stiffness on the cellular level, as well as cell-scaffold adhesive interaction (chemical functionality). This study has successfully varied scaffold mechanical properties without affecting their surface chemistry. In vitro tests indicate that the micromechanical compatibility between cells and scaffolds has been significantly correlated with mechanosensitive gene expression markers and osteogenic differentiation markers of DFSCs. The agreement between experimental observations and the thermodynamic model affirms that the cellular response to the mechanical environment, though biological in nature, follows the laws of the energy interchange to achieve its self-regulating behavior. More importantly, this study provides systematic evidence, through extensive and rigorous experimental studies, for the first time that rationalizes that micromechanical compatibility is indeed important to the efficacy of regenerative medicine.

Healing of ruptured tendons remains a clinical challenge because of its slow progress and relatively weak mechanical force at an early stage. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have therapeutic potential for tissue regeneration. In this study, we isolated EVs from adipose-derived stem cells (ADSCs) and evaluated their ability to promote tendon regeneration. Our results indicated that ADSC-EVs significantly enhanced the proliferation and migration of tenocytes in vitro. To further study the roles of ADSC-EVs in tendon regeneration, ADSC-EVs were used in Achilles tendon repair in rabbits. The mechanical strength, histology, and protein expression in the injured tendon tissues significantly improved 4 weeks after ADSC-EV treatment. Decorin and biglycan were significantly upregulated in comparison to the untreated controls. In summary, ADSC-EVs stimulated the proliferation and migration of tenocytes and improved the mechanical strength of repaired tendons, suggesting that ADSC-EV treatment is a potential highly potent therapeutic strategy for tendon injuries.

The present study demonstrated the protective effects of low-molecular-weight adipose-derived stem cell-conditioned medium (LADSC-CM) in a mouse model of dry eye syndrome. Mice subjected to desiccating stress and benzalkonium chloride had decreased tear secretion, impaired corneal epithelial tight junction with microvilli, and decreased conjunctival goblet cells. Topical application of adipose-derived stem cell-conditioned medium (ADSC-CM) stimulated lacrimal tear secretion, preserved tight junction and microvilli of the corneal epithelium, and increased the density of goblet cells and MUC16 expression in the conjunctiva. The low-molecular-weight fractions (< 10 kDa and < 3 kDa) of ADSC-CM (LADSC-CM) provided better protections than the > 10 kDa or > 3 kDa fractions of ADSC-CM. In the in vitro study, desiccation for 10 min or hyperosmolarity (490 osmols) for 24 h caused decreased viability of human corneal epithelial cells, which were reversed by LADSC-CM. The active ingredients in the LADSC-CM were lipophobic and stable after heating and lyophilization. Our study demonstrated that LADSC-CM had beneficial effects on experimental dry eye. It is worthy of further exploration for the active ingredient(s) and the mechanism., (© 2021. The Author(s).)

Talin is a cell adhesion molecule that is indispensable for the development and function of multicellular organisms. Despite its central role for many cell biological processes, suitable methods to investigate the nanoscale organization of talin in its native environment are missing. Here, we overcome this limitation by combining single-molecule resolved PAINT (points accumulation in nanoscale topography) imaging with the IRIS (image reconstruction by integrating exchangeable single-molecule localization) approach, enabling the quantitative analysis of genetically unmodified talin molecules in cells. We demonstrate that a previously reported peptide can be utilized to specifically label the two major talin isoforms expressed in mammalian tissues with a localization precision of <10 nm. Our experiments show that the methodology performs equally well as state-of-the-art single-molecule localization techniques, and the first applications reveal a thus far undescribed cell adhesion structure in differentiating stem cells. Furthermore, we demonstrate the applicability of this peptide-PAINT technique to mouse tissues paving the way to single-protein imaging of endogenous talin proteins under physiologically relevant conditions., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Administration of plerixafor with granulocyte-colony stimulating factor (G-CSF) mobilizes CD34+ cells much more effectively than G-CSF alone, but cost generally limits plerixafor use to patients at high risk of insufficient CD34+ cell collection based on low peripheral blood (PB) CD34+ counts following 4 days of G-CSF. We analyzed costs associated with administering plerixafor to patients with higher day 4 CD34+ cell counts to decrease apheresis days and explored the use of a fixed split dose of plerixafor instead of weight-based dosing. We analyzed 235 patients with plasma cell disorders or non-Hodgkin's lymphoma who underwent progenitor cell mobilization and autologous hematopoietic cell transplantation (AHCT) between March 2014 and December 2017. Two hundred ten (89%) received G-CSF plus Plerixafor and 25 (11%) received G-CSF alone. Overall, 180 patients (77%) collected in 1 day, 53 (22%) in 2 days and 2 (1%) in 3 days. Based on our data, we present a probabilistic algorithm to identify patients likely to require more than one day of collection using G-CSF alone. CD34+ cell yield, ANC and platelet recovery were not significantly different between fixed and standard dose plerixafor. Plerixafor enabled collection in 1 day and with estimated savings of $5000, compared to patients who did not receive plerixafor and required collection for three days. While collection and processing costs and patient populations vary among institutions, our results suggest re-evaluation of current algorithms., (© 2021 Wiley Periodicals LLC.)

Context: Healthy hyperplasic (many but smaller fat cells) white adipose tissue (WAT) expansion is mediated by recruitment, proliferation and/or differentiation of new fat cells. This process (adipogenesis) is controlled by transcriptional programs that have been mostly identified in rodents., Objective: A systemic investigation of adipogenic human transcription factors (TFs) that are relevant for metabolic conditions has not been revealed previously., Methods: TFs regulated in WAT by obesity, adipose morphology, cancer cachexia, and insulin resistance were selected from microarrays. Their role in differentiation of human adipose tissue-derived stem cells (hASC) was investigated by RNA interference (RNAi) screen. Lipid accumulation, cell number, and lipolysis were measured for all screened factors (148 TFs). RNA (RNAseq), protein (Western blot) expression, insulin, and catecholamine responsiveness were examined in hASC following siRNA treatment of selected target TFs., Results: Analysis of TFs regulated by metabolic conditions in human WAT revealed that many of them belong to adipogenesis-regulating pathways. The RNAi screen identified 39 genes that affected fat cell differentiation in vitro, where 11 genes were novel. Of the latter JARID2 stood out as being necessary for formation of healthy fat cell metabolic phenotype by regulating expression of multiple fat cell phenotype-specific genes., Conclusion: This comprehensive RNAi screening in hASC suggests that a large proportion of WAT TFs that are impacted by metabolic conditions might be important for hyperplastic adipose tissue expansion. The screen also identified JARID2 as a novel TF essential for the development of functional adipocytes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Rett syndrome (RTT) is a rare neurodevelopmental disorder that results in multiple disabilities. Exosomal microRNA (miRs) from urine-derived stem cells (USCs) have been shown to induce neurogenesis and aid in functional recovery from brain ischemia. In the present study, we sought to determine whether that exosomal miR-21-5p from USCs could promote early neural formation in a model of RTT. USCs were isolated and evaluated by flow cytometry. Exosomes were analyzed by transmission electron microscopy, tunable resistive pulse sensing (TRPS), and western blotting. PKH26 fluorescent dyes were used to observe intake of exosomes in vivo and in vitro. An RTT mouse model was treated with exosomes for behavioral studies. Dual-luciferase report gene assays were conducted to evaluate the relationship between miR-21-5p and Eph receptor A4 (EphA4). In vitro, treatment with exosomes from human urine-derived stem cells (USC-Exos) increased the percentage of neuron-specific class III beta-tubulin (Tuj1) + nerve cells as well as the transcription levels of β-III tubulin and doublecortin (DCX). A higher level of miR-21-5p was observed in USC-Exos, which promoted differentiation in NSCs by targeting the EPha4/TEK axis. In vivo, exosomal miR-21-5p improved the behavior, motor coordination, and cognitive ability of mice, facilitated the differentiation of NSCs in the subventricular zone of the lateral ventricle and promoted a marked rise in the number of DCX + cells. Our data provide evidence that exosomal miR-21-5p from human USCs facilitate early nerve formation by regulating the EPha4/TEK axis.

