Background: The process of prenatal hematopoiesis occurs in various anatomical locations, including the placenta. The placenta is not merely a temporary hematopoietic reservoir, but it is one of the key sites for the synthesis of hematopoietic stem cells (HSCs). This study aimed to investigate the presence, distribution, and immunoprofiles of HSCs in the human placenta during different gestational periods. Materials and Methods: Placental samples of different gestational ages (first, second, and third trimesters) were analyzed using classical hematoxylin and eosin staining and immunohistochemical staining for CD34, CD117, and CD41 markers, with HSC quantification through numerical areal density (NA). Results: Highly immunoreactive CD34 HSCs were present in placentas throughout gestation, while highly immunoreactive CD117 and CD41 HSCs were observed during the first two trimesters. In the first trimester, HSCs were found within the lumen of blood vessels and as individual cells in the mesenchyme of chorionic villi. With advancing gestation, the number of HSCs in the mesenchyme of chorionic villi increased. Conclusions: Immunoreactive CD34, CD117, and CD41 cells are present in significant proportions in various parts of the placenta throughout gestation, indicating that the placenta provides a substantial proportion of HSCs for hematopoiesis.

Lizards are distinguished as the only amniotes, and closest relatives of mammals, capable of multilineage epimorphic regeneration. Tail blastemas of green anole lizards (Anolis carolinensis) consist of col3a1+ fibroblastic connective tissue cells enclosed in krt5+ wound epidermis (WE), both of which are required for regeneration. Blastema and WE formation are known to be closely associated with phagocytic cell populations, including macrophages and osteoclasts. However, it remains unclear what specific phagocytic cell types are required to stimulate regeneration. Here, we explicitly assess the roles of osteoclast activity during blastema and WE formation in regenerating lizard tails. First, probe sequencing was performed at regenerative timepoints on fibroblasts isolated based on col3a1 expression toward establishing pathways involved in stimulating blastema formation and subsequent tail regrowth. Next, treatments with osteoclast inhibitor zoledronic acid (ZA) were used to assess the roles of osteoclast activity in lizard tail regeneration and fibroblast signaling. ZA treatment stunted lizard tail regrowth, suggesting osteoclast activity was required for blastema formation and regeneration. Transcriptomic profiling of fibroblasts isolated from ZA-treated and control lizards linked inhibition of osteoclast activity with limitations in fibroblasts to form pro-regenerative extracellular matrix and support WE formation. These results suggest that crosstalk between osteoclasts and fibroblasts regulates blastema and WE formation during lizard tail regeneration.

Secondary immunodeficiencies in allo-HSCT (allogeneic hematopoietic stem cell transplantation) recipients increase the risk of viral reactivation, making vaccinations a vital issue. There is a paucity of data on the use of recombinant vaccine against herpes zoster (RZV) after allo-HSCT.

Mesenchymal stem/stromal cells (MSCs) are non-hematopoietic, plastic-adherent, and self-renewing cells capable of in vitro trilineage differentiation into fat, bone, and cartilage tissue. Suggestively, MSCs have additional plasticity, as demonstrated by their ability to differentiate in vitro into myocytes, neuron-like cells, and hepatocytes. MSCs are ideal for therapeutic application owing to their numerous advantages; they exhibit limited growth and differentiation abilities, leading to heterogeneous cell populations with inconsistent functions. However, highly purified MSCs, namely, rapidly expanding clones (RECs) that are isolated by single-cell sorting, display uniform functionality. RECs have the potential to offer many benefits, such as transplantable cells for treating several disorders of bone, heart, peripheral nerves, brain, and other organs. This study aimed to assess the effects of RECs on the pheochromocytoma (PC12) cell line, a well-known neuronal cell model.

