Stem Cell Differentiation and Applications

The differentiation of ESCs into cardiomyocytes in vitro is an excellent and reliable model system for studying normal cardiomyocyte development in mammals, modeling cardiac diseases, and for use in drug screening. Mouse ESC differentiation still provides relevant biological information about cardiac development. However, the current methods for efficiently differentiating ESCs into cardiomyocytes are limiting. Here, we describe the “WNT Switch” method to efficiently commit mouse ESCs into cardiomyocytes using the small molecule WNT signaling modulators CHIR99021 and XAV939 in vitro. This method significantly improves the yield of beating cardiomyocytes, reduces number of treatments, and is less laborious. Full article

Human embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DANs), and Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson’s disease (PD) patients. Here, we obtained well-characterized DANs from hESCs and transplanted them into two parkinsonian monkeys to assess their behavioral and imaging changes. DANs from hESCs expressed dopaminergic markers, generated action potentials, and released dopamine (DA) in vitro. These neurons were transplanted bilaterally into the putamen of parkinsonian NHPs, and using magnetic resonance imaging techniques, we calculated the fractional anisotropy (FA) and mean diffusivity (MD), both employed for the first time for these purposes, to detect in vivo axonal and cellular density changes in the brain. Likewise, positron-emission tomography scans were performed to evaluate grafted DANs. Histological analyses identified grafted DANs, which were quantified stereologically. After grafting, animals showed signs of partially improved motor behavior in some of the HALLWAY motor tasks. Improvement in motor evaluations was inversely correlated with increases in bilateral FA. MD did not correlate with behavior but presented a negative correlation with FA. We also found higher 11C-DTBZ binding in positron-emission tomography scans associated with grafts. Higher DA levels measured by microdialysis after stimulation with a high-potassium solution or amphetamine were present in grafted animals after ten months, which has not been previously reported. Postmortem analysis of NHP brains showed that transplanted DANs survived in the putamen long-term, without developing tumors, in immunosuppressed animals. Although these results need to be confirmed with larger groups of NHPs, our molecular, behavioral, biochemical, and imaging findings support the integration and survival of human DANs in this pre-clinical PD model. Full article

The airway epithelium is a critical component of the respiratory system, serving as a barrier against inhaled pathogens and toxins. It is composed of various cell types, each with specific functions essential to proper airway function. Chronic respiratory diseases can disrupt the cellular composition of the airway epithelium, leading to a decrease in multiciliated cells (MCCs) and an increase in secretory cells (SCs). Basal cells (BCs) have been identified as the primary stem cells in the airway epithelium, capable of self-renewal and differentiation into MCCs and SCs. This review emphasizes the role of transcription factors in the differentiation process from BCs to MCCs and SCs. Recent advancements in single-cell RNA sequencing (scRNAseq) techniques have provided insights into the cellular composition of the airway epithelium, revealing specialized and rare cell types, including neuroendocrine cells, tuft cells, and ionocytes. Understanding the cellular composition and differentiation processes within the airway epithelium is crucial for developing targeted therapies for respiratory diseases. Additionally, the maintenance of BC populations and the involvement of Notch signaling in BC self-renewal and differentiation are discussed. Further research in these areas could provide valuable insights into the mechanisms underlying airway epithelial homeostasis and disease pathogenesis. Full article

The transcription factors Oct4, Sox2, Klf4, and c-Myc enable the reprogramming of somatic cells into induced pluripotent cells. Reprogramming generates newly differentiated cells for potential therapies in cancer, neurodegenerative diseases, and rejuvenation processes. In cancer therapies, these transcription factors lead to a reduction in the size and aggressiveness of certain tumors, such as sarcomas, and in neurodegenerative diseases, they enable the production of dopaminergic cells in Parkinson’s disease, the replacement of affected neuronal cells in olivopontocerebellar atrophy, and the regeneration of the optic nerve. However, there are limitations, such as an increased risk of cancer development when using Klf4 and c-Myc and the occurrence of abnormal dyskinesias in the medium term, possibly generated by the uncontrolled growth of differentiated dopaminergic cells and the impairment of the survival of the new cells. Therefore, the Yamanaka transcription factors have shown therapeutic potential through cell reprogramming for some carcinomas, neurodegenerative diseases, and rejuvenation. However, the limitations found in the studies require further investigation before the use of these transcription factors in humans. Full article