Finding a simple and effective way for transferring cells to the brain lesion site with minimum side effects mounts a challenge in cell therapy. Cell delivery via nasal route using the bypassing the blood-brain barrier (BBB) property is a simple and non-invasive strategy without serious complications such as trauma. Therefore, it is a suitable technique to treat neurodegenerative disorders like Parkinson's disease (PD). Olfactory ectomesenchymal stem cells (OE-MSCs) located in the lamina propria of olfactory mucosa could be differentiated into dopaminergic neurons under in vitro and in vivo conditions. Thus, OE-MSCs represent a good source of Parkinson's stem cell-based therapy. In this research, we studied thirty male rats (n = 10 in each group) in three control (Ctl), lesion (LE), and intranasal administration (INA) groups to investigate the therapeutic effect of intranasal injection of OE-MSCs in the Parkinson's animal models. To do so, we examined the homing variation of OE-MSCs in different brain regions such as olfactory bulb (OB), cortex, striatum (Str), hippocampus (HPC), and substantia nigra (SN). The results of real-time PCR and immunohistochemistry (IHC) analysis showed the expression of dopaminergic neuron markers such as PITX3, PAX2, PAX5 (as dopaminergic neurons markers), tyrosine hydroxylase (TH), and dopamine transporter (DAT) 2 months after INA of 1 × 10 6 OE-MSCs. The results confirmed that IN OE-MSCs delivery into the central nervous system (CNS) was powerful enough to improve the behavioral functions in the animal models of PD.

Background: Chronic wounds constitute a significant medical and social problem. Chronic wound treatment may be supported by various techniques, such as negative pressure therapy, phototherapy or stem cells therapy, yet most of those supporting therapies need more evidence to be used for standard wound care. Current study covers the use of sonicated Antlerogenic Stem Cells (ASC) extract on chronic wounds., Methods: Study was performed on 20 dermatological patients with venous leg ulcers, divided into two groups - treated with and without ASC extract respectively. The area and circumference of the wounds during the follow-up visits were measured on the wound imprint. Dynamics of wound healing was determined and compared between control and study group; statistics includes changes in absolute values (wound area, circumference), as well as relative (percentage of wound decrease, circumference/area ratio) and their change in time. For the purpose of Ki-67 immunohistochemical staining, sections were sampled from the wound edge at distinct check-points during therapy. Results of both groups were compared with Student test or Mann-Whitney test, depending on results distribution., Results: Besides Ki-67 expression, all tested wound healing parameters (including relative and absolute wound decrease and changes in circumference/area ratio) were statistically significant more favorable in experimental group., Conclusion: ASC extract significantly supported standard chronic wound treatment. Due to small population of study the results should be considered preliminary, yet promising for further research.

Regenerative medicine offers the potential to repair or substitute defective tissues by constructing active tissues to address the scarcity and demands for transplantation. The method of forming 3D constructs made up of biomaterials, cells, and biomolecules is called bioprinting. Bioprinting of stem cells provides the ability to reliably recreate tissues, organs, and microenvironments to be used in regenerative medicine. 3D bioprinting is a technique that uses several biomaterials and cells to tailor a structure with clinically relevant geometries and sizes. This technique's promise is demonstrated by 3D bioprinted tissues, including skin, bone, cartilage, and cardiovascular, corneal, hepatic, and adipose tissues. Several bioprinting methods have been combined with stem cells to effectively produce tissue models, including adult stem cells, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and differentiation techniques. In this review, technological challenges of printed stem cells using prevalent naturally derived bioinks (e.g., carbohydrate polymers and protein-based polymers, peptides, and decellularized extracellular matrix), recent advancements, leading companies, and clinical trials in the field of 3D bioprinting are delineated.

Limbal stem cells (LSCs) reside in the basal layer of limbal epithelial cells (LECs). They are crucial for maintenance of corneal epithelium homeostasis and corneal wound healing. Their stemness is determined by their gene expression pattern. Despite of several positive identifiers have been reported, the unique biomarker for LSCs still remain elusive. Differentially expressed genes (DEGs) between stem cells and differentiated cells affect the fate of stem cells via specific signaling pathway. In order to understand the DEGs in the LSCs, RNA-seq was firstly conducted using a mouse model. A total of 1907 up-regulated DEGs and 395 down-regulated DEGs were identified in the limbus (L) compared to central cornea (CC) and conjunctiva (Cj). Reliability of the expression of genes from RNA-seq analysis was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The expression pattern of putative biomarkers was considered to be age-related. In up-regulated DEGs GO analysis, 570 gene ontology (GO) terms were significantly enriched. Five groups of genes related with biological processes from these significantly enriched GO terms comprised ionic transport, regulation of tissue development, muscle contraction, visual perception, and cell adhesion, which were clustered as a weighted similar network. Whereas, in down-regulated DEGs GO analysis, 61 GO terms were significantly enriched and only one group of ATP biosynthesis and metabolic process were clustered. Furthermore, we identified 55 signaling pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database based on up-regulated genes and 14 KEGG pathways based on down-regulated genes. In this study, we provide a landscape of the expression of putative LSCs biomarkers and stemness-related signaling pathways in a mouse model. Our findings could aid in the identification of LSC niche factors that may be related to the stemness of the LSCs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Degeneration of the cartilage endplate (CEP) induces intervertebral disc degeneration (IVDD). Nucleus pulposus cell (NPC) apoptosis is also an important exacerbating factor in IVDD, but the cascade mechanism in IVDD is not clear. We investigated the apoptosis of NPCs and IVDD when stimulated by normal cartilage endplate stem cell (CESC)-derived exosomes (N-Exos) and degenerated CESC-derived exosomes (D-Exos) in vitro and in vivo. Tert-butyl hydroperoxide (TBHP) was used to induce inflammation of CESCs. The bioinformatics differences between N-Exos and D-Exos were analyzed using mass spectrometry, heat map, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. NPC apoptosis was examined using TUNEL staining. The involvement of the AKT and autophagy signaling pathways was investigated using the signaling inhibitor LY294002. Magnetic resonance imaging, Western blotting, and immunofluorescence staining were used to evaluate the therapeutic effects of N-Exos in rats with IVDD. TBHP effectively induced inflammation and the degeneration of CEP in rat. N-Exos were more conducive to autophagy activation than D-Exos. The apoptotic rate of NPCs decreased obviously after treatment with N-Exos compared to D-Exos. N-Exos inhibited NPCs apoptosis and attenuated IVDD in rat via activation of the AKT and autophagy pathways. These results are the first findings to confirm that CEP delayed the progression of IVDD via exosomes. The therapeutic effects of N-Exos on NPC apoptosis inhibition and the slowing of IVDD progression were more effective than D-Exos due to activation of the PI3K/AKT/autophagy pathway, which explained the increase in the incidence of IVDD after inflammation of the CEP., (©2021 The Authors. Stem Cells published by Wiley Periodicals LLC on behalf of AlphaMed Press 2020.)