Background: Podocytes are essential for kidney function, and their dysfunction can result in nephrotic syndrome, such as minimal change disease (MCD). Oxidative stress contributes to podocyte damage. We investigated the therapeutic potential of intravenously infused mesenchymal stem cells (MSCs) in a puromycin aminonucleoside (PAN)-induced rodent MCD model, focusing on oxidative stress modulation. Methods: Sprague-Dawley rats were divided into three groups: intact, PAN-Vehicle, and PAN-MSC. MCD was induced through subcutaneous PAN injection. MSCs were infused intravenously in the PAN-MSC group on day 7. Urinary albumin, serum albumin, and creatinine levels were assessed. Histological analysis of the renal cortex was performed. Podocyte protein (NPHS1, NPHS2, and PODXL) and antioxidant enzyme (SOD1, SOD2, and GPX1) levels were measured using quantitative real-time reverse-transcription PCR (qRT-PCR). Results: MSC infusion significantly reduced proteinuria and restored podocyte structure in the PAN-MSC group. Electron microscopy revealed that infused MSCs could inhibit the fusion of the foot process induced by PAN injection. qRT-PCR showed that intravenous infusion of MSCs rescued the inhibition of GPX1 expression. GFP-labeled MSCs accumulated at the podocyte injury sites. Conclusion: Systemic MSC infusion mitigates PAN-induced MCD by reducing proteinuria, preserving podocyte structure, and modulating oxidative stress via the GPX1 pathway, offering a potential therapeutic approach for nephrotic syndrome.

Background/Objectives: X-inactive-specific transcript (XIST) is a factor that plays a role in neuroinflammation. This study investigated the role of XIST in neuronal development, neuroinflammation, myelination, and therapeutic responses within cerebral organoids in the context of Multiple Sclerosis (MS) pathogenesis. Methods: Human cerebral organoids with oligodendrocytes were produced from XIST-silenced H9 cells, and the mature organoids were subsequently treated with either FTY720 or DMF. Gene expression related to inflammation and myelination was subsequently analyzed via qRT-PCR. Immunofluorescence staining was used to assess the expression of proteins related to inflammation, myelination, and neuronal differentiation. Alpha-synuclein protein levels were also checked via ELISA. Finally, transcriptome analysis was conducted on the organoid samples. Results: XIST-silenced organoids presented a 2-fold increase in the expression of neuronal stem cells, excitatory neurons, microglia, and mature oligodendrocyte markers. In addition, XIST silencing increased IL-10 mRNA expression by 2-fold and MBP and PLP1 expression by 2.3- and 0.6-fold, respectively. Although XIST silencing tripled IBA1 protein expression, it did not affect organoid MBP expression. FTY720, but not DMF, distinguished MBP and IBA1 expression in XIST-silenced organoids. Furthermore, XIST silencing reduced the concentration of alpha-synuclein from 300 to 100 pg/mL, confirming its anti-inflammatory role. Transcriptomic and gene enrichment analyses revealed that the differentially expressed genes are involved in neural development and immune processes, suggesting the role of XIST in neuroinflammation. The silencing of XIST modified the expression of genes associated with inflammation, myelination, and neuronal growth in cerebral organoids, indicating a potential involvement in the pathogenesis of MS. Conclusions: XIST may contribute to the MS pathogenesis as well as neuroinflammatory diseases such as and Alzheimer's and Parkinson's diseases and may be a promising therapeutic target.

Adipocyte differentiation is a complex process in which pluripotent mesenchymal stem cells (MSCs) differentiate and develop into mature fat cells, also known as adipocytes. This process is controlled by various transcription factors, hormones, and signaling molecules that regulate the development of these cells. Recently, an increasing number of non-coding RNAs (ncRNAs), especially microRNAs (miRNAs), have been established to be involved in the regulation of many biological processes, including adipocyte differentiation, development, metabolism, and energy homeostasis of white and brown adipose tissue. Several in vitro and in vivo studies reported the significant role of ncRNAs in either promoting or inhibiting adipocyte differentiation into white or brown fat cells by targeting specific transcription factors and regulating the expression of key adipogenic genes. Identifying the function of ncRNAs and their subsequent targets contributes to our understanding of how these molecules can be used as potential biomarkers and tools for therapies against obesity, diabetes, and other diseases related to obesity. This could also contribute to advancements in tissue-engineering based treatments. In this review, we intended to present an up-to-date comprehensive literature overview of the role of ncRNAs, including miRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), focusing particularly on miRNAs, in regulating the differentiation and development of cells into white and brown adipose tissue. In addition, we further discuss the potential use of these molecules as biomarkers for the development of novel therapeutic strategies for future personalized treatment options for patients.