Leiomyomas (fibroids) are monoclonal tumors in which myometrial stem cells (MSCs) turn tumorigenic after mutation, abnormal methylation, or aberrant signaling. Several factors contribute to metabolic dysfunction in obesity, including abnormal cellular proliferation, oxidative stress, and DNA damage. The present study aims to determine how adipocytes and adipocyte-secreted factors affect changes in MSCs in a manner that promotes the growth of uterine leiomyomas. Myometrial stem cells were isolated from the uteri of patients by fluorescence-activated cell sorting (FACS) using CD44/Stro1 antibodies. Enzyme-linked immunosorbent assay (ELISA), Western blot, and immunocytochemistry assays were performed on human adipocytes (SW872) co-cultured with MSCs and treated with leptin or adiponectin to examine the effects of proliferation, extracellular matrix (ECM) deposition, oxidative damage, and DNA damage. Co-culture with SW872 increased MSC proliferation compared to MSC culture alone, according to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) results. The expressions of PCNA and COL1A increased significantly with SW872 co-culture. In addition, the expression of these markers was increased after leptin treatment and decreased after adiponectin treatment in MSCs. The Wnt/β-catenin and TGF-β/SMAD signaling pathways promote proliferation and ECM deposition in uterine leiomyomas. The expression of Wnt4, β-catenin, TGFβ3, and pSMAD2/3 of MSCs was increased when co-cultured with adipocytes. We found that the co-culture of MSCs with adipocytes resulted in increased NOX4 expression, reactive oxygen species production, and γ-H2AX expression. Leptin acts by binding to its receptor (LEP-R), leading to signal transduction, resulting in the transcription of genes involved in cellular proliferation, angiogenesis, and glycolysis. In MSCs, co-culture with adipocytes increased the expression of LEP-R, pSTAT3/STAT3, and pERK1/2/ERK/12. Based on the above results, we suggest that obesity may mediate MSC initiation of tumorigenesis, resulting in leiomyomas. Full article

Au decorated with type I collagen (Col) was used as a core material to cross-link with stromal cell-derived factor 1α (SDF1α) in order to investigate biological performance. The Au-based nanoparticles were subjected to physicochemical determination using scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet–visible (UV-Vis) and Fourier-transform infrared spectroscopy (FTIR). Mesenchymal stem cells (MSCs) were used to evaluate the biocompatibility of this nanoparticle using the MTT assay and measuring reactive oxygen species (ROS) production. Also, the biological effects of the SDF-1α-conjugated nanoparticles (Au-Col-SDF1α) were assessed and the mechanisms were explored. Furthermore, we investigated the cell differentiation-inducing potential of these conjugated nanoparticles on MSCs toward endothelial cells, neurons, osteoblasts and adipocytes. We then ultimately explored the process of cell entry and transportation of the nanoparticles. Using a mouse animal model and retro-orbital sinus injection, we traced in vivo biodistribution to determine the biosafety of the Au-Col-SDF1α nanoparticles. In summary, our results indicate that Au-Col is a promising drug delivery system; it can be used to carry SDF1α to improve MSC therapeutic efficiency. Full article

