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A common genetic variant within the T cell receptor alpha (TCRA)-T cell receptor delta (TCRD) locus (rs2204985) has been recently found to associate with thymic function. Aim of this study was to investigate the potential impact of donor rs2204985 genotype on patient’s outcome after unrelated hematopoietic stem cell transplantation (uHSCT). 2016 adult patients were retrospectively analyzed. rs2204985 genotyping was performed by next generation sequencing, p < 0.05 was considered significant and donor rs2204985 GG/AG genotypes were set as reference vs. the AA genotype. Multivariate analysis of the combined cohort regarding the impact of donor’s rs2204985 genotype indicated different risk estimates in 10/10 and 9/10 HLA matched transplantations. A subanalysis on account of HLA incompatibility revealed that donor AA genotype in single HLA mismatched cases (n = 624) associated with significantly inferior overall- (HR: 1.48, p = 0.003) and disease-free survival (HR: 1.50, p = 0.001). This effect was driven by a combined higher risk of relapse incidence (HR: 1.40, p = 0.026) and non-relapse mortality (HR: 1.38, p = 0.042). This is the first study to explore the role of rs2204985 in a clinical uHSCT setting. Our data suggest that donor rs2204985 AA genotype in combination with single HLA mismatches may adversely impact post-HSCT outcome and should thus be avoided., publishedVersion

There is paucity of data on COVID-19 vaccine hesitancy amongst parents of pediatric (age ≤ 17 years) hematopoietic cell transplant (HCT) recipients. We conducted a cross-sectional study to determined COVID-19 vaccine hesitancy, and COVID-19 impact on family and related distress in this population. A national group (n = 80) was recruited via social media (Facebook) from February−May 2021. With vaccine approval for ≥12 years in July 2021, a second group (n = 37) was recruited locally. Parents completed surveys including the Vaccine Hesitancy Questionnaire and COVID-19 Exposure and Family Impact Scale (CEFIS). Nonparametric statistics were used to analyze results and factors impacting Vaccine Hesitancy Scores (VHS). The majority of parents were non-Hispanic White (≥90%) and children ≥3 months post-HCT (85%). Mean CEFIS score (scale 0–60) was 41.11 (SD = 8.24), with higher scores indicating negative impact of the pandemic. Mean (± standard deviation) VHS was 2.87 (±0.79) on a scale of 1–4, with 1 indicating higher and 4 lower hesitancy. Concerns about vaccine related side effects, lower parental age, child age, household income, and education were associated with lower VHS. Receiving reliable information and recommendations by providers was associated with higher VHS. Improving vaccine acceptance in this population is critical in protecting pediatric HCT recipients.

Fine particulate matter (PM) has a small diameter but a large surface area; thus, it may have broad toxic effects that subsequently damage many tissues of the human body. Interestingly, many studies have suggested that the recent decline in female fertility could be associated with increased PM exposure. However, the precise mechanisms underlying the negative effects of PM exposure on female fertility are still a matter of debate. A previous study demonstrated that resident stem cell deficiency limits the cyclic regenerative capacity of the endometrium and subsequently increases the pregnancy failure rate. Therefore, we hypothesized that PM exposure induces endometrial tissue damage and subsequently reduces the pregnancy rate by inhibiting various beneficial functions of local endometrial stem cells. Consistent with our hypothesis, we showed for the first time that PM exposure significantly inhibits various beneficial functions of endometrial stem cells, such as their self-renewal, transdifferentiation, and migratory capacities, in vitro and in vivo through the PM target gene SERPINB2, which has recently been shown to be involved in multiple stem cell functions. In addition, the PM-induced inhibitory effects on the beneficial functions of endometrial stem cells were significantly diminished by SERPINB2 depletion. Our findings may facilitate the development of promising therapeutic strategies for improving reproductive outcomes in infertile women., Reproductive health: Pollution is problematic for female fertility Airborne pollutants may reduce female fertility through their debilitating effects on the stem cells that maintain the endometrium, the interior lining of the uterus. Recent evidence suggests that toxic byproducts from fossil fuels known as ‘particulate matter’ represent a danger to women’s reproductive health. South Korean researchers led by Ji-Won Jung, Korea Centers for Disease Control and Prevention, and In-Sun Hong, Gachon University, Incheon, have investigated this risk by exposing cultured human endometrial stem cells to diesel-derived particulate matter. These stem cells normally maintain the endometrium, allowing embryonic implantation to take place, but exposure to particulate matter greatly impaired the cells’ regenerative function. Mice exposed to particulate matter exhibited similar impairments of endometrial maintenance. The researchers identified a molecular pathway associated with this response that could guide development of fertility-restoring treatments.

Novel therapies for the treatment of early steroid-induced osteonecrosis of the femoral head (SONFH) are urgently needed in orthopedics. Transplantation of bone marrow mesenchymal stem cells (BMSCs) provides new strategies for treating this condition at the early stage. However, stress-induced apoptosis of BMSCs transplanted into the femoral head necrotic area limits the efficacy of BMSC transplantation. Inhibiting BMSC apoptosis is key to improving the efficacy of this procedure. In our previous studies, we confirmed that Parkinson disease protein 7 (PARK7) is active in antioxidant defense and can clear reactive oxygen species (ROS), protect the mitochondria, and impart resistance to stress-induced apoptosis in BMSCs. In this study, we investigated the mechanism driving this PARK7-mediated resistance to apoptosis in BMSCs. Our results indicate that PARK7 promoted the disintegration of nuclear factor (erythroid-derived 2)–like 2 (Nrf2)/Kelch-like echinacoside–associated protein 1 (Keap1) complex. The free Nrf2 then entered the nucleus and activated the genetic expression of manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), and other antioxidant enzymes that clear excessive ROS, thereby protecting BMSCs from stress-induced apoptosis. To further explore whether PARK7-mediated resistance to stress-induced apoptosis could improve the efficacy of BMSC transplantation in early-stage SONFH, we transplanted BMSCs-overexpressing PARK7 into rats with early-stage SONFH. We then evaluated the survival of transplanted BMSCs and bone regeneration in the femoral head necrotic area of these rats. The results indicated that PARK7 promoted the survival of BMSCs in the osteonecrotic area and improved the transplantation efficacy of BMSCs on early-stage SONFH. This study provides new ideas and methods for resisting the stress-induced apoptosis of BMSCs and improving the transplantation effect of BMSCs on early-stage SONFH.

Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive tumor with poor clinical outcomes due to recurrence, metastasis, and treatment resistance. Cancer stem cells (CSCs), a small population among tumor cells, are proposed to be responsible for tumor initiation, progression, metastasis, drug resistance, and recurrence. Here we show that high LSD1 expression was a predictor of poor prognosis for HNSCC patients. We found that high expression of LSD1 is essential for the maintenance of the CSC properties by regulating Bmi-1 expression. Moreover, tumor LSD1 ablation suppresses CSC-like characteristics in vitro and inhibits tumorigenicity in vivo in immune-deficient xenografts. However, this deletion induces the upregulation of PDL1 levels, which compromises antitumor immunity and reduces antitumor efficacy in an immune-competent mouse model. Functionally, the combination of LSD1 inhibitor and anti-PD-1 monoclonal antibody can overcome tumor immune evasion and greatly inhibit tumor growth, which was associated with reduced Ki-67 level and augmented CD8+ T cell infiltration in immunocompetent tumor-bearing mouse models. In summary, these findings provide a novel and promising combined strategy for the treatment of HNSCC using a combination of LSD1 inhibition and PD-1 blockade.