Male germline stem cells are relevant for stem cell researchers but also for andrologists as they are crucial for testis function and initiation/maintenance of spermatogenesis. They are also considered a target for fertility preservation in the male; e.g. germ cell transplantation or testicular grafting rely on spermatogonial stem cells (SSCs) and may soon become clinical tools to recover fertility. In the current review, we report new insights into genesis of spermatogonia, germline plasticity, and models of spermatogonial expansion. These insights and an array of novel cellular and molecular tools have provided great technological advances and new knowledge and therefore the field of SSCs needs an up-to-date review.

Mesenchymal stem cells (MSCs) are widely used for tissue regeneration due to their multilineage differentiation potential and ability to secrete paracrine factors with immunomodulatory and angiogenic functions. Standard MSC differentiation protocols typically rely on two-dimensional (2D) or pellet culture models that are simple to use but not well-suited for translational or clinical applications. To promote better cell survival, tissue deposition, and differentiation of MSCs, a wide variety of three-dimensional (3D) biomaterial scaffolds and platforms have been developed that provide structural support and present a carefully defined set of biochemical and biophysical cues to cells. While biomaterials can guide cell behavior and promote desirable tissue regeneration outcomes, one remaining challenge in the field is inherent donor-to-donor variability in MSC behavior, phenotype, and differentiation capacity. Although MSCs are promising tools for regeneration, the influence of donor variability on MSC differentiation across culture models remains poorly understood. Previous studies typically use cells from a single donor or rely solely on standard culture models. To address these gaps, we compared MSCs from six human donors and assessed differentiation across chondrogenic, osteogenic, and adipogenic lineages using both standard (pellet or 2D) and 3D biomaterial-based culture models. Alginate hydrogels were used to assess chondrogenesis, while gelatin microribbon (µRB) hydrogels were used to evaluate osteogenesis and adipogenesis in 3D. Significant donor-to-donor variability was observed in differentiation outcomes across all three lineages and within both 2D and 3D culture models. By directly comparing donor variability in 2D and 3D, we provide evidence that standard 2D models cannot predict MSC differentiation capacity in 3D biomaterials. Therefore, to improve therapeutic efficacy and advance biomaterial-based strategies for tissue regeneration, it is critical to understand how donor variability affects MSC differentiation patterns across 3D biomaterial-based culture models.

Neural stem cell therapies hold great promise for improving stroke recovery, but the hostile stroke microenvironment can hinder the initial graft survival. It has long been well documented that the microenvironment evolves over time, making it crucial to identify the optimal transplantation window to maximize therapeutic efficacy. However, it remains uncertain whether acute or delayed local cell transplantations better supports graft viability after stroke. Here, it is shown that delayed intracerebral transplantation of neural progenitor cells (NPCs) derived from human induced pluripotent cells (iPSCs) at 7 days post stroke significantly enhances graft proliferation and survival, and promotes axonal sprouting, compared to acute transplantation at 1 day post stroke, in a mouse model of large cortical stroke. Using in vivo bioluminescence imaging over a 6-week period post-transplantation, a more than fivefold increase is observed in bioluminescence signal in mice that received delayed NPC therapy, compared to those that underwent acute NPC transplantation. The increased number of cell grafts in mice receiving delayed NPC transplantation is driven by increased proliferation rates early after transplantation, which subsequently declines to similarly low levels in both groups. Notably, it is found that the majority of transplanted NPCs differentiate into neurons after 6 weeks, with no significant differences in the neuron-to-glia ratio between acute and delayed transplantation groups. These findings suggest that delayed NPC transplantation improves early graft survival and proliferation, which could help identify the optimal therapeutic window for maximizing the effectiveness of NPC-based therapies in stroke.