Reactive oxygen species (ROS) are currently recognized as a key driver of several physiological processes. Increasing evidence indicates that ROS levels can affect myogenic differentiation, but the molecular mechanisms still need to be elucidated. Protein kinase C (PKC) epsilon (PKCe) promotes muscle stem cell differentiation and regeneration of skeletal muscle after injury. PKCs play a tissue-specific role in redox biology, with specific isoforms being both a target of ROS and an up-stream regulator of ROS production. Therefore, we hypothesized that PKCe represents a molecular link between redox homeostasis and myogenic differentiation. We used an in vitro model of a mouse myoblast cell line (C2C12) to study the PKC–redox axis. We demonstrated that the transition from a myoblast to myotube is typified by increased PKCe protein content and decreased ROS. Intriguingly, the expression of the antioxidant enzyme superoxide dismutase 2 (SOD2) is significantly higher in the late phases of myogenic differentiation, mimicking PKCe protein content. Furthermore, we demonstrated that PKCe inhibition increases ROS and reduces SOD2 protein content while SOD2 silencing did not affect PKCe protein content, suggesting that the kinase could be an up-stream regulator of SOD2. To support this hypothesis, we found that in C2C12 cells, PKCe interacts with Nrf2, whose activation induces SOD2 transcription. Overall, our results indicate that PKCe is capable of activating the antioxidant signaling preventing ROS accumulation in a myotube, eventually promoting myogenic differentiation. Full article

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger. Full article

Odontoblastic differentiation of human dental pulp stem cells (hDPSCs) is crucial for the intricate formation and repair processes in dental pulp. Until now, the literature is not able to demonstrate the role of ubiquitination in the odontoblastic differentiation of hDPSCs. This study investigated the role of F-box-only protein 32 (FBXO32), an E3 ligase, in the odontoblastic differentiation of hDPSCs. The mRNA expression profile was obtained from ribonucleic acid sequencing (RNA-Seq) data and analyzed. Immunofluorescence and immunohistochemical staining identify the FBXO32 expression in human dental pulp and hDPSCs. Small-hairpin RNA lentivirus was used for FBXO32 knockdown and overexpression. Odontoblastic differentiation of hDPSCs was determined via alkaline phosphatase activity, Alizarin Red S staining, and mRNA and protein expression levels were detected using real-time quantitative polymerase chain reaction and Western blotting. Furthermore, subcutaneous transplantation in nude mice was performed to evaluate the role of FBXO32 in mineralization in vivo using histological analysis. FBXO32 expression was upregulated in the odontoblast differentiated hDPSCs as evidenced by RNA-Seq data analysis. FBXO32 was detected in hDPSCs and the odontoblast layer of the dental pulp. Increased FBXO32 expression in hDPSCs during odontoblastic differentiation was confirmed. Through lentivirus infection method, FBXO32 downregulation in hDPSCs attenuated odontoblastic differentiation in vitro and in vivo, whereas FBXO32 upregulation promoted the hDPSCs odontoblastic differentiation, without affecting proliferation and migration. This study demonstrated, for the first time, the promotive role of FBXO32 in regulating the odontoblastic differentiation of hDPSCs, thereby providing novel insights into the regulatory mechanisms during odontoblastic differentiation in hDPSCs. Full article

Neuropilin 1 (NRP1), a non-tyrosine kinase receptor for several ligands, is highly expressed in many kinds of mesenchymal stem cells (MSCs), but its function is poorly understood. In this study, we explored the roles of full-length NRP1 and glycosaminoglycan (GAG)-modifiable NRP1 in adipogenesis in C3H10T1/2 cells. The expression of full-length NRP1 and GAG-modifiable NRP1 increased during adipogenic differentiation in C3H10T1/2 cells. NRP1 knockdown repressed adipogenesis while decreasing the levels of Akt and ERK1/2 phosphorylation. Moreover, the scaffold protein JIP4 was involved in adipogenesis in C3H10T1/2 cells by interacting with NRP1. Furthermore, overexpression of non-GAG-modifiable NRP1 mutant (S612A) greatly promoted adipogenic differentiation, accompanied by upregulation of the phosphorylated Akt and ERK1/2. Taken together, these results indicate that NRP1 is a key regulator that promotes adipogenesis in C3H10T1/2 cells by interacting with JIP4 and activating the Akt and ERK1/2 pathway. Non-GAG-modifiable NRP1 mutant (S612A) accelerates the process of adipogenic differentiation, suggesting that GAG glycosylation is a negative post-translational modification of NRP1 in adipogenic differentiation. Full article