Background: Extracellular vesicles (EVs) are a diverse group of membrane-bound nanovesicles potentially released by every cell. With the liver's unique ensemble of cells and its fundamental physiological tasks, elucidating the role of EV-mediated hepatic cellular crosstalk and their role in different pathologies has been gaining the attention of many scientists., Scope of Review: The present review shifts the perspective into practice: we aim to critically discuss the methods used to purify and to biochemically analyse EVs from specific liver resident cells, including hepatocytes, hepatic stellate cells, cholangiocytes, liver sinusoidal endothelial cells, Kupffer cells, liver stem cells. The review offers a reference guide to current approaches., Major Conclusions: Strategies for EV isolation and characterization are as varied as the research groups performing them. We present main advantages and disadvantages for the methods, highlighting common causes for concern, such as FBS handling, reporting of cell viability, EV yield and storage, differences in differential centrifugations, suboptimal method descriptions, and method transferability. We both looked at how adaptable the research between human and rodent cells in vitro is, and also assessed how well either of them translates to ex vivo settings., General Significance: We reviewed methodological practices for the isolation and analysis of liver-derived EVs, making a cell type specific user guide that shows where to start, what has worked so far and to what extent. We critically discussed room for improvement, placing a particular focus on working towards a potential standardization of methods., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Genetic lineage tracing is accomplished using bi-transgenic mice, where one allele is altered to express Cre recombinase, and another allele encodes a Cre-dependent genetic reporter protein. Once Cre is activated (constitutive or in response to tamoxifen), the marker gene-expressing cells become indelibly labeled by the reporter protein. Therefore, daughter cells derived from labeled cells are permanently labeled even if the marker gene that drove Cre recombinase expression is no longer expressed in these cells. This system is commonly used to label putative progenitor cells and determine the fate of their progeny. Here, we describe the use of c-kit-based genetic lineage-tracing mouse line as an example and discuss caveats for performing these types of experiments.

Stem cells and their derivatives are novel pharmaceutics that have the potential for use as tissue replacement therapies. However, the heterogeneous characteristics of stem cell cultures have hindered their biomedical applications. In theory and practice, when cell type-specific or stage-specific cell surface proteins are targeted by unique antibodies, they become highly efficient in detecting and isolating specific cell populations. There is a growing demand to identify reliable and actionable cell surface markers that facilitate purification of particular cell types at specific developmental stages for use in research and clinical applications. The identification of these markers as very important members of plasma membrane proteins, ion channels, transporters, and signaling molecules has directly benefited from proteomics and tools for proteomics-derived data analyses. Here, we review the methodologies that have played a role in the discovery of cell surface markers and introduce cutting edge single cell proteomics as an advanced tool. We also discuss currently available specific cell surface markers for stem cells and their lineages, with emphasis on the nervous system, heart, pancreas, and liver. The remaining gaps that pertain to the discovery of these markers and how single cell proteomics and identification of surface markers associated with the progenitor stages of certain terminally differentiated cells may pave the way for their use in regenerative medicine are also discussed.

Stroke is one of the most common causes of disability and death, and currently, ideal clinical treatment is lacking. Stem cell transplantation is a widely-used treatment approach for stroke. When compared with other types of stem cells, bone marrow mesenchymal stem cells (BMSCs) have been widely studied because of their many advantages. The paracrine effect is the primary mechanism for stem cells to play their role, and exosomes play an essential role in the paracrine effect. When compared with cell therapy, cell-free exosome therapy can prevent many risks and difficulties, and therefore, represents a promising and novel approach for treatment. In this study, we reviewed the research progress in the application of BMSCs-derived exosomes (BMSCs-exos) and BMSCs in the treatment of stroke. In addition, the advantages and disadvantages of cell therapy and cell-free exosome therapy were described, and the possible factors that hinder the introduction of these two treatments into the clinic were analyzed. Furthermore, we reviewed the current optimization methods of cell therapy and cell-free exosome therapy. Taken together, we hypothesize that cell-free exosome therapy will have excellent research prospects in the future, and therefore, it is worth further exploring. There are still some issues that need to be further addressed. For example, differences between the in vivo microenvironment and in vitro culture conditions will affect the paracrine effect of stem cells. Most importantly, we believe that more preclinical and clinical design studies are required to compare the efficacy of stem cells and exosomes., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Human trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether trophoblast stem cell-derived exosomes (TSC-Exos) can protect cardiomyocytes from doxorubicin (Dox)-induced injury remains unclear. In the present study, TSC-Exos were isolated from the supernatants of human trophoblasts using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. In vitro, primary cardiomyocytes were subjected to Dox and treated with TSC-Exos, miR-200b mimic or miR-200b inhibitor. Cellular apoptosis was observed by flow cytometry and immunoblotting. In vivo, mice were intraperitoneally injected into Dox to establish a heart failure model. Then, different groups of mice were administered either PBS, adeno-associated virus (AAV)-vector, AAV-miR-200b-inhibitor or TSC-Exos via tail vein injection. Then, the cardiac function, cardiac fibrosis and cardiomyocyte apoptosis in each group were evaluated, and the downstream molecular mechanism was explored. TSC-Exos and miR-200b inhibitor both decreased primary cardiomyocyte apoptosis. Similarly, mice receiving TSC-Exos and AAV-miR-200b inhibitor exhibited improved cardiac function, accompanied by reduced apoptosis and inflammation. The bioinformatic prediction and luciferase reporter results confirmed that Zeb1 was a downstream target of miR-200b and had an antiapoptotic effect. TSC-Exos attenuated doxorubicin-induced cardiac injury by playing antiapoptotic and anti-inflammatory roles. The underlying mechanism could be an increase in Zeb1 expression by the inhibition of miR-200b expression. In summary, this study sheds new light on the application of TSC-Exos as a potential therapeutic tool for heart failure.