The aberrant cellular senescence in chronic wounds presents a significant barrier to healing. Mitochondrial dysfunction is critical in initiating and maintaining cellular senescence, underscoring therapeutic potential in restoring mitochondrial function by delivering healthy mitochondria to wound cells. However, approaches for delivering mitochondria to achieve optimized wound repair remain lacking. Herein, enucleated MSCs-derived microvesicles containing functional mitochondria (Mito@euMVs) via simple extrusion are developed. By controlling the size of microvesicles within a small micron-scale range, the mitochondrial encapsulation efficiency is optimized. Mito@euMVs effectively delivered mitochondria into fibroblasts and HUVECs, inhibiting and rejuvenating hyperglycemia-induced cellular senescence. To enhance the clinical applicability, soluble PVA microneedle patches for the transdermal Mito@euMVs delivery are utilized. In diabetic rats with pressure sores, the senescence-inhibiting and -rescuing properties of Mito@euMVs are further validated, along with their therapeutic efficacy, demonstrating their potential for chronic wound repair. Moreover, as a versatile delivery vehicle for mitochondria, Mito@euMVs hold promising for treating mitochondrial dysfunction and aging-related conditions.

The aim of this study was to investigate the treatment effect of an artificial nerve graft produced with polylactic acid (PLA) and polyvinylpyrrolidone (PVP) by electrospinning method, in combination with rat allogeneic adipose tissue-derived mesenchymal stem cells (rAT-MSCs), on axonal regeneration in a rat sciatic nerve injury. A 10 mm long gap in the right sciatic nerve was created in rats which were divided into three groups and the gap was repaired with autologous nerve graft (autograft group), PLA/PVP conduit alone (PLA/PVP group), and PLA/PVP conduit with rAT-MSCs inside (PLA/PVP with rAT-MSC group). At the end of the eighth week, the results were evaluated functionally, electrophysiologically, and histologically. In functional evaluation, sciatic functional index (SFI) was used and all groups showed improvement over time. The results were seen in the autograft and PLA/PVP with rAT-MSC groups, with no statistically significant difference between them. In electrophysiological evaluation, action potential was seen in all three groups, with the best results seen in the PLA/PVP with rAT-MSC group with statistical significance. Histological evaluation showed axonal regeneration in all three groups. These results show that PLA/PVP conduit contributes to regeneration even when used alone, and increases regeneration when used together with rAT-MSCs.

Study DesignSystematic review and meta-analysis.ObjectivesThe objective of this study was to conduct a systematic review and meta-analysis of the literature regarding the therapeutic effect of embryonic and mesenchymal stem cells on the treatment of traumatic spinal cord injury (SCI) in humans. Primary outcome measures were overall American Spinal Injury Association (ASIA) scores, ASIA motor and sensory scores, urinary and bowel function, pain, and adverse events.MethodsStudies with human patients ages 18-80 years receiving embryonic, induced pluripotent, or mesenchymal stem cells for SCI were included. Study quality was assessed using the Cochrane risk of bias 2 tool and the Newcastle Ottawa scale for randomized and non-randomized studies, respectively. Primary outcomes were overall ASIA grade, ASIA motor scores, ASIA sensory scores, bladder and bowel function, pain, and adverse events.ResultsThirty total studies with 656 patients were included, with 43.3% of patients experiencing improvement in ASIA grade, 49.4% in motor function, and 73.6% in sensory function. Qualitative analysis of bladder and bowel outcomes suggests overall improved sensation and control. No serious adverse events were reported. The most common side effects were mild and resolved within hours to weeks without requiring additional medical treatment.ConclusionsStem cell transplantation for SCI appears to offer moderate improvements in overall ASIA grade, motor, sensory, bladder, and bowel function, accompanied by a relatively mild and transient side effect profile. Further research, particularly high-quality, blinded, randomized controlled trials, is essential to optimize treatment protocols and achieve more consistent and improved clinical outcomes.

Developing highly fluorescent biogenic carbon dots (CDs) as molecular probes and cellular imaging agents with excellent cellular biocompatibility and water dispersibility can advance the scope of CDs in various biomedical applications. The present work aims to produce CDs from biological precursors such as natural amino acids. Due to their molecular recognition properties, the resultant CDs exhibit biocompatibility, polar functional groups, and selective molecule sensing. Microwave (MW)-assisted carbonization of L-tryptophan, L-glutamic acid, and their mixture led to the formation of CDs. However, the CDs derived from the mix of amino acids exhibited strong emission at 450 nm, with a quantum yield of 31.3%, and showed excellent stability under wide pH conditions. These CDs have a crystalline graphitic core, predominantly derived from the carbonization of the aromatic moiety intrinsic to L-tryptophan amino acid, while the partial carbonization of L-glutamic acid led to anchor polar functional groups on the CDs surface. These CDs were established as molecular probes for sensing the biomolecule cysteamine with enhanced sensitivity and remarkable cross-selectivity among the interfering metal ions, anions, and other thiols. To demonstrate these materials' cellular biocompatibility, the cell viability of MCF-7 and fibroblast cells was studied following their exposure to CDs. The studies revealed that the fluorescent CDs did not elicit cytotoxic effects on the cells at elevated concentrations. The CDs from the mixture of amino acids were efficiently internalized by MCF-7 cells, enabling efficient imaging of live cells. Overall, this study developed biogenic and cellular biocompatible CDs with robust luminescent properties and demonstrated their applications in biomolecular sensing, cellular uptake, and imaging.