Background: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the HBB gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. Methods: iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of HBB and HBG2. Results: Both 2D and 3D differentiation protocols led to the induction of CD34⁺/CD43⁺ HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71⁺/CD235a⁺ cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a⁺/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal HBG2 compared to adult HBB. Conclusion: A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development. Full article

Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into hepatocyte-like cells (HLCs) to alleviate acute liver injury (ALI). Herpetfluorenone (HPF), as an active ingredient in the dried, mature seeds Herpetospermum caudigerum Wall, used in Tibetan medicine, has been proven to effectively alleviate ALI. Therefore, the purpose of this study was to determine whether HPF can promote the differentiation of BMSCs into HLCs and promote ALI recovery. Mouse BMSCs were isolated, and the BMSCs’ differentiation into HLCs was induced by HPF and hepatocyte growth factor (HGF). Under the induction of HPF and HGF, the expression of hepatocellular specific markers and the accumulation of glycogen and lipids in the BMSCs increased, indicating that BMSCs successfully differentiated into HLCs. Then, the ALI mouse model was established, using carbon tetrachloride, followed by an intravenous injection of BMSCs. Then, only HPF was injected intraperitoneally, in order to verify the effect of HPF in vivo. In vivo imaging was used to detect the homing ability of HPF–BMSCs, and it was detected that HPF–BMSCs significantly increased the levels of serum AST, ALT and ALP in the liver of ALI mice, and alleviated liver cell necrosis, oxidative stress and liver pathology. In conclusion, HPF can promote the differentiation of BMSCs into HLCs and promote the recovery of ALI in mice. Full article

Pluripotent neural stem or progenitor cells (NSC/NPC) have been reported in the brains of adult preclinical models for decades, as have mesenchymal stem/stromal cells (MSC) been reported in a variety of tissues from adults. Based on their in vitro capabilities, these cell types have been used extensively in attempts to repair/regenerate brain and connective tissues, respectively. In addition, MSC have also been used in attempts to repair compromised brain centres. However, success in treating chronic neural degenerative conditions such as Alzheimer’s disease, Parkinson’s disease, and others with NSC/NPC has been limited, as have the use of MSC in the treatment of chronic osteoarthritis, a condition affecting millions of individuals. However, connective tissues are likely less complex than neural tissues regarding cell organization and regulatory integration, but some insights have been gleaned from the studies regarding connective tissue healing with MSC that may inform studies attempting to initiate repair and regeneration of neural tissues compromised acutely or chronically by trauma or disease. This review will discuss the similarities and differences in the applications of NSC/NPC and MSC, where some lessons have been learned, and potential approaches that could be used going forward to enhance progress in the application of cellular therapy to facilitate repair and regeneration of complex structures in the brain. In particular, variables that may need to be controlled to enhance success are discussed, as are different approaches such as the use of extracellular vesicles from stem/progenitor cells that could be used to stimulate endogenous cells to repair the tissues rather than consider cell replacement as the primary option. Caveats to all these efforts relate to whether cellular repair initiatives will have long-term success if the initiators for neural diseases are not controlled, and whether such cellular initiatives will have long-term success in a subset of patients if the neural diseases are heterogeneous and have multiple etiologies. Full article