Circular RNAs (circRNAs) are crucial elements of non-coding RNA, that regulate various biological processes. To date, expression patterns and functional roles of circRNAs during osteogenic differentiation of human umbilical cord mesenchymal stromal cells (hUCMSCs) remain unknown. In this study, we analyzed RNA-sequence data to reveal expression profiles of circRNAs during osteogenesis of hUCMSCs, then elucidated the underlying mechanisms of action. We identified a total of 5457 circRNAs in hUCMSCs, of which 34 and 33 were upregulated and downregulated, respectively. We applied Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses to determine functions and related pathways of differentially expressed circRNAs. Moreover, we applied bioinformatics tools to construct competing endogenous RNA networks, comprising 10 circRNAs, 46 micro RNAs and 413 mRNAs. Furthermore, we predicted protein-coding potential of the upregulated circRNAs then constructed a co-expression network comprising the top 5 upregulated circRNAs and 75 RNA-binding proteins. Next, we validated 6 differentially-expressed circRNAs and found that overexpressing circ‐CTTN could promote osteogenesis of hUCMSCs. Overall, our findings indicate that clusters of circRNAs are aberrantly expressed in hUCMSCs during osteogenic differentiation, hence lay a foundation for future research into promoting hUCMSCs osteogenic differentiation and bone regeneration.

Although functional interplay between intestinal microbiota and distant sites beyond the gut has been identified, the influence of microbiota-derived metabolites on hematopoietic stem cells (HSCs) remains unclear. This study investigated the role of microbiota-derived lactate in hematopoiesis using mice deficient in G-protein-coupled receptor (Gpr) 81 (Gpr81−/−), an established lactate receptor. We detected significant depletion of total HSCs in the bone marrow (BM) of Gpr81−/− mice compared with heterogenic (Gpr81+/−) mice in a steady state. Notably, the expression levels of stem cell factor (SCF), which is required for the proliferation of HSCs, decreased significantly in leptin receptor-expressing (LepR+) mesenchymal stromal cells (MSCs) around the sinusoidal vessels of the BM from Gpr81−/− mice compared with Gpr81+/− mice. Hematopoietic recovery and activation of BM niche cells after irradiation or busulfan treatment also required Gpr81 signals. Oral administration of lactic acid-producing bacteria (LAB) activated SCF secretion from LepR+ BM MSCs and subsequently accelerated hematopoiesis and erythropoiesis. Most importantly, LAB feeding accelerated the self-renewal of HSCs in germ-free mice. These results suggest that microbiota-derived lactate stimulates SCF secretion by LepR+ BM MSCs and subsequently activates hematopoiesis and erythropoiesis in a Gpr81-dependent manner., Blood cell formation: Support from gut bacteria Lactic acid produced by microbes in the gut has been implicated in supporting the production of blood cells, suggesting oral administration of lactic acid-producing bacteria might be useful for treating blood disorders, including anemia. Researchers in South Korea and China, led by Mi-Na Kweon at the University of Ulsan in Seoul, explored the significance of lactic acid using mice deficient in the gene for a protein receptor that allows lactic acid to influence various cellular processes. Without the benefit of this receptor the levels of blood cell-forming stem cells in bone marrow were reduced. The normal effect of lactic acid was linked to production of a protein called stem cell factor in specific cells. Oral administration of lactic acid-producing bacteria restored blood cell formation, indicating its therapeutic potential.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has placed a global public burden on health authorities. Although the virological characteristics and pathogenesis of COVID-19 has been largely clarified, there is currently no specific therapeutic measure. In severe cases, acute SARS-CoV-2 infection leads to immune disorders and damage to both the adaptive and innate immune responses. Having roles in immune regulation and regeneration, mesenchymal stem cells (MSCs) serving as a therapeutic option may regulate the over-activated inflammatory response and promote recovery of lung damage. Since the outbreak of the COVID-19 pandemic, a series of MSC-therapy clinical trials has been conducted. The findings indicate that MSC treatment not only significantly reduces lung damage, but also improves patient recovery with safety and good immune tolerance. Herein, we summarize the recent progress in MSC therapy for COVID-19 and highlight the challenges in the field.

Cellular therapy exerts profound therapeutic potential for curing a broad spectrum of diseases. Adult stem cells reside within a specified dynamic niche in vivo, which is essential for continuous tissue homeostatic maintenance through balancing self-renewal with lineage selection. Meanwhile, adult stem cells may be multipotent or unipotent, and are present in both quiescent and actively dividing states in vivo of the mammalians, which may switch to each other state in response to biophysical cues through mitochondria-mediated mechanisms, such as alterations in mitochondrial respiration and metabolism. In general, stem cells facilitate tissue repair after tissue-specific homing through various mechanisms, including immunomodulation of local microenvironment, differentiation into functional cells, cell “empowerment” via paracrine secretion, immunoregulation, and intercellular mitochondrial transfer. Interestingly, cell-source-specific features have been reported between different tissue-derived adult stem cells with distinct functional properties due to the different microenvironments in vivo, as well as differential functional properties in different tissue-derived stem cell-derived extracellular vehicles, mitochondrial metabolism, and mitochondrial transfer capacity. Here, we summarized the current understanding on roles of mitochondrial dynamics during stem cell homeostasis and aging, and lineage-specific differentiation. Also, we proposed potential unique mitochondrial molecular signature features between different source-derived stem cells and potential associations between stem cell aging and mitochondria–endoplasmic reticulum (ER) communication, as well as potential novel strategies for anti-aging intervention and healthy aging.

Spinal cord injury (SCI) is a salient traumatic disease that often leads to permanent disability, and motor and sensory impairments. Human umbilical cord mesenchymal stem cells (HucMSCs) have a wide application prospect in the treatment of SCI. This study explored the repair effect of HucMSCs-derived extracellular vesicles (HucMSCs-EVs) on SCI. HucMSCs and HucMSCs-EVs were cultured and identified. The rat model of SCI was established, and SCI rats were treated with HucMSCs-EVs. The motor function of SCI rats and morphology of spinal cord tissues were evaluated. Levels of NeuN, GFAP, and NF200 in spinal cord tissues were detected and cell apoptosis was measured. SCI rats were treated with EVs extracted from miR-29b-3p inhibitor-transfected HucMSCs. The downstream gene and pathway of miR-29b-3p were examined. HucMSCs-EVs-treated rats showed obvious motor function recovery and reduced necrosis, nuclear pyknosis, and cavity. HucMSCs-EVs alleviated spinal cord neuronal injury. miR-29b-3p was poorly expressed in SCI tissues, but highly expressed in EVs and SCI rats treated with EVs. miR-29b-3p targeted PTEN. Inhibition of miR-29b-3p or overexpression of PTEN reversed the repair effect of EVs on SCI. EVs activated the AKT/mTOR pathway via the miR-29b-3p/PTEN. In conclusion, HucMSCs-EVs reduced pathological changes, improved motor function, and promoted nerve function repair in SCI rats via the miR-29b-3p/PTEN/Akt/mTOR axis.