Background: Endometrial regenerative cells (ERCs), a novel type of mesenchymal-like stem cells, were identified as an attractive candidate for immunoregulation and induction of cardiac allograft tolerance. However, the underlying mechanisms of ERCs in immune regulation still remain largely unclear. The present study is designed to determine whether the expression of Galectin-9 (Gal-9), a soluble tandem-repeat member of the galectin family, is crucial for ERC-based immunomodulation., Methods: In this study, we measured Gal-9 expression on ERCs and then co-cultured Gal-9-ERCs, ERCs, and ERCs+lactose (Gal-9 blocker) with activated C57BL/6-derived splenocytes. Furthermore, we performed mouse heart transplantation between BALB/c (H-2 d ) donor and C57BL/6 (H-2 b ) recipient. ERCs were administrated 24 h after the surgery, either alone or in combination with rapamycin., Results: Our data demonstrate that ERCs express Gal-9, and this expression is increased by IFN-γ stimulation in a dose-dependent manner. Moreover, both in vitro and in vivo results show that Gal-9-ERC-mediated therapy significantly suppressed Th1 and Th17 cell response, inhibited CD8 + T cell proliferation, abrogated B cell activation, decreased donor-specific antibody production, and enhanced the Treg population. The therapeutic effect of ERCs was further verified by their roles in prolonging cardiac allograft survival and alleviating graft pathological changes., Conclusions: Taken together, these data indicate that Gal-9 is required for ERC-mediated immunomodulation and prevention of allograft rejection.

Considerable knowledge has been acquired in inorganic nanoparticles' synthesis and nanoparticles' potential use in biomedical applications. Among different materials, iron oxide nanoparticles remain unrivaled for several reasons. Not only do they respond to multiple physical stimuli (e.g., magnetism, light) and exert multifunctional therapeutic and diagnostic actions but also they are biocompatible and integrate endogenous iron-related metabolic pathways. With the aim to optimize the use of (magnetic) iron oxide nanoparticles in biomedicine, different biophysical phenomena have been recently identified and studied. Among them, the concept of a "nanoparticle's identity" is of particular importance. Nanoparticles' identities evolve in distinct biological environments and over different periods of time. In this Account, we focus on the remodeling of magnetic nanoparticles' identities following their journey inside cells. For instance, nanoparticles' functions, such as heat generation or magnetic resonance imaging, can be highly impacted by endosomal confinement. Structural degradation of nanoparticles was also evidenced and quantified in cellulo and correlates with the loss of magnetic nanoparticle properties. Remarkably, in human stem cells, the nonmagnetic products of nanoparticles' degradation could be subsequently reassembled into neosynthesized, endogenous magnetic nanoparticles. This stunning occurrence might account for the natural presence of magnetic particles in human organs, especially the brain. However, mechanistic details and the implication of such phenomena in homeostasis and disease have yet to be completely unraveled.This Account aims to assess the short- and long-term transformations of magnetic iron oxide nanoparticles in living cells, particularly focusing on human stem cells. Precisely, we herein overview the multiple and ever-evolving chemical, physical, and biological magnetic nanoparticles' identities and emphasize the remarkable intracellular fate of these nanoparticles.

Local transplantation of epidermal stem cells (ESCs) exerts a therapeutic effect on burn wounds. However, cell viability can impede their clinical application. HOX antisense intergenic RNA (HOTAIR) is involved in regulating adult tissue stem cells, as well as in developmental patterning and pluripotency. However, little is known about its role in regulating ESCs. The present study was performed to investigate the effects of HOTAIR in the modulation of ESCs and wound repair. Firstly, reverse transcription‑quantitative PCR was used to detect the relative expression of HOTAIR during burn wound healing in mice to determine whether HOTAIR is associated with wound healing. Subsequently, ESCs derived from mouse skin were transfected with a lentiviral vector to overexpress or knockdown HOTAIR. The effects of HOTAIR on cell proliferation and differentiation were measured by 5‑bromodeoxyuridine and MTT assays, and by assessing NANOG mRNA expression. Lastly, mice with burns were administered a subcutaneous injection of HOTAIR‑overexpressing ESCs. Images were captured and histological analyses were performed to evaluate wound healing. The results revealed that the expression of HOTAIR gradually increased and peaked at day 7 post‑burn and maintained at relatively high levels until day 14 post‑burn during wound healing. Furthermore, overexpression of HOTAIR promoted ESC proliferation and maintained the stem cell state in vitro. By contrast, suppression of HOTAIR inhibited cell proliferation and cell stemness. It was also identified that HOTIR‑overexpressing ESCs accelerated re‑epithelialization and facilitated burn wound repair. In conclusion, the present findings confirmed an essential role of HOTAIR in the regulation of ESC proliferation and stemness. Therefore, targeting HOTAIR in ESCs may be a potentially promising therapy for burn wound healing.

The purpose of this study was to analyze in vitro the biological effects on human dental pulp stem cells triggered in response to substances leached or dissolved from two experimental cements for dental pulp capping. The experimental materials, based on extracts from Copaifera reticulata Ducke (COP), were compared to calcium hydroxide [Ca(OH) 2 ] and mineral trioxide aggregate (MTA), materials commonly used for direct dental pulp capping in restorative dentistry. For this, human dental pulp stem cells were exposed to COP associated or not with Ca(OH) 2 or MTA. Cell cytocompatibility, migration, and differentiation (mineralized nodule formation (Alizarin red assay) and gene expression (RT-qPCR) of OCN , DSPP , and HSP-27 (genes regulated in biomineralization events)) were evaluated. The results showed that the association of COP reduced the cytotoxicity of Ca(OH) 2 . Upregulations of the OCN , DSPP , and HSP-27 genes were observed in response to the association of COP to MTA, and the DSPP and HSP-27 genes were upregulated in the Ca(OH) 2 + COP group. In up to 24 h, cell migration was significantly enhanced in the MTA + COP and Ca(OH) 2 + COP groups. In conclusion, the combination of COP with the currently used materials for dental pulp capping [Ca(OH )2 and MTA] improved the cell activities related to pulp repair (i.e., cytocompatibility, differentiation, mineralization, and migration) including a protective effect against the cytotoxicity of Ca(OH) 2 ., Competing Interests: The authors declare no conflicts of interest.