Aims: In Friedreich ataxia (FRDA), early motor discoordination stems from dysfunctional sensory neurons in the spinal cord driven by epigenetic dysregulation, frataxin (FXN) deficiency, oxidative stress, and inflammation. Omaveloxolone, a nuclear factor erythroid 2-related factor-2 (NRF2) inducer, is the only treatment available. In various chronic disease models, sulforaphane (SF) can target NRF2 and the above processes. This study compared the effects of SF with omaveloxolone and dimethyl fumarate (DMF) in sensory neurons generated from FRDA patient-induced pluripotent stem cells and their isogenic control. Results: The successful generation of the FRDA and isogenic control sensory neurons was confirmed by the positive expression of β-III TUBULIN, BRN3A, ISLET1, PERIPHERIN, and tropomyosin receptor kinase C. In comparison with the isogenic control, FRDA sensory neurons displayed an aberrant gene expression profile alike to that reported in patients. None of the drugs affected the viability of the isogenic control sensory neurons. SF treatment improved the viability of FRDA sensory neurons by up to 61% versus the untreated control. DMF treatment showed a modest 35% increase, while omaveloxolone lacked an effect. SF-treated FRDA sensory neurons demonstrated increased reduced glutathione/oxidized glutathione ratio and expression of FXN and redox markers, and a reduced expression of selected epigenetic enzymes and inflammatory cytokines, at the respective gene and protein levels. DMF and omaveloxolone treatments only modulated some of these biomarkers. Innovation: We revealed the therapeutic potential of SF and how it performs in comparison with omaveloxolone and DMF, in a physiologically and genetically relevant in vitro FRDA model. Conclusion: SF offers a multipronged approach to alleviating the different cellular events underlying FRDA. Antioxid. Redox Signal. 00, 000-000. [Figure: see text].

The international guidelines outlining the mandatory developmental toxicology studies of new molecules on pre-implantation, post-implantation and organogenesis phases, require a minimum of 60 pregnant female rats for each molecule to be tested. To date, available in vitro methods still have many limitations, resulting in poor translational power.

Management of chronic non-healing wounds in cats requires a comprehensive approach. This report describes the treatment of a severe open skin wound on the skull using a combined approach involving allogeneic adipose-derived mesenchymal stem cells (MSCs) and autologous platelet-rich plasma (PRP). A 12-year-old neutered male mixed-breed domestic cat presented with a non-healing chronic wound on the skull. The wound extended from the orbital to the occipital area and from the left to the right temporal region. Laboratory test results were positive for feline immunodeficiency virus and impaired kidney function. Sensitivity tests revealed resistance to several antibiotics. Due to limited skin reconstruction options, MSCs were administered subcutaneously at the wound edge once a month for three months. PRP was collected one month after the initial MSC administration and injected at the wound edge monthly between MSC treatments. The wound diameter was measured daily during saline cleaning. The cat received protein-supplemented food daily. Wound healing was observed two weeks after the MSC administration, gradually decreasing in size and closing completely within 5 months. This case demonstrates the successful application of MSCs and PRP for treating chronic wounds in cats.