The post-transplant scoring system (PTSS), developed by the Francophone Society of Bone Marrow Transplantation and Cellular Therapy, is based on three independent post-transplant risk factors: grade of acute graft-versus-host disease, lack of platelet recovery before day 100, and relapse before day 100; discriminating low- (0), intermediate- (1–3), and high-risk (4–8) patients. We investigated the prognostic value of the PTSS in a cohort of patients with MDS who underwent myeloablative CD34-selected TCD transplants. From 2008 to 2018, 109 patients underwent a first TCD-HCT for MDS at our center. We used Cox proportional hazards models and different landmark analyses to evaluate the association of categorized PTSS score risk groups with overall survival (OS). Patients with an intermediate/ high risk PTSS score had decreased OS at day 180 (univariate HR 3.25 [95% CI 1.60, 6.60], p = 0.001) and at day 365 (univariate HR 5.42 [95% CI 2.21, 13.3], p

Long non-coding RNA (lncRNA) is receiving increasing attention in embryonic stem cells (ESCs) research. However, the roles of lncRNA in the differentiation of ESCs into pacemaker-like cells are still unclear. Therefore, the present study aims to explore the roles and mechanisms of lncRNA in the differentiation of ESCs into pacemaker-like cells. ESCs were cultured and induced differentiation to pacemaker-like cells. RNA sequencing was used to identify the differential expression lncRNAs during the differentiation of ESCs into pacemaker-like cells. Cell morphology observation, flow cytometry, quantitative real-time polymerase chain reaction, western blot, and immunofluorescence were used to detect the differentiation of ESCs into pacemaker-like cells. LncRNA and genes overexpression or knockdown through transfected adenovirus in the differentiation process. The fluorescence in situ hybridization (FISH) detected the lncRNA location in the differentiated ESCs. Luciferase reporter gene assay, methylation-specific PCR, chromatin immunoprecipitation assay, and RNA immunoprecipitation assay were performed to reveal the mechanism of lncRNA-regulating HCN4 expression. Rescue experiments were used to confirm that lncRNA regulates the differentiation of ESCs into pacemaker-like cells through HCN4. We cultured the ESCs and induced the differentiation of ESCs into pacemaker-like cells successfully. The expression of lncRNA RCPCD was significantly decreased in the differentiation of ESCs into pacemaker-like cells. Overexpression of RCPCD inhibited the differentiation of ESCs into pacemaker-like cells. RCPCD inhibited the expression of HCN4 by increasing HCN4 methylation at the promoter region through DNMT1, DNMT2, and DNMT3. RCPCD inhibited the differentiation of ESCs into pacemaker-like cells by inhibiting the expression of HCN4. Our results confirm the roles and mechanism of lncRNA RCPCD in the differentiation of ESCs into pacemaker-like cells, which could pave the path for the development of a cell-based biological pacemaker.

The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34+ progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells.

Human FOXP3+ regulatory T (Treg) cells are central to immune tolerance. However, their heterogeneity and differentiation remain incompletely understood. Here we use single-cell RNA and T cell receptor sequencing to resolve Treg cells from healthy individuals and patients with or without acute graft-versus-host disease (aGVHD) who undergo stem cell transplantation. These analyses, combined with functional assays, separate Treg cells into naïve, activated, and effector stages, and resolve the HLA-DRhi, LIMS1hi, highly suppressive FOXP3hi, and highly proliferative MKI67hi effector subsets. Trajectory analysis assembles Treg subsets into two differentiation paths (I/II) with distinctive phenotypic and functional programs, ending with the FOXP3hi and MKI67hi subsets, respectively. Transcription factors FOXP3 and SUB1 contribute to some Path I and Path II phenotypes, respectively. These FOXP3hi and MKI67hi subsets and two differentiation pathways are conserved in transplanted patients, despite having functional and migratory impairments under aGVHD. These findings expand the understanding of Treg cell heterogeneity and differentiation and provide a single-cell atlas for the dissection of Treg complexity in health and disease., Human Treg cells are central to immune tolerance, yet their heterogeneity and differentiation remain incompletely understood. Here the authors perform single-cell RNA and T cell receptor sequencing to resolve Treg cells from healthy individuals and patients with or without acute graft-versus-host disease revealing Treg complexity in health and disease.

Mesenchymal stromal/stem cells (MSCs) are currently being used in clinical trials as proposed treatments for a large range of genetic, immunological, orthopaedic, cardiovascular, endocrine and neurological disorders. MSCs are potent anti-inflammatory mediators which are considered immune evasive and employ a large range of secreted vesicles to communicate and repair damaged tissue. Despite their prolific use in therapy, sex specific mechanism of action is rarely considered as a potential confounding factor for use. The purpose of this study was to examine the potency and functionality of both female and male adipose derived MSCs in order to gain further insights into donor selection. Methods MSC were expanded to passage 4, secretome was harvested and stored at − 80c. To assess potency MSC were also primed and assessed via functional immune assays, ELISA, multiplex and immunophenotyping. Results Female MSCs (fMSC), consistently suppressed Peripheral blood mononuclear cell (PBMC) proliferation significantly (p

The adult heart is a vital and highly specialized organ of the human body, with limited capability of self-repair and regeneration in case of injury or disease. Engineering biomimetic cardiac tissue to regenerate the heart has been an ambition in the field of tissue engineering, tracing back to the 1990s. Increased understanding of human stem cell biology and advances in process engineering have provided an unlimited source of cells, particularly cardiomyocytes, for the development of functional cardiac muscle, even though pluripotent stem cell-derived cardiomyocytes poorly resemble those of the adult heart. This review outlines key biology-inspired strategies reported to improve cardiomyocyte maturation features and current biofabrication approaches developed to engineer clinically relevant cardiac tissues. It also highlights the potential use of this technology in drug discovery science and disease modeling as well as the current efforts to translate it into effective therapies that improve heart function and promote regeneration.

Periodontal ligament (PDL) is a uniquely differentiated tissue that anchors the tooth to the alveolar bone socket and plays key roles in oral function. PDL cells can respond rapidly to mechanical stimuli, resulting in accelerated tissue remodeling. Cell proliferation is an initial event in tissue remodeling and participates in maintaining the cell supply; therefore, analyzing cell-proliferative activity might provide a comprehensive view of cellular dynamics at the tissue level. In this study, we investigated proliferating cells in mouse molar PDL during orthodontic tooth movement (OTM)-induced tissue remodeling. Our results demonstrated that the mechanical stimuli evoked a dynamic change in the proliferative-cell profile at the entire PDL. Additionally, cell-tracing analysis revealed that the proliferated cells underwent further division and subsequently contributed to tissue remodeling. Moreover, OTM-induced proliferating cells expressed various molecular markers that most likely arise from a wide range of cell types, indicating the lineage plasticity of PDL cells in vivo. Although further studies are required, these findings partially elucidated the global views of the cell trajectory in mouse molar PDL under mechanical-loading conditions, which is vital for understanding the cellular dynamics of the PDL and beneficial for dental treatment in humans.

Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreER RosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-β) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases., How endothelial to mesenchymal transition is regulated in endovascular progenitors is unclear. Here, the authors show that blocking Sox9 expression in murine endovascular progenitors regulates this transition on skin wounding, affecting the size of scarring, with changes in Rbpj having the opposite effect.

The bone marrow microenvironment (BMME) plays a key role in the pathophysiology of myelodysplastic syndromes (MDS), clonal blood disorders affecting the differentiation, and maturation of hematopoietic stem and progenitor cells (HSPCs). In lower-risk MDS patients, ineffective late-stage erythropoiesis can be restored by luspatercept, an activin receptor type IIB ligand trap. Here, we investigated whether luspatercept can modulate the functional properties of mesenchymal stromal cells (MSCs) as key components of the BMME. Luspatercept treatment inhibited Smad2/3 phosphorylation in both healthy and MDS MSCs and reversed disease-associated alterations in SDF-1 secretion. Pre-treatment of MDS MSCs with luspatercept restored the subsequent clonogenic potential of co-cultured HSPCs and increased both their stromal-adherence and their expression of both CXCR4 and ß3 integrin. Luspatercept pre-treatment of MSCs also increased the subsequent homing of co-cultured HSPCs in zebrafish embryos. MSCs derived from patients who had received luspatercept treatment had an increased capacity to maintain the colony forming potential of normal but not MDS HSPCs. These data provide the first evidence that luspatercept impacts the BMME directly, leading to a selective restoration of the ineffective hematopoiesis that is a hallmark of MDS.

Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche., Short term systemic expression of the reprogramming factors Oct-3/4, Sox2, Klf4, c-Myc (OSKM) rejuvenates aging cells and promotes tissue regeneration. Here the authors show that myofiber-specific expression of OSKM accelerates muscle regeneration by reducing secretion of muscle stem cell quiescence promoting Wnt4.

Stem cell therapy holds great promise for tissue regeneration and cancer treatment, although its efficacy is still inconclusive and requires further understanding and optimization of the procedures. Non-invasive cell tracking can provide an important opportunity to monitor in vivo cell distribution in living subjects. Here, using a combination of positron emission tomography (PET) and in vitro 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) direct cell labelling, the feasibility of engrafted stem cell monitoring was tested in multiple animal species. Human mesenchymal stem cells (MSCs) were incubated with phosphate-buffered saline containing [18F]FDG for in vitro cell radiolabelling. The pre-labelled MSCs were administrated via peripheral vein in a mouse (n = 1), rats (n = 4), rabbits (n = 4) and non-human primates (n = 3), via carotid artery in rats (n = 4) and non-human primates (n = 3), and via intra-myocardial injection in rats (n = 5). PET imaging was started 10 min after cell administration using a dedicated small animal PET system for a mouse and rats. A clinical PET system was used for the imaging of rabbits and non-human primates. After MSC administration via peripheral vein, PET imaging revealed intense radiotracer signal from the lung in all tested animal species including mouse, rat, rabbit, and non-human primate, suggesting administrated MSCs were trapped in the lung tissue. Furthermore, the distribution of the PET signal significantly differed based on the route of cell administration. Administration via carotid artery showed the highest activity in the head, and intra-myocardial injection increased signal from the heart. In vitro [18F]FDG MSC pre-labelling for PET imaging is feasible and allows non-invasive visualization of initial cell distribution after different routes of cell administration in multiple animal models. Those results highlight the potential use of that imaging approach for the understanding and optimization of stem cell therapy in translational research.

Regeneration is an elegant and complex process informed by both local and long-range signals. Many current studies on regeneration are largely limited to investigations of local modulators within a canonical cohort of model organisms. Enhanced genetic tools increasingly enable precise temporal and spatial perturbations within these model regenerators, and these have primarily been applied to cells within the local injury site. Meanwhile, many aspects of broader spatial regulators of regeneration have not yet been examined with the same level of scrutiny. Recent studies have shed important insight into the significant effects of environmental cues and circulating factors on the regenerative process. These observations highlight that consideration of more systemic and possibly more broadly acting cues will also be critical to fully understand complex tissue regeneration. In this review, we explore the ways in which systemic cues and circulating factors affect the initiation of regeneration, the regenerative process, and its outcome. As this is a broad topic, we conceptually divide the factors based on their initial input as either external cues (for example, starvation and light/dark cycle) or internal cues (for example, hormones); however, all of these inputs ultimately lead to internal responses. We consider studies performed in a diverse set of organisms, including vertebrates and invertebrates. Through analysis of systemic mediators of regeneration, we argue that increased investigation of these “systemic factors” could reveal novel insights that may pave the way for a diverse set of therapeutic avenues.

Extracellular vesicles (EVs) are cell derivatives containing diverse cellular molecules, have various physiological properties and are also present in stem cells used for regenerative therapy. We selected a “multiplexed target” that demonstrates multiple effects on various cardiovascular cells, while functioning as a cargo of EVs. We screened various microRNAs (miRs) and identified miR-210 as a candidate target for survival and angiogenic function. We confirmed the cellular and biological functions of EV-210 (EVs derived from ASCmiR-210) secreted from adipose-derived stem cells (ASCs) transfected with miR-210 (ASCmiR-210). Under hypoxic conditions, we observed that ASCmiR-210 inhibits apoptosis by modulating protein tyrosine phosphatase 1B (PTP1B) and death-associated protein kinase 1 (DAPK1). In hypoxic endothelial cells, EV-210 exerted its angiogenic capacity by inhibiting Ephrin A (EFNA3). Furthermore, EV-210 enhanced cell survival under the control of PTP1B and induced antiapoptotic effects in hypoxic H9c2 cells. In cardiac fibroblasts, the fibrotic ratio was reduced after exposure to EV-210, but EVs derived from ASCmiR-210 did not communicate with fibroblasts. Finally, we observed the functional restoration of the ischemia/reperfusion-injured heart by maintaining the intercommunication of EVs and cardiovascular cells derived from ASCmiR-210. These results suggest that the multiplexed target with ASCmiR-210 is a useful tool for cardiovascular regeneration., Heart disease: Boosting microRNA activity may restore heart function A therapy designed to enhance the activity of a small regulatory RNA molecule could prompt the restoration of cellular communication and improve heart function during heart disease. Heart cells struggle to function and regenerate under the reduced oxygen conditions caused by narrowed or blocked blood vessels. Stem cell-derived therapies may restore damaged heart tissues, and can be designed to secrete specific factors that boost regeneration. To identify factors that may improve heart recovery chances, Woochul Chang at Pusan National University, Busan, Korea and co-workers examined stem cell-derived extracellular vesicles (EVs), cellular membrane sacs that carry proteins and RNAs to surrounding tissues and boost intercellular communication. The team identified microRNA-210 as a key factor that inhibits heart cell death, reduces fibrosis, and enhances regeneration of heart cell populations. EVs secreting microRNA-210 may prove a useful therapeutic tool.