Synthetic nanoparticles are extensively utilized in various biomedical engineering fields because of their unique physicochemical properties. However, their exogenous characteristics result in synthetic nanosystem invaders that easily induce the passive immune clearance mechanism, thereby increasing the retention effect caused by reticuloendothelial system (RES), resulting in low therapeutic efficacy and toxic effects. Recently, a cell membrane cloaking has been emerging technique as a novel interfacing approach from the biological/immunological perspective. This has been considered as useful technique for improving the performance of synthetic nanocarriers in vivo. By cell membrane cloaking, nanoparticles acquire the biological functions of natural cell membranes due to the presence of membrane-anchored proteins, antigens, and immunological moieties as well as physicochemical property of natural cell membrane. Due to cell membrane cloaking, the derived biological properties and functions of nanoparticles such as their immunosuppressive capability, long circulation time, and disease targeting ability have enhanced their future potential in biomedicine. Here, we review the cell membrane-cloaked nanosystems, highlight their novelty, introduce the preparation and characterization methods with relevant biomedical applications, and describe the prospects for using this novel biomimetic system that was developed from a combination of cell membranes and synthetic nanomaterials., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk.

Background: Crohn's disease (CD) is characterized by persistent inflammation and ulceration of the small or large bowel, and expansion of mesenteric adipose tissue, termed creeping fat (CF). We previously demonstrated that human adipose-derived stem cells (hASCs) from CF of patients with CD exhibit dysfunctional phenotypes, including a pro-inflammatory profile, high phagocytic capacity, and weak immunosuppressive properties. Importantly, these phenotypes persist in patients in remission and are found in all adipose depots explored including subcutaneous fat. We hypothesized that changes in hASCs are a consequence of epigenetic modifications., Methods: We applied epigenome-wide profiling with a methylation array (Illumina EPIC/850k array) and gene expression analysis to explore the impact of CD on the methylation signature of hASCs isolated from the subcutaneous fat of patients with CD and healthy controls (n = 7 and 5, respectively; cohort I). Differentially methylated positions (p value cutoff < 1 × 10 -4 and ten or more DMPs per gene) and regions (inclusion threshold 0.2, p value cutoff < 1 × 10 -2 and more than 2 DMRs per gene) were identified using dmpfinder and Bumphunter (minfi), respectively. Changes in the expression of differentially methylated genes in hASCs were validated in a second cohort (n = 10/10 inactive and active CD and 10 controls; including patients from cohort I) and also in peripheral blood mononuclear cells (PBMCs) of patients with active/inactive CD and of healthy controls (cohort III; n = 30 independent subjects)., Results: We found a distinct DNA methylation landscape in hASCs from patients with CD, leading to changes in the expression of differentially methylated genes involved in immune response, metabolic, cell differentiation, and development processes. Notably, the expression of several of these genes in hASCs and PBMCs such as tumor necrosis factor alpha (TNFA) and PR domain zinc finger protein 16 (PRDM16) were not restored to normal (healthy) levels after disease remission., Conclusions: hASCs of patients with CD exhibit a unique DNA methylation and gene expression profile, but the expression of several genes are only partially restored in patients with inactive CD, both in hASCs and PBMCs. Understanding how CD shapes the functionality of hASCs is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies. Human adipose-stem cells isolated from subcutaneous fat of patients with Crohn's disease exhibit an altered DNA methylation pattern and gene expression profile compared with those isolated from healthy individuals, with immune system, cell differentiation, metabolic and development processes identified as the main pathways affected. Interestingly, the gene expression of several genes involved in these pathways is only partially restored to control levels in patients with inactive Crohn's disease, both in human adipose-stem cells and peripheral blood mononuclear cells. Understanding how Crohn's disease shapes the functionality of human adipose-stem cells is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies.

Genetic lineage tracing is widely used to study organ development and tissue regeneration. Multicolor reporters are a powerful platform for simultaneously tracking discrete cell populations. Here, combining Dre-rox and Cre-loxP systems, we generated a new dual-recombinase reporter system, called Rosa26 traffic light reporter ( R26-TLR ), to monitor red, green, and yellow fluorescence. Using this new reporter system with the three distinct fluorescent reporters combined on one allele, we found that the readouts of the two recombinases Cre and Dre simultaneously reflect Cre + Dre - , Cre - Dre + , and Cre + Dre + cell lineages. As proof of principle, we show specific labeling in three distinct progenitor/stem cell populations, including club cells, AT2 cells, and bronchoalveolar stem cells, in Sftpc-DreER;Scgb1a1-CreER;R26-TLR mice. By using this new dual-recombinase reporter system, we simultaneously traced the cell fate of these three distinct cell populations during lung repair and regeneration, providing a more comprehensive picture of stem cell function in distal airway repair and regeneration. We propose that this new reporter system will advance developmental and regenerative research by facilitating a more sophisticated genetic approach to studying in vivo cell fate plasticity., (© 2020 Liu et al.)

Alternative splicing (AS) generates functionally distinct transcripts and is involved in multiple cellular processes, including stem cell differentiation. Several epithelial-to-mesenchymal transition-related splicing factors have also been associated with pluripotency. Concomitantly with the interest in studying AS in stem cell biology, the advent of next-generation sequencing of RNA (RNA-seq) has increased the public availability of transcriptomic data and enabled genome-wide AS studies. To facilitate performing such analyses in large publicly available or user-provided transcriptomics datasets, the psichomics R package provides an easy-to-use interface and efficient data visualization tools for AS quantification and integrative analyses of AS and gene expression data.psichomics is employed herein to study AS changes between human stem cells and fibroblasts, based on dimensionality reduction, and median- and variance-based differential AS and gene expression analyses. Putative RNA-binding protein regulators involved in those alterations are then identified based on correlation analyses in large cohorts of human tissue transcriptomes. We identified several alterations, both novel and previously reported, in alternative splicing events and in the expression of their candidate regulators that are associated with stem cell differentiation into fibroblasts.

Differentiation of stem cells into chondrocytes has been studied for the engineering of cartilage tissue. However, stem cells cultured two-dimensionally have limited ability to differentiate into chondrocytes, which led to the development of three-dimensional culture systems. A recently developed microtechnological method uses microwells as a tool to form uniformly sized spheroids. In this study, we fabricated an array (10 × 10) of goblet-shaped microwells based on polydimethylsiloxane for spheroid culture. A central processing unit (CPU) was used to form holes, and metallic beads were used to form hemispherical microwell geometry. The holes were filled with Pluronic F-127 to prevent cells from sinking through the holes and allowing the cells to form spheroids. Viability and chondrogenic differentiation of human adipose-derived stem cells were assessed. The fabrication method using a micro-pin mold and metallic beads is easy and cost-effective. Our three-dimensional spheroid culture system optimizes the efficient differentiation of cells and has various applications, such as drug delivery, cell therapy, and tissue engineering.

Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.