SOX9 and RUNX2 are lineage defining transcription factors that drive differentiation of chondrocyte and osteoblast lineages respectively from osteochondral progenitors. In limb development, these progenitors are specified first by SOX9 expression required for mesenchymal stem cell (MSC) condensation prior to RUNX2 activation and osteochondral differentiation to chondrocyte and osteoblast lineages. Unlike limb development, the anterior craniofacial skeleton arises from cranial neural crest (cNCC) stem cells. To examine the temporal activation of SOX9 and RUNX2 within cNCCs, we utilized a combination of immunofluorescence to detect endogenous proteins and genetic reporters to label SOX9 and RUNX2 expressing cells. We find that RUNX2 is expressed broadly throughout cNCC stem cells of the first branchial arch that will give rise to developing mandibular tissue at a timepoint prior to osteochondral lineage determination. Substantial SOX9 expression is activated subsequently within differentiating chondrocytes. These findings were validated by fluorescent reporters inserted in the 3' untranslated regions (3'UTRs) of Sox9 and Runx2 . Although the GFP based Runx2 reporter did not delete any 3'UTR sequences, homozygous Runx2 GFP/GFP pups develop postnatal deficiencies in intramembranous and endochondral ossification that correlate with enhanced expression of RUNX2 protein in osteoblasts and hypertrophic chondrocytes. Runx2 GFP/GFP phenotypes model the human disorder, Metaphyseal Dysplasia with Maxillary Hypoplasia and Brachydactyly (MDMHB), resulting from RUNX2 enhanced activity due to intragenic duplications. Altogether, this reporter model provides a valuable tool for studying RUNX2 function in early cNCC-derived stem cell lineages and highlights the high sensitivity of ossification pathways to RUNX2 dosage.

The ability to reprogram mature, differentiated cells into induced pluripotent stem cells (iPSCs) using exogenous pluripotency factors opened up unprecedented opportunities for their application in biomedicine. iPSCs are already successfully used in cell and regenerative therapy, as various drug discovery platforms and for in vitro disease modeling. However, even though already 20 years have passed since their discovery, the production of iPSC-based therapies is still associated with a number of hurdles due to low reprogramming efficiency, the complexity of accurate characterization of the resulting colonies, and the concerns associated with the safety of this approach. However, significant progress in many areas of molecular biology facilitated the production, characterization, and thorough assessment of the safety profile of iPSCs. The number of iPSC-based studies has been steadily increasing in recent years, leading to the accumulation of significant knowledge in this area. In this review, we aimed to provide a comprehensive analysis of methods used for reprogramming and subsequent characterization of iPSCs, discussed barriers towards achieving these goals, and various approaches to improve the efficiency of reprogramming of different cell populations. In addition, we focused on the analysis of iPSC application in preclinical and clinical studies. The accumulated breadth of data helps to draw conclusions about the future of this technology in biomedicine.

Hepatocellular carcinoma is the seventh most common kind of cancer worldwide and the second largest cause of cancer-related deaths in males, behind lung cancer. Globally, 866,000 people were diagnosed with hepatocellular carcinoma (HCC) in 2022, and nearly 42,240 new cases will be identified in 2025 in the United States. Using stem cells obtained from bone marrow can effectively reduce the number of malignant tumor cells through the induction of an epigenetic impact. We obtained bone marrow-derived mesenchymal stem cells (BM-MSCs) from mice and collected the conditioned medium (CM) from cultured cells with 90% confluency. The effect of the CM was identified using both 2D and 3D sphere cultures of wild-type human liver cancer cell line (HepG2), considering variations in sphere size and percentage. A cell death study was conducted using the cell cytotoxicity (MTT) kit, while the quantity of stem cells was determined by immunohistochemistry and gene expression analysis. The effectiveness of our therapy was demonstrated by an in vivo assessment of BM-MSCs through intravenous injection and the currently available anticancer drug cisplatin. In vitro, the combination treatment resulted in a synergetic effect, leading to 74% cell death in both adherent and spherical cultures when treated with 25 µM of cisplatin and 90%CM. In vivo, the histological study indicated a decrease in tumor size and number following treatment with cisplatin and BM-MSCs. The study lasted 18 weeks and revealed that the body weight of mice improved across all treatment groups, with the combination group exhibiting the most significant improvement. Both in vitro and in vivo studies showed the synergetic effect of cisplatin and isolated conditioned medium. Our study aimed to identify more efficient therapeutic approaches utilizing stem cells and existing marketed medications to minimize adverse effects with better efficacy.