Using the nematode C. elegans germline as a model system, we previously reported that PUF-8 (a PUF RNA-binding protein) and LIP-1 (a dual-specificity phosphatase) repress sperm fate at 20 °C and the dedifferentiation of spermatocytes into mitotic cells (termed “spermatocyte dedifferentiation”) at 25 °C. Thus, double mutants lacking both PUF-8 and LIP-1 produce excess sperm at 20 °C, and their spermatocytes return to mitotically dividing cells via dedifferentiation at 25 °C, resulting in germline tumors. To gain insight into the molecular competence for spermatocyte dedifferentiation, we compared the germline phenotypes of three mutant strains that produce excess sperm—fem-3(q20gf), puf-8(q725); fem-3(q20gf), and puf-8(q725); lip-1(zh15). Spermatocyte dedifferentiation was not observed in fem-3(q20gf) mutants, but it was more severe in puf-8(q725); lip-1(zh15) than in puf-8(q725); fem-3(q20gf) mutants. These results suggest that MPK-1 (the C. elegans ERK1/2 MAPK ortholog) activation in the absence of PUF-8 is required to promote spermatocyte dedifferentiation. This idea was confirmed using Resveratrol (RSV), a potential activator of MPK-1 and ERK1/2 in C. elegans and human cells, respectively. Notably, spermatocyte dedifferentiation was significantly enhanced by RSV treatment in the absence of PUF-8, and its effect was blocked by mpk-1 RNAi. We, therefore, conclude that PUF-8 and MPK-1 are essential regulators for spermatocyte dedifferentiation and tumorigenesis. Since these regulators are broadly conserved, we suggest that similar regulatory circuitry may control cellular dedifferentiation and tumorigenesis in other organisms, including humans. Full article

Adipose stem cells (ASCs) are reported to play a role in normal physiology as well as in inflammation and disease. The objective of this work was to elucidate inter-individual differences in growth, gene expression and response to inflammatory stimuli in ASCs from different donors. Human ASC1 (male donor) and ASC2 (female donor) were purchased from Lonza (Walkersville, MD). Cell proliferation was determined by the sulforhodamine B assay. After time-dependent treatment of ASCs with or without bacterial lipopolysaccharide (LPS), marker gene mRNAs for proliferation, steroid hormones, and xenobiotic and immune pathways were determined using RT-PCR, and secreted cytokine levels in media were measured using the Bio-Plex cytokine assay kit. ASCs from both donors expressed androgen receptors but not estrogen receptors. ASC2 had a 2-fold higher proliferation rate and a 6-fold higher level of proliferation marker Ki67 mRNA than ASC1. ASC2 exhibited significantly greater fold induction of TNF-α and CCL2 by LPS compared to ASC1. TNF-α and GM-CSF protein levels were also significantly higher in the LPS-induced ASC2 media, but IL-6 secretion was higher in the LPS-induced ASC1 media. Our findings suggest that inter-individual variability and/or possible sex differences exist in ASCs, which may serve as a key determinant to inflammatory responses of ASCs. Full article

Calorie restriction enhances stem cell self-renewal in various tissues, including the mammary gland. We hypothesized that similar to their intestinal counterparts, mammary epithelial stem cells are insulated from sensing changes in energy supply, depending instead on niche signaling. The latter was investigated by subjecting cultures of mammary epithelial stem cells for 8 days to in vivo paracrine calorie-restriction signals collected from a 4-day-conditioned medium of individual mammary cell populations. Conditioned medium from calorie-restricted non-epithelial cells induced latent cell propagation and mammosphere formation—established markers of stem cell self-renewal. Combined RNA-Seq, immunohistochemistry and immunofluorescence analyses of the non-epithelial population identified macrophages and secreted CSF1 as the energy sensor and paracrine signal, respectively. Calorie restriction-induced pStat6 expression in macrophages suggested that skewing to the M2 phenotype contributes to the sensing mechanism. Enhancing CSF1 signaling with recombinant protein and interrupting the interaction with its highly expressed receptor in the epithelial stem cells by neutralizing antibodies were both affected stem cell self-renewal. In conclusion, combined in vivo, in vitro and in silico studies identified macrophages and secreted CSF1 as the energy sensor and paracrine transmitter, respectively, of the calorie restriction-induced effect on mammary stem cell self-renewal. Full article