Human clinical specimens are a valuable source of tissue-resident stem cells, but such cells need to be collected immediately after tissue collection. To extend the timescale for collection from fresh human samples, we developed a new extracellular fluid (ECF)-type preservation solution based on a high-sodium and low-potassium solution containing low-molecular-weight dextran and glucose, which is used for preservation of organs for transplantation. In this study, we compared the preservation of tissue-resident stem cells using our ECF solution with that using three other solutions: PBS, Dulbecco's modified Eagle's medium and Euro-Collins solution. These solutions represent a common buffer, a common culture medium and a benchmark organ-preservation solution, respectively. Lung tissues were removed from mice and preserved for 72 h under low-temperature conditions. Of the solutions tested, only preservation in the ECF-type solution could maintain the proliferation and differentiation capacity of mouse lung tissue-resident stem cells. In addition, the ECF solution could preserve the viability and proliferation of human alveolar epithelial progenitor cells when stored for more than 7 days at 4 °C. The mean viability of human alveolar type II cells at 2, 5, 8 and 14 days of low-temperature preservation was 90.9%, 84.8%, 85.7% and 66.3%, respectively, with no significant differences up to 8 days. Overall, our findings show that use of our ECF-type preservation solution may maintain the viability and function of tissue-resident stem cells. Use of this preservation solution may facilitate the investigation of currently unobtainable human tissue specimens for human stem cell biology., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Three-dimensional (3D) bioprinting holds the promise to fabricate tissue and organ substitutes for regenerative medicine. However, the lack of bioactive inks to fabricate and support functional living constructs is one of the main limitations hindering the progress of this technology. In this study, a biofunctional human-based nanocomposite bioink (HUink) composed of platelet lysate hydrogels reinforced by cellulose nanocrystals is reported. When combined with suspended bioprinting technologies, HUink allows the biofabrication of 3D freeform constructs with high resolution and integrity, mimicking the hierarchical nano-to-macro fibrillary composition of native tissues. Remarkably, HUink supports bioprinting of stem cells with high viability immediately after extrusion and over long-term cell culture without the need for additional biochemical or animal-derived media supplementation. As opposed to typical polymer-based bioinks, the pool of growth factors, cytokines and adhesion proteins in HUink boosts cell spreading and proliferation, stimulating the fast production of cell-secreted extracellular matrix. This innovative bioprinting platform with unpaired biofunctionality allows the fabrication of complex freeform cell-laden constructs that can ultimately be applied in the development of xeno-free 3D tissue models for in vitro research or to develop tissue and organ surrogates for clinical applications.

Piwi-interacting RNAs (piRNAs) are important for repressing transposable elements (TEs) and modulating gene expression in germ cells, thereby maintaining genome stability and germ cell function. Although they are also important for maintaining germline stem cells (GSCs) in the Drosophila ovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. Here, we show that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identify more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3' UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, we show that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development., (© 2019 Story et al.)

Rationale: Articular cartilage injury is extremely common in congenital joint dysplasia patients. Genetic studies have identified Growth differentiation factor 5 (GDF5) as a shared gene in joint dysplasia and OA progression across different populations. However , few studies have employed GDF5 in biological regeneration for articular cartilage repair. Methods & Results: In the present study, we report identified genetic association between GDF5 loci and hip joint dysplasia with genome-wide association study (GWAS). GWAS and replication studies in separate populations achieved significant signals for GDF5 loci. GDF5 expression was dysregulated with allelic differences in hip cartilage of DDH and upregulated in the repaired cartilage in a rabbit cartilage defect model. GDF5 in vitro enhanced chondrogenesis and migration of bone marrow stem cells (BMSCs), GDF5 was tested in ectopic cartilage generation with BMSCs by GDF5 in nude mice in vivo. Genetically inspired, we further generated functional knee articular cartilage construct for cartilage repair by 3d-bioprinting a GDF5-conjugated BMSC-laden scaffold. GDF5-conjugated scaffold showed better cartilage repairing effects compared to control. Meanwhile, transplantation of the 3D-bioprinted GDF5-conjugated BMSC-laden scaffold in rabbit knees conferred long-term chondroprotection. Conclusions: In conclusion, we report identified genetic association between GDF5 and DDH with combined GWAS and replications, which further inspired us to generate a ready-to-implant GDF5-conjugated BMSC-laden scaffold with one-step 3d-bioprinting for cartilage repair., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The tremendous importance of microbiota in microbial homoeostasis, alterations in metabolism and both innate and adaptive immune systems has been well established. A growing body of evidence support that dysbiosis or compositional changes in gut microbiota is linked to the ageing of stem cells in terms of dysregulations of metabolism, aberrant activation of the immune system as well as promoting epigenetic instability of stem cell. In this concise review, we elucidate recent emerging topics on microbiotic alterations and underlying mechanisms in stem cell ageing., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Aneurysmal subarachnoid hemorrhage (aSAH), characterized by the extravasation of blood into the subarachnoid space caused by an intracranial aneurysm rupture, may lead to neurocognitive impairments and permanent disability and usually carries poor outcome. Dental or gingiva-derived stem cells have been shown to contribute to immune modulation and neuroregeneration, but the underlying mechanisms are unclear. In the present study, we sought to investigate whether dental pulp stem cells (DPSCs) secrete certain factor(s) that can ameliorate the neural damage and other manifestations in a rat aSAH model. Twenty-four hours after the induction of aSAH, microthrombosis, cortical vasoconstriction, and the decrease in microcirculation and tissue oxygen pressure were detected. Intrathecal administration of DPSC-derived conditioned media (DPSC-CM) ameliorated aSAH-induced vasoconstriction, neuroinflammation, and improved the oxygenation in the injured brain. Rotarod test revealed that the aSAH-induced cognitive and motor impairments were significantly improved by this DPSC-CM administration. Cytokine array indicated the major constituent of DPSC-CM was predominantly insulin growth factor-1 (IGF-1). Immunohistochemistry staining of injured brain tissue revealed the robust increase in Iba1-positive cells that were also ameliorated by DPSC-CM administration. Antibody-mediated neutralization of IGF-1 moderately deteriorated the rescuing effect of DPSC-CM on microcirculation, Iba1-positive cells in the injured brain area, and the cognitive/motor impairments. Taken together, the DPSC-derived secretory factors showed prominent therapeutic potential for aSAH. This therapeutic efficacy may include improvement of microcirculation, alleviation of neuroinflammation, and microglial activation; partially through IGF-1-dependent mechanisms.