Retinal degenerative disorders, including age-related macular degeneration and retinitis pigmentosa (RP), are characterized by the irreversible loss of photoreceptor cells and retinal pigment epithelial (RPE) cells; however, the long-term effect of implanting both human induced pluripotent stem cell (hiPSC)-derived RPE and photoreceptor for retinal regeneration has not yet been investigated. In this study, we evaluated the long-term effects of hiPSC-derived RPE and photoreceptor cell transplantation in Pde6b knockout rats to study RP; cells were injected into the subretinal space of the right eyes of rats before the appearance of signs of retinal degeneration at 2–3 weeks of age. Ten months after transplantation, we evaluated the cells using fundus photography, optical coherence tomography, and histological evaluation, and no abnormal cell proliferation was observed. A relatively large number of transplanted cells persisted during the first 4 months; subsequently, the number of these cells decreased gradually. Notably, immunohistochemical analysis revealed that the hiPSC-derived retinal cells showed characteristics of both RPE cells and photoreceptors of human origin after transplantation. Functional analysis of vision by scotopic electroretinogram revealed significant preservation of vision after transplantation. Our study suggests that the transplantation of hiPSC-derived retinal cells, including RPE cells and photoreceptors, has a potential therapeutic effect against irreversible retinal degenerative diseases., Retinal disease: Cell transplants offer hope Cells with the potential to regenerate retinal cells could become a useful treatment for serious eye diseases such as age-related macular degeneration and retinitis pigmentosa. The retina has only limited self-regenerating potential. Joo Yong Lee and colleagues at the University of Ulsan in Seoul, South Korea, studied the long-term effects of implanting cells derived from human stem cells into the eyes of rats that had been genetically modified to act as a model of retinal degenerative disease. The implanted cells have the features of the photoreceptor cells that detect light and those cells that generate a layer of the retina called the retinal pigment epithelium. A substantial population of the cells persisted for at least four months, significantly preserving the animals’ vision. The results justify further exploration of the therapeutic possibilities.

Poorly differentiated tumors usually exhibit phenotypes similar to that of their developmental precursor cells. Tumor cells that acquire the lineage progenitor cells feature usually exploit developmental signaling to potentiate cancer progression. However, the underlying molecular events remain elusive. In this study, based on analysis of an in vitro hepatocyte differentiation model, the maternal factor PGC7 (also known as DPPA3, STELLA) was found closely associated with liver development and tumor differentiation in hepatocellular carcinoma (HCC). Expression of PGC7 decreased during hepatocyte maturation and increased progressively from well-differentiated HCCs to poorly differentiated HCCs. Whole-genome methylation sequencing found that PGC7 could induce promoter demethylation of genes related to development. Pathway-based network analysis indicated that downstream targets of PGC7 might form networks associated with developmental transcription factor activation. Overexpression of PGC7 conferred progenitor-like features of HCC cells both in vitro and in vivo. Mechanism studies revealed that PGC7 could impede nuclear translocation of UHRF1, and thus facilitate promoter demethylation of GLI1 and MYCN, both of which are important regulators of HCC self-renewal and differentiation. Depletion or inhibition of GLI1 effectively downregulated MYCN, abolished the effect of PGC7, and sensitized HCC cells to sorafenib treatment. In addition, we found a significant correlation of PGC7 with GLI1/MYCN and lineage differentiation markers in clinical HCC patients. PGC7 expression might drive HCC toward a “dedifferentiated” progenitor lineage through facilitating promoter demethylation of key developmental transcription factors; further inhibition of PGC7/GLI1/MYCN might reverse poorly differentiated HCCs and provide novel therapeutic strategies.

Oxysterols play vital roles in the human body, ranging from cell cycle regulation and progression to dopaminergic neurogenesis. While naïve human mesenchymal stem cells (hMSCs) have been explored to have neurogenic effect, there is still a grey area to explore their regenerative potential after in vitro differentiation. Hence, in the current study, we have investigated the neurogenic effect of 22(R)-hydroxycholesterol (22-HC) on hMSCs obtained from bone marrow, adipose tissue and dental pulp. Morphological and morphometric analysis revealed physical differentiation of stem cells into neuronal cells. Detailed characterization of differentiated cells affirmed generation of neuronal cells in culture. The percentage of generation of non-DA cells in the culture confirmed selective neurogenic potential of 22-HC. We substantiated the efficacy of these cells in neuro-regeneration by transplanting them into Parkinson’s disease Wistar rat model. MSCs from dental pulp had maximal regenerative effect (with 80.20 ± 1.5% in vitro differentiation efficiency) upon transplantation, as shown by various behavioural examinations and immunohistochemical tests. Subsequential analysis revealed that 22-HC yields a higher percentage of functional DA neurons and has differential effect on various tissue-specific primary human MSCs. 22-HC may be used for treating Parkinson’s disease in future with stem cells.

Mesenchymal stem cell (MSC)-based therapy has emerged as a novel strategy to treat many degenerative diseases. Accumulating evidence shows that the function of MSCs declines with age, thus limiting their regenerative capacity. Nonetheless, the underlying mechanisms that control MSC ageing are not well understood. We show that compared with bone marrow-MSCs (BM-MSCs) isolated from young and aged samples, NADH dehydrogenase (ubiquinone) iron-sulfur protein 6 (Ndufs6) is depressed in aged MSCs. Similar to that of Ndufs6 knockout (Ndufs6−/−) mice, MSCs exhibited a reduced self-renewal and differentiation capacity with a tendency to senescence in the presence of an increased p53/p21 level. Downregulation of Ndufs6 by siRNA also accelerated progression of wild-type BM-MSCs to an aged state. In contrast, replenishment of Ndufs6 in Ndufs6−/−-BM-MSCs significantly rejuvenated senescent cells and restored their proliferative ability. Compared with BM-MSCs, Ndufs6−/−-BM-MSCs displayed increased intracellular and mitochondrial reactive oxygen species (ROS), and decreased mitochondrial membrane potential. Treatment of Ndufs6−/−-BM-MSCs with mitochondrial ROS inhibitor Mito-TEMPO notably reversed the cellular senescence and reduced the increased p53/p21 level. We provide direct evidence that impairment of mitochondrial Ndufs6 is a putative accelerator of adult stem cell ageing that is associated with excessive ROS accumulation and upregulation of p53/p21. It also indicates that manipulation of mitochondrial function is critical and can effectively protect adult stem cells against senescence.