(1) Background: Reconstruction of Achilles tendon defects and prevention of postoperative tendon adhesions were two serious clinical problems. In the treatment of Achilles tendon defects, decellularized matrix materials and mesenchymal stem cells (MSCs) were thought to address both problems. (2) Methods: In vitro, cell adhesion, proliferation, and tenogenic differentiation of tendon-derived stem cells (TDSCs) on small intestinal submucosa (SIS) were evaluated. RAW264.7 was induced by culture medium of TDSCs and TDSCs–SIS scaffold groups. A rat Achilles tendon defect model was used to assess effects on tendon regeneration and antiadhesion in vivo. (3) Results: SIS scaffold facilitated cell adhesion and tenogenic differentiation of TDSCs, while SIS hydrogel coating promoted proliferation of TDSCs. The expression of TGF-β and ARG-1 in the TDSCs-SIS scaffold group were higher than that in the TDSCs group on day 3 and 7. In vivo, the tendon regeneration and antiadhesion capacity of the implanted TDSCs–SIS scaffold was significantly enhanced. The expression of CD163 was significantly highest in the TDSCs–SIS scaffold group; meanwhile, the expression of CD68 decreased more significantly in the TDSCs–SIS scaffold group than the other two groups. (4) Conclusion: This study showed that biologically prepared SIS scaffolds synergistically promote tendon regeneration with TDSCs and achieve antiadhesion through M2 polarization of macrophages. Full article

Insulin-producing cells derived from induced pluripotent stem cells (iPSCs) are promising candidates for β cell replacement in type 1 diabetes. However, the risk of teratoma formation due to residual undifferentiated iPSCs contaminating the differentiated cells is still a critical concern for clinical application. Here, we hypothesized that pretreatment of iPSC-derived insulin-producing cells with an anti-CD30 antibody–drug conjugate could prevent in vivo teratoma formation by selectively killing residual undifferentiated cells. CD30 is expressed in all human iPSCs clones tested by flow cytometry (n = 7) but not in iPSC-derived β cells (iβs). Concordantly, anti-CD30 treatment in vitro for 24 h induced a dose-dependent cell death (up to 90%) in human iPSCs while it did not kill iβs nor had an impact on iβ identity and function, including capacity to secrete insulin in response to stimuli. In a model of teratoma assay associated with iβ transplantation, the pretreatment of cells with anti-CD30 for 24 h before the implantation into NOD-SCID mice completely eliminated teratoma development (0/10 vs. 8/8, p < 0.01). These findings suggest that short-term in vitro treatment with clinical-grade anti-CD30, targeting residual undifferentiated cells, eliminates the tumorigenicity of iPSC-derived β cells, potentially providing enhanced safety for iPSC-based β cell replacement therapy in clinical scenarios. Full article

Current protocols for the differentiation of human-induced pluripotent stem cells (hiPSC) into cardiomyocytes only generate a small amount of cardiac pacemaker cells. In previous work, we reported the generation of high amounts of cardiac pacemaker cells by co-culturing hiPSC with mouse visceral endoderm-like (END2) cells. However, potential medical applications of cardiac pacemaker cells generated according to this protocol, comprise an incalculable xenogeneic risk. We thus aimed to establish novel protocols maintaining the differentiation efficiency of the END2 cell-based protocol, yet eliminating the use of END2 cells. Three protocols were based on the activation and inhibition of the Wingless/Integrated (Wnt) signaling pathway, supplemented either with retinoic acid and the Wnt activator CHIR99021 (protocol B) or with the NODAL inhibitor SB431542 (protocol C) or with a combination of all three components (protocol D). An additional fourth protocol (protocol E) was used, which was originally developed by the manufacturer STEMCELL Technologies for the differentiation of hiPSC or hESC into atrial cardiomyocytes. All protocols (B, C, D, E) were compared to the END2 cell-based protocol A, serving as reference, in terms of their ability to differentiate hiPSC into cardiac pacemaker cells. Our analysis revealed that protocol E induced upregulation of 12 out of 15 cardiac pacemaker-specific genes. For comparison, reference protocol A upregulated 11, while protocols B, C and D upregulated 9, 10 and 8 cardiac pacemaker-specific genes, respectively. Cells differentiated according to protocol E displayed intense fluorescence signals of cardiac pacemaker-specific markers and showed excellent rate responsiveness to adrenergic and cholinergic stimulation. In conclusion, we characterized four novel and END2 cell-independent protocols for the differentiation of hiPSC into cardiac pacemaker cells, of which protocol E was the most efficient. Full article