Cardiovascular disease (CVD) is a major health problem worldwide. Since adult cardiomyocytes irreversibly withdraw from the cell cycle soon after birth, it is hard for cardiac cells to proliferate and regenerate after myocardial injury, such as that caused myocardial infarction (MI). Live cell-based therapies, which we term as first generation of therapeutic strategies, have been widely used for the treatment of many diseases, including CVD. However, cellular approaches have the problems of poor retention of the transplanted cells and the significant entrapment of the cells in the lungs when delivered intravenously. Another big problem is the low storage/shipping stability of live cells, which limits the manufacturability of living cell products. The field of chemical engineering focuses on designing large-scale processes to convert chemicals, raw materials, living cells, microorganisms, and energy into useful forms and products. By definition, chemical engineers conceive and design processes to produce, transform, and transport materials. This matches the direction that cell therapies are heading toward: "produce", from live cells to synthetic artificial cells; "transform", from bare cells to cell/matrix/factor combinations; and "transport". from simple systemic injections to targeted delivery. Thus, we hereby introduce the "chemical engineering of cell therapies" as a concept. In this Account, we summarize our recent efforts to develop chemical engineering approaches to repair injured hearts. To address the limitations of poor cellular retention and integration, the first step was the artificial manipulation of stem cells before injections (we term this the second generation of therapeutic strategies). For example, we took advantage of the natural infarct-targeting ability of platelet membranes by fusing them onto the surface of cardiac stromal/stem cells (CSCs). By doing so, we improved the rate at which they were delivered through the vasculature to sites of MI. In addition to modifying natural CSCs, we described a bioengineering approach that involved the encapsulation of CSCs in a polymeric microneedle patch for myocardium regeneration. The painless microneedle patches were used as an in situ delivery device, which directly transported the loaded CSCs to the MI heart. In addition to low cell retention, there are some other barriers that need to be addressed before further clinical application is viable, including the storage/shipping stability of and the evident safety concerns about live cells. Therefore, we developed the third generation of therapeutic strategies, which utilize cell-free approaches for cardiac cell therapies. Numerous studies have indicated that paracrine mechanisms reasonably explain stem cell based heart repair. By imitating or adapting natural stem cells, as well as their secretions, and using them in conjunction with biocompatible materials, we can simulate the function of natural stem cells while avoiding the complications association with the first and second generation therapeutic options. Additionally, we can develop approaches to capture endogenous stem cells and directly transport them to the infarct site. Using these third generation therapeutic strategies, we can provide unprecedented opportunities for cardiac cell therapies. We hope that our designs will promote the use of chemical engineering approaches to transform, transport, and fabricate cell-free systems as novel cardiac cell therapeutic agents for clinical applications.

Functional reprogramming of a differentiated cell toward pluripotent cell may have long-term applications in numerous aspects, especially in regenerative medicine. Evidences accumulating from recent studies suggest that cellular extracts from stem cells or pluripotent cells can induce epigenetic reprogramming and facilitate pluripotency in otherwise highly differentiated cell types. Epigenetic reprogramming using cellular extracts has gained increasing attention and applied to recognize the functional factors, acquire the target cell types, and explain the mechanism of reprogramming. Now, more and more researches have proved that cellular extract treatment is an important strategy of cellular reprogramming. Thus, this review mainly focused on the progresses and potential mechanisms in epigenetic reprogramming using cellular extracts.

Most bones in mammals display a limited capacity for natural large-scale repair. The ribs are a notable exception, yet the source of their remarkable regenerative ability remains unknown. Here, we identify a Sox9 -expressing periosteal subpopulation that orchestrates large-scale regeneration of murine rib bones. Deletion of the obligate Hedgehog co-receptor, Smoothened , in Sox9 -expressing cells prior to injury results in a near-complete loss of callus formation and rib bone regeneration. In contrast to its role in development, Hedgehog signaling is dispensable for the proliferative expansion of callus cells in response to injury. Instead, Sox9-positive lineage cells require Hh signaling to stimulate neighboring cells to differentiate via an unknown signal into a skeletal cell type with dual chondrocyte/osteoblast properties. This type of callus cell may be critical for bridging large bone injuries. Thus despite contributing to only a subset of callus cells, Sox9 -positive progenitors play a major role in orchestrating large-scale bone regeneration., Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., Competing Interests: SK, MS, VV, NT, NH, JL, JC, FM No competing interests declared, (© 2019, Kuwahara et al.)

Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells. A similar technique that has raised recent interest is nanoinjection. The pipettes used here are much smaller and allow for the precise deposition of molecules into single cells via electrokinetics with minimal influence on the cells' health. Unfortunately, the equipment utilized for nanoinjection originates from scanning ion conductance microscopy (SICM) and is therefore expensive and not portable, but usually fixed to a specific microscope setup. The level of precision that these systems achieve is much higher than what is needed for the more robust nanoinjection process. We present Mobile Nanoinjection (MoNa), a portable, cost-efficient and easy to build system for the injection of single cells. Sacrificing unnecessary sub-nanometer accuracy and low ion current noise levels, we were able to inject single living cells with high accuracy. We determined the noise of the MoNa system and investigated the injection conditions for 16 prominent fluorescent labels and fluorophores. Further, we performed proof of concepts by injection of ATTO655-Phalloidin and MitoTracker Deep Red to living human osteosarcoma (U2OS) cells and of living adult human inferior turbinate stem cells (ITSC's) following neuronal differentiation with the MoNa system. We achieved significant cost reductions of the nanoinjection technology and gained full portability and compatibility to most optical microscopes.

Accumulating evidence revealed that hypoxia contributed to many human diseases, including ischemic myocardium and heart failure (HF). In recent years, the roles of hypoxia in stem cell survival and cardiac biology have been studied extensively. However, the underlying molecular mechanisms remain to be elucidated. As a leading cause of HF, ischemic heart disease was correlated with hypoxia. In this study, we firstly constructed the hypoxia cell model by CoCl 2 in cardiac stem cells (CSCs) and found that hypoxia induced the cell proliferation and migration potential in CSCs. Then, we demonstrated that the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was promoted in CoCl 2 -induced CSCs hypoxia model. Furthermore, we found that knockdown of MALAT1 inhibited the cell proliferation and migration in CoCl 2 -induced CSCs hypoxia model. In addition, we revealed that MALAT1 regulated the microRNA-155 (miR-155) expression in CSCs under both the normal and hypoxia conditions and further, manipulation of the miR-155 expression affected the role of MALAT1 in CoCl 2 -induced CSCs hypoxia cell model. We then illustrated that miR-155 regulated the myocyte enhancer factor 2A (MEF2A) expression in CSCs under both the normal and hypoxia conditions and further, changing the expression of MEF2A affected the role of miR-155. Finally, we demonstrated that MALAT1 regulated the MEF2A expression and exerted its role via modulation of the MALAT1/miR-155/MEF2A pathway. Taken together, our study illustrated that MALAT1 promoted the cell proliferation and migration in CoCl 2 -induced CSCs hypoxia model, acting mechanistically by promoting MEF2A expression via "sponging" miR-155., (© 2018 Wiley Periodicals, Inc.)