The number of publications on the governance of research with human embryo-like structures (hELS), i.e., 3D aggregates of human (induced) pluripotent stem cells made to model early human development, is growing rapidly. Public involvement is called for in many of these publications, but studies on public perspectives towards this emerging field remain lacking due to its novelty. To reduce the gap in the literature and contribute to the ongoing scholarly debate, we conducted interviews with Dutch lay citizens, health law and health care professionals, and interviewees reasoning from prominent worldviews in the Netherlands. This article reports on these participants’ views about the conceptual and moral qualification of hELS. With regard to the conceptual qualification of hELS, participants believed it should provide a shorthand for their (dis)similarity to human embryos, but differences remained with regard to the features upon which this (dis)similarity should be based. With regard to the moral qualification of hELS, participants believed this should depend on whether or not hELS possessed the features they considered morally relevant, among which those associated with sentience and a potential for continuous human development. Taken together, these findings align well with the arguments and positions traditionally found in related ethical debates and the recently proposed recommendations for the governance of research with hELS specifically. As such, they may also help allay concerns about lay publics not being able to meaningfully participate in debates about the ethical ramifications of (novel) scientific developments.

Photoreceptor apoptosis is recognized as one key pathogenesis of retinal degeneration, the counteraction of which represents a promising approach to safeguard visual function. Recently, mesenchymal stem cell transplantation (MSCT) has demonstrated immense potential to treat ocular disorders, in which extracellular vesicles (EVs), particularly exosomes, have emerged as effective ophthalmological therapeutics. However, whether and how MSCT protects photoreceptors against apoptotic injuries remains largely unknown. Here, we discovered that intravitreal MSCT counteracted photoreceptor apoptosis and alleviated retinal morphological and functional degeneration in a mouse model of photoreceptor loss induced by N-methyl-N-nitrosourea (MNU). Interestingly, effects of MSCT were inhibited after blockade of exosomal generation by GW4869 preconditioning. Furthermore, MSC-derived exosomal transplantation (EXOT) effectively suppressed MNU-provoked photoreceptor injury. Notably, therapeutic efficacy of MSCT and EXOT on MNU-induced retinal degeneration was long-lasting as photoreceptor preservance and retinal maintenance were detected even after 1–2 months post to injection for only once. More importantly, using a natural occurring retinal degeneration model caused by a nonsense mutation of Phosphodiesterase 6b gene (Pde6bmut), we confirmed that MSCT and EXOT prevented photoreceptor loss and protected long-term retinal function. In deciphering therapeutic mechanisms regarding potential exosome-mediated communications, we identified that miR-21 critically maintained photoreceptor viability against MNU injury by targeting programmed cell death 4 (Pdcd4) and was transferred from MSC-derived exosomes in vivo for functional regulation. Moreover, miR-21 deficiency aggravated MNU-driven retinal injury and was restrained by EXOT. Further experiments revealed that miR-21 mediated therapeutic effects of EXOT on MNU-induced photoreceptor apoptosis and retinal dysfunction. These findings uncovered the efficacy and mechanism of MSCT-based photoreceptor protection, indicating exosomal miR-21 as a therapeutic for retinal degeneration.

A detailed description of pathophysiological effects that viruses exert on their host is still challenging. For the first time, we report a highly controllable viral expression model based on an iPS-cell line from a healthy human donor. The established viral model system enables a dose-dependent and highly localized RNA-virus expression in a fully controllable environment, giving rise for new applications for the scientific community.

In modern biology, the correct identification of cell types is required for the developmental study of tissues and organs and the production of functional cells for cell therapies and disease modeling. For decades, cell types have been defined on the basis of morphological and physiological markers and, more recently, immunological markers and molecular properties. Recent advances in single-cell RNA sequencing have opened new doors for the characterization of cells at the individual and spatiotemporal levels on the basis of their RNA profiles, vastly transforming our understanding of cell types. The objective of this review is to survey the current progress in the field of cell-type identification, starting with the Human Cell Atlas project, which aims to sequence every cell in the human body, to molecular marker databases for individual cell types and other sources that address cell-type identification for regenerative medicine based on cell data guidelines., Regenerative medicine: Precise cell identification will improve future therapies The characterization of cells into more precise groups will improve the chances of selecting the best types of cells to use for regenerative medicine. Traditionally, cell types are defined by physiological and morphological markers and molecular properties. However, sequencing technologies are enabling researchers to classify cells into increasingly distinct subgroups. Wataru Fujibuchi at Kyoto University, Japan, and co-workers reviewed progress in cell-type identification to help guide regenerative medicine. They examined the insights gained from the Human Cell Atlas project, an international collaboration of over 1000 institutes across 71 countries. Participants in this project are sequencing cellular RNA and categorizing hundreds of thousands of individual cells, demonstrating that many assumptions about cells are too simplistic. Such data will inform regenerative therapies, for example, by selecting the “best” platelet-producing stem cells for blood donation.

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidate for regenerative medicine to repair non-healing bone defects due to their high and easy availability. However, the limited osteogenic differentiation potential greatly hinders the clinical application of ADSCs in bone repair. Accumulating evidences demonstrate that circular RNAs (circRNAs) are involved in stem/progenitor cell fate determination, but their specific role in stem/progenitor cell osteogenesis, remains mostly undescribed. Here, we show that circRNA-vgll3 originating from the vgll3 locus markedly enhances osteogenic differentiation of ADSCs; nevertheless, silencing of circRNA-vgll3 dramatically attenuates ADSC osteogenesis. Furthermore, we validate that circRNA-vgll3 functions in ADSC osteogenesis through a circRNA-vgll3/miR-326-5p/integrin α5 (Itga5) pathway. Itga5 promotes ADSC osteogenic differentiation and miR-326-5p suppresses Itga5 translation. CircRNA-vgll3 directly sequesters miR-326-5p in the cytoplasm and inhibits its activity to promote osteogenic differentiation. Moreover, the therapeutic potential of circRNA-vgll3-modified ADSCs with calcium phosphate cement (CPC) scaffolds was systematically evaluated in a critical-sized defect model in rats. Our results demonstrate that circRNA-vgll3 markedly enhances new bone formation with upregulated bone mineral density, bone volume/tissue volume, trabeculae number, and increased new bone generation. This study reveals the important role of circRNA-vgll3 during new bone biogenesis. Thus, circRNA-vgll3 engineered ADSCs may be effective potential therapeutic targets for bone regenerative medicine.

Allogeneic hematopoietic stem cell transplantation (HSCT) is curative for a variety of nonmalignant disorders including osteopetrosis, bone marrow failures, and immune deficiencies. Haploidentical HSCT is a readily available option in the absence of a matched donor, but engraftment failure and other post-transplant complications are a concern. Post-transplant cyclophosphamide (PT-Cy) regimens are gaining popularity and recent reports show promising results. We report our experience with nine pediatric patients with nonmalignant diseases who were transplanted from a haploidentical donor with PT-Cy. From 2015 to 2019, nine children with nonmalignant diseases underwent haploidentical HSCT with PT-Cy, two as a second transplant and seven as primary grafts after upfront serotherapy and busulfan-based myeloablative conditioning. Patient’s diseases included osteopetrosis (n = 5), congenital amegakaryocytic thrombocytopenia (n = 2), hemophagocytic lymphohistiocytosis (n = 1), and Wiskott Aldrich syndrome (n = 1). Two patients failed to engraft following upfront PT-Cy transplants, one was salvaged with a second PT-Cy transplant, and the other with a CD34+ selected graft. None of the patients suffered from graft-versus-host disease. Three patients died from early posttransplant infectious complications and six patients are alive and well. In conclusion, haploidentical HSCT with PT-Cy is a feasible option for pediatric patients with nonmalignant diseases lacking a matched donor.

Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nuclease AsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of the RET SNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding gene COL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.

Intensive upfront therapy in newly-diagnosed multiple myeloma (MM) including induction therapy (IT), high-dose melphalan (MEL200), and autologous blood stem cell transplantation (ASCT) followed by consolidation and/or maintenance is mostly restricted to patients up to 65 years of age. Prospective phase III trial data in the era of novel agents for patients up to 70 years of age are not available. The GMMG-MM5 trial included 601 patients between 18 and 70 years of age, divided in three groups for the present analysis: ≤60 years (S1, n = 353), 61–65 years (S2, n = 107) and 66–70 years (S3, n = 141). Treatment consisted of a bortezomib-containing IT, MEL200/ASCT, consolidation, and maintenance with lenalidomide. Adherence to treatment was similar among patients of the three age groups. Overall toxicity during all treatment phases was increased in S2 and S3 compared to S1 (any adverse event/any serious adverse event: S1:81.7/41.8% vs. S2:90.7/56.5% vs. S3:87.2/68.1%, p = 0.05/p = 0.73), overall survival (log-rank p = 0.54) as well as time-to-progression (Gray’s p = 0.83) and non-relapse mortality (Gray’s p = 0.25), no differences were found between the three age groups. Our results imply that an intensive upfront therapy with a bortezomib-containing IT, MEL200/ASCT, lenalidomide consolidation, and maintenance should be applied to transplant-eligible MM patients up to 70 years of age.

Biological self-assembly is crucial in the processes of development, tissue regeneration, and maturation of bioprinted tissue-engineered constructions. The cell aggregates—spheroids—have become widely used model objects in the study of this phenomenon. Existing approaches describe the fusion of cell aggregates by analogy with the coalescence of liquid droplets and ignore the complex structural properties of spheroids. Here, we analyzed the fusion process in connection with structure and mechanical properties of the spheroids from human somatic cells of different phenotypes: mesenchymal stem cells from the limbal eye stroma and epithelial cells from retinal pigment epithelium. A nanoindentation protocol was applied for the mechanical measurements. We found a discrepancy with the liquid drop fusion model: the fusion was faster for spheroids from epithelial cells with lower apparent surface tension than for mesenchymal spheroids with higher surface tension. This discrepancy might be caused by biophysical processes such as extracellular matrix remodeling in the case of mesenchymal spheroids and different modes of cell migration. The obtained results will contribute to the development of more realistic models for spheroid fusion that would further provide a helpful tool for constructing cell aggregates with required properties both for fundamental studies and tissue reparation.

Bony defects are a common problem in musculoskeletal surgery. Replacement with autologous bone grafts is limited by availability of transplant material. Sterilized cancellous bone, while being osteoconductive, has limited osteoinductivity. Nanofiber scaffolds are currently used for several purposes due to their capability of imitating the extracellular matrix. Furthermore, they allow modification to provide functional properties. Previously we showed that electrospun nanofiber scaffolds can be used for bone tissue regeneration. While aiming to use the osteoinductive capacities of collagen type-I nanofibers we saw reduced scaffold pore sizes that limited cellular migration and thus colonization of the scaffolds. Aim of the present study was the incorporation of mesenchymal stem cells into the electrospinning process of a nanofiber scaffold to produce cell-seeded nanofiber scaffolds for bone replacement. After construction of a suitable spinning apparatus for simultaneous electrospinning and spraying with independently controllable spinning and spraying devices and extensive optimization of the spinning process, in vitro and in vivo evaluation of the resulting scaffolds was conducted. Stem cells isolated from rat femora were incorporated into PLLA (poly-l-lactide acid) and PLLA-collagen type-I nanofiber scaffolds (PLLA Col I Blend) via simultaneous electrospinning and –spraying. Metabolic activity, proliferation and osteoblastic differentiation were assessed in vitro. For in vivo evaluation scaffolds were implanted into critical size defects of the rat scull. After 4 weeks, animals were sacrificed and bone healing was analyzed using CT-scans, histological, immunhistochemical and fluorescence evaluation. Successful integration of mesenchymal stem cells into the scaffolds was achieved by iteration of spinning and spraying conditions regarding polymer solvent, spinning distance, the use of a liquid counter-electrode, electrode voltage and spinning duration. In vivo formation of bone tissue was achieved. Using a PLLA scaffold, comparable results for the cell-free and cell-seeded scaffolds were found, while the cell-seeded PLLA-collagen scaffolds showed significantly better bone formation when compared to the cell-free PLLA-collagen scaffolds. These results provide support for the future use of cell-seeded nanofiber scaffolds for large bony defects.

Effective engineering approaches for cartilage regeneration involve a combination of cells and biomaterial scaffolds. Multipotent mesenchymal stem cells (MSCs) are important sources for cartilage regeneration. Atelocollagen provides a suitable substrate for MSC attachment and enhancing chondrogenic differentiation. Here, we assessed the chondrogenic potential of adipose tissue derived human MSCs (hMSCs) mixed with atelocollagen gel. We observed cell attachment, viability, and microstructures by electron microscopy over 21 days. The levels of Sox9, type II collagen, aggrecan, type I collagen, Runx2, type X collagen, ALP, Osterix, and MMP13 were measured by RT-qPCR. Cartilage matrix-related proteins were assessed by enzyme-linked immunosorbent assay (ELISA), histology, and immunohistochemistry. hMSCs of all groups exhibited well-maintained cell survival, distribution and morphology. Abundant type II collagen fibers developed on day 21; while Sox9, type II collagen, and aggrecan expression increased over time in the atelocollagen group. However, type I collagen, RUNX2, type X collagen (CoL10A1), Osterix, and ALP were not expressed. These results corroborated the protein expression detected by ELISA. Further, histological analysis revealed lacunae-like structures, while staining demonstrated glycosaminoglycan accumulation. Cumulatively, these results indicate that atelocollagen scaffolds improve hMSC chondrogenic differentiation and are a potential approach for cartilage regeneration.

Acute graft-versus-host disease (aGVHD) is a serious complication after stem cell transplantation and is associated with high non-relapse mortality. If steroid treatment as first-line therapeutic approach fails, treatment options are limited. In retrospective studies, ruxolitinib, a selective Janus kinase 1/2 inhibitor as well as extracorporeal photopheresis (ECP) could show high efficacy in treatment of steroid refractory acute and chronic GVHD. Here, we report single-center experience of combining JAK-inhibitor treatment with ECP in 18 patients with severe steroid refractory aGVHD of lower GI-tract. The treatment was well tolerated and no severe cytopenia (grade IV) occurred, in three patients grade III cytopenia could be observed. Response was complete or partial in 44% and 11%, respectively, resulting in an estimated 2 year overall survival of 56%. Steroids were tapered rapidly with a median time of 2 days for halving of dosage avoiding additional steroid-associated side effects. Under treatment with ruxolitinib and ECP, an increased level of regulatory T cells could be observed elucidating direct effects of this treatment on immune response.