SOX2 positive pituitary stem cells (PSCs) are specified embryonically and persist throughout life, giving rise to all pituitary endocrine lineages. We have previously shown the activation of the STK/LATS/YAP/TAZ signalling cascade in the developing and postnatal mammalian pituitary. Here, we investigate the function of this pathway during pituitary development and in the regulation of the SOX2 cell compartment. Through loss- and gain-of-function genetic approaches, we reveal that restricting YAP/TAZ activation during development is essential for normal organ size and specification from SOX2+ PSCs. Postnatal deletion of LATS kinases and subsequent upregulation of YAP/TAZ leads to uncontrolled clonal expansion of the SOX2+ PSCs and disruption of their differentiation, causing the formation of non-secreting, aggressive pituitary tumours. In contrast, sustained expression of YAP alone results in expansion of SOX2+ PSCs capable of differentiation and devoid of tumourigenic potential. Our findings identify the LATS/YAP/TAZ signalling cascade as an essential component of PSC regulation in normal pituitary physiology and tumourigenesis., Competing Interests: EL, AS, JR, PX, TJ, RJ, ST, SB, CA No competing interests declared, (© 2019, Lodge et al.)

Background: The pathogenesis of immunological tolerance caused by avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, is largely unknown., Results: In this study, the development, differentiation, and immunological capability of B cells and their progenitors infected with ALV-J were studied both morphologically and functionally by using a model of ALV-J congenital infection. Compared with posthatch infection, congenital infection of ALV-J resulted in severe immunological tolerance, which was identified as the absence of detectable specific antivirus antibodies. In congenitally infected chickens, immune organs, particularly the bursa of Fabricius, were poorly developed. Moreover, IgM-and IgG-positive cells and total immunoglobulin levels were significantly decreased in these chickens. Large numbers of bursa follicles with no differentiation into cortex and medulla indicated that B cell development was arrested at the early stage. Flow cytometry analysis further confirmed that ALV-J blocked the differentiation of CD117 + chB6 + B cell progenitors in the bursa of Fabricius. Furthermore, both the humoral immunity and the immunological capability of B cells and their progenitors were significantly suppressed, as assessed by (a) the antibody titres against sheep red blood cells and the Marek's disease virus attenuated serotype 1 vaccine; (b) the proliferative response of B cells against thymus-independent antigen lipopolysaccharide (LPS) in the spleen germinal centres; and (c) the capacities for proliferation, differentiation and immunoglobulin gene class-switch recombination of B cell progenitors in response to LPS and interleukin-4(IL-4) in vitro., Conclusions: These findings suggested that the anergy of B cells in congenitally infected chickens is caused by the developmental arrest and dysfunction of B cell progenitors, which is an important factor for the immunological tolerance induced by ALV-J.

Two-dimensional electrophoresis is useful for separation of individual proteins that can be easily isolated and processed with mass spectrometry for their identification. Here we describe a simplified method of 2D electrophoresis with aim to help beginners in optimization of protein separation protocol, which can be further modified to enhance protein spot resolution. Current protocol can be used in different fields of biology including progenitor cells.

Tissue engineering by self-assembly allows for the fabrication of living tissue surrogates by taking advantage of the cell's inherent ability to produce and deposit tissue-specific extracellular matrix. However, the long culture periods required to build a tissue substitute in conducive to phenotypic drift in vitro microenvironments result in phenotype and function losses. Although several biophysical microenvironmental modulators (e.g., surface topography, substrate stiffness, mechanical stimulation) have been used to address these issues, slow extracellular matrix deposition remains a limiting factor in clinical translation and commercialization of such therapies. Macromolecular crowding is an alternative in vitro microenvironment modulator that has been shown to accelerate extracellular matrix deposition by several orders of magnitude, thereby decreasing culture periods required for the development of an implantable device, while maintaining cell phenotype and function. Herein, we provide protocols for the production of tissue surrogates rich in extracellular matrix from human dermal fibroblasts, equine tenocytes, and equine adipose-derived stem cells using the principles of macromolecular crowding and the subsequent characterization thereof by means of immunofluorescent staining and complementary fluorescence intensity analysis.

Background: Wnt-β-catenin signaling is essential for homeostasis of intestinal stem cells in mice and is thought to promote intestinal crypt fission., Aims: The aim of this study was to investigate Wnt-β-catenin signaling in intestinal crypts of human infants., Methods: Duodenal biopsies from nine infants (mean, range 0.9 years, 0.3-2 years) and 11 adults (mean, range 43 years, 34-71 years) were collected endoscopically. Active β-catenin signaling was assessed by cytoplasmic and nuclear β-catenin, nuclear c-Myc, and cytoplasmic Axin-2 expression in the base of crypts. Tissues were stained by an immunoperoxidase staining technique and quantified as pixel energy using cumulative signal analysis. Data were expressed as mean ± SD and significance assessed by Student's t test., Results: Crypt fission was significantly higher in infants compared to adults (16 ± 8.6% versus 0.7 ± 0.6%, respectively, p < 0.0001). Expression of cytoplasmic and nuclear β-catenin was 1.8-fold (p < 0.0001) and 2.9-fold (p < 0.0001) higher in infants, respectively, while cytoplasmic Axin-2 was 3.1-fold (p < 0.0001) increased in infants. c-Myc expression was not significantly different between infants and adults. Expression was absent in Paneth cells but present in the transit amplifying zone of crypts. Crypt base columnar cells, which were intercalated between Paneth cells, expressed c-Myc., Conclusions: Wnt-β-catenin signaling was active in crypt base columnar cells (i.e., intestinal stem cells) in human infants. This signaling could promote crypt fission during infancy. Wnt-β-catenin signaling likely acts in concert with other pathways to promote postnatal growth.

Bioengineering strategies to enhance the natural targeting function of nanocarriers would expand their therapeutic applications. Here, we designed bioengineered stem cell membrane-functionalized nanocarriers (BSMNCs) harboring C-X-C chemokine receptor type 4 (CXCR4) to achieve robust targeting and also to increase their retention time in ischemic tissue. Stem cell membrane coated nanocarrier (SMNCs) or poly (lactic-co-glycolic acid) (PLGA) nanocarriers (PNCs) and BSMNCs were prepared by functionalizing PNCs with human adipose-derived stem cells (hASCs) membranes and hASCs engineered to overexpress CXCR4-receptor, respectively. The functionalization of PNCs with stem cell membranes derived from hASCs significantly enhance the nanocarrier penetration across endothelial cell barrier compare to PNCs. In addition, stem cell membrane functionalization on PNCs also significantly decreased the nanoparticles uptake in J774 (murine) and THP (human) macrophages respectively from 84% to 76%-29% and 24%. Interestingly, BSMNCs showed much higher level of accumulation in ischemic tissue than SMNCs. Systemic retro-orbital injection of BSMNCs loaded with VEGF into mice with hindlimb ischemia resulted substantially enhancement of blood reperfusion, muscle repair, and limb salvage compared to animals treated with SMNCs loaded with similar concentration of VEGF. The reported strategy could be used to create biocompatible and custom-tailored biomimetic nanoparticles with various hybrid functionalities, which may overcome the limitations of current nanoparticle-based therapeutic and imaging platforms., (Copyright © 2018 Elsevier Ltd. All rights reserved.)