Cryopreservation procedures negatively affect the quality traits of sperm, causing certain changes at structural and molecular levels due to thermal, mechanical, osmotic, and oxidative damage. The objective of this study was to examine the potential of canine adipose-derived mesenchymal stem cells (Ad-MSCs) for providing protection to the dog sperm against cryo-damage. Canine Ad-MSCs were selected on the basis of the significantly higher gene expression for different proteins actively involved in the cell repair including annexin 1 (ANX1), histone H3 (H3) and high mobility group B (HMGB) protein compared to skin fibroblasts. Semen was collected from four healthy dogs by digital manipulation. The washed pooled ejaculates were diluted with buffer 2 (extender) supplemented without Ad-MSCs (Control), with 2.5 × 106 Ad-MSCs/mL (Group 1) or with 5 × 106 Ad-MSCs/mL (Group 2). Group 1 exhibited significantly higher post-thaw motility, live sperm, intact plasma membrane and normal acrosomes than the other groups. Additionally, Group 1 showed significantly higher expression levels of genes related to the repair of membranes (ANX1, dysferlin; DYSF, and fibronectin; FN1) and chromatin material (H3 and HMGB). Protein expression of ANX1, H 3, and FN1 was also statistically more in Group 1 than in Control. The results confirm that canine Ad-MSCs can effectively preserve the quality of frozen-thawed sperm by a reduction in cryoinjury. At an appropriate concentration, Ad-MSCs significantly improve the quality of post-thaw dog sperm.

Human embryonic stem cells (ESCs) offer a promising therapeutic approach for osteoarthritis (OA). The unlimited source of cells capable of differentiating to chondrocytes has potential for repairing damaged cartilage or to generate disease models via gene editing. However their use is limited by the efficiency of chondrogenic differentiation. An improved understanding of the transcriptional and post-transcriptional regulation of chondrogenesis will enable us to improve hESC chondrogenic differentiation protocols. Small RNA-seq and whole transcriptome sequencing was performed on distinct stages of hESC-directed chondrogenesis. This revealed significant changes in the expression of several microRNAs including upregulation of known cartilage associated microRNAs and those transcribed from the Hox complexes, and the downregulation of pluripotency associated microRNAs. Integration of miRomes and transcriptomes generated during hESC-directed chondrogenesis identified key functionally related clusters of co-expressed microRNAs and protein coding genes, associated with pluripotency, primitive streak, limb development and extracellular matrix. Analysis identified regulators of hESC-directed chondrogenesis such as miR-29c-3p with 10 of its established targets identified as co-regulated ‘ECM organisation’ genes and miR-22-3p which is highly co-expressed with ECM genes and may regulate these genes indirectly by targeting the chondrogenic regulators SP1 and HDAC4. We identified several upregulated transcription factors including HOXA9/A10/D13 involved in limb patterning and RELA, JUN and NFAT5, which have targets enriched with ECM associated genes. We have developed an unbiased approach for integrating transcriptome and miRome using protein-protein interactions, transcription factor regulation and miRNA target interactions and identified key regulatory networks prominent in hESC chondrogenesis.

Histological and cytological observations of the human anterior cruciate ligament (ACL) had been described, but the differentiation potency based on their location is still unknown. To determine and compare proliferation and differentiation potential of cells derived from distal and middle thirds of the ACL remnant, ACL remnant was initially marked at the distal third (within 10 mm from the tibial insertion) and middle third (between 10–20 mm from the tibial insertion) and then dissected. Both the middle and distal third regions of ACL remnant were analyzed using CD34+ cell counting. Cell proliferation rate did not differ in both middle and distal third regions of ACL remnant, but they showed different characteristics in cell differentiation depending on their location. The distal third region of the ACL remnant had a tendency for chondrogenic differentiation with higher expression of CD34+ cells. On the other hand, the middle third region of ACL remnant had a strong tendency for osteogenic and ligamentous differentiation. Characteristics of the ACL remnant tissues should be considered when performing remnant-preserving or harvesting ACL remnants for tissue engineering.

Due to the curative potential and improvement in progression-free survival (PFS), high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is considered the standard of care for several hematologic malignancies, such as multiple myeloma, and lymphomas. ASCT typically involves support with blood product transfusion. Thus, difficulties arise when Jehovah’s Witness patients refuse blood transfusions. In order to demonstrate the safety of performing “bloodless” ASCT (BL-ASCT), we performed a retrospective analysis of 66 Jehovah's Witnesses patients who underwent BL-ASCT and 1114 non-Jehovah’s Witness patients who underwent transfusion-supported ASCT (TF-ASCT) at Cedars-Sinai Medical Center between January 2000 and September 2018. Survival was compared between the two groups. Transplant-related complications, mortality, engraftment time, length of hospital stay, and number of ICU transfers were characterized for the BL-ASCT group. One year survival was found to be 87.9% for both groups (P = 0.92). In the BL-ASCT group, there was one death prior to the 30 days post transplant due to CNS hemorrhage, and one death prior to 100 days due to sepsis. Based on our data, BL-ASCT can be safely performed with appropriate supportive measures, and we encourage community oncologists to promptly refer JW patients for transplant evaluation when ASCT is indicated.

Beta 3 (β3) integrin plays an important role in the initiation of myogenesis in adult muscle. Protein disulfide isomerases (PDIs) can activate β3 integrin in various cells to promote cell migration, adhesion and fusion. However, the effect of PDIs on myogenesis during muscle regeneration has not been elucidated. Here, we report that PDIA3 expression is induced in regenerating myofibers. The inhibition of PDIA3 in muscle injuries in mice disrupts myoblast differentiation, impairs muscle regeneration, and ultimately aggravates muscle damage. Moreover, PDIA3 expression is upregulated and observed on the cell surfaces of myoblasts during differentiation and fusion. The inhibition of extracellular PDIA3 with an anti-PDIA3 monoclonal antibody attenuates β3 integrin/AKT/mTOR signal activity, inhibits myoblast differentiation, and blocks the fusion of myoblasts. Thus, PDIA3 may be a mediator of myoblast differentiation and fusion during muscle regeneration., Muscle development: Regeneration after injury A regulatory protein secreted into the extracellular medium helps promote formation of new skeletal muscle tissue after injury. This regenerative process is enabled by muscle cell progenitors called satellite cells, which respond to damage by developing into myoblasts that subsequently fuse into muscle fibers. Researchers led by Xi Peng of the Third Military Medical University in Chongqing, China, have identified the protein PDIA3 as an important intermediary in this process. After chemically inducing muscle damage in mice, the researchers noted that tissue regeneration was associated with a sharp increase in PDIA3 expression. They subsequently linked PDIA3 with activation of a signaling pathway that drives muscle fiber formation and fusion, and demonstrated that inhibition of this protein impeded healing. PDIA3 was not strongly expressed in satellite cells, indicating that it acts relatively late in the maturation process.