Hematopoietic stem and progenitor cell (HSPC) maintenance and the differentiation of various lineages is a highly complex but precisely regulated process. Multiple signaling pathways and an array of transcription factors influence HSPC maintenance and the differentiation of individual lineages to constitute a functional hematopoietic system. Nuclear factor of activated T cell (NFAT) family transcription factors have been studied in the context of development and function of multiple mature hematopoietic lineage cells. However, until now their contribution in HSPC physiology and HSPC differentiation to multiple hematopoietic lineages has remained poorly understood. Here, we show that NFAT proteins, specifically NFATc1, play an indispensable role in the maintenance of HSPCs. In the absence of NFATc1, very few HSPCs develop in the bone marrow, which are functionally defective. In addition to HSPC maintenance, NFATc1 also critically regulates differentiation of lymphoid, myeloid, and erythroid lineage cells from HSPCs. Deficiency of NFATc1 strongly impaired, while enhanced NFATc1 activity augmented, the differentiation of these lineages, which further attested to the vital involvement of NFATc1 in regulating hematopoiesis. Hematopoietic defects due to lack of NFATc1 activity can lead to severe pathologies such as lymphopenia, myelopenia, and a drastically reduced lifespan underlining the critical role NFATc1 plays in HSPC maintenance and in the differentaion of various lineages. Our findings suggest that NFATc1 is a critical component of the myriad signaling and transcriptional regulators that are essential to maintain normal hematopoiesis. Full article

Chitosan (Chi) is a natural polymer that has been demonstrated to have potential as a promoter of neural regeneration. In this study, Chi was prepared with various amounts (25, 50, and 100 ppm) of gold (Au) nanoparticles for use in in vitro and in vivo assessments. Each as-prepared material was first characterized by UV-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), and Dynamic Light Scattering (DLS). Through the in vitro experiments, Chi combined with 50 ppm of Au nanoparticles demonstrated better biocompatibility. The platelet activation, monocyte conversion, and intracellular ROS generation was remarkably decreased by Chi–Au 50 pm treatment. Furthermore, Chi–Au 50 ppm could facilitate colony formation and strengthen matrix metalloproteinase (MMP) activation in mesenchymal stem cells (MSCs). The lower expression of CD44 in Chi–Au 50 ppm treatment demonstrated that the nanocomposites could enhance the MSCs undergoing differentiation. Chi–Au 50 ppm was discovered to significantly induce the expression of GFAP, β-Tubulin, and nestin protein in MSCs for neural differentiation, which was verified by real-time PCR analysis and immunostaining assays. Additionally, a rat model involving subcutaneous implantation was used to evaluate the superior anti-inflammatory and endothelialization abilities of a Chi–Au 50 ppm treatment. Capsule formation and collagen deposition were decreased. The CD86 expression (M1 macrophage polarization) and leukocyte filtration (CD45) were remarkably reduced as well. In summary, a Chi polymer combined with 50 ppm of Au nanoparticles was proven to enhance the neural differentiation of MSCs and showed potential as a biosafe nanomaterial for neural tissue engineering. Full article

The success of regenerative medicine in various clinical applications depends on the appropriate selection of the source of mesenchymal stem cells (MSCs). Indeed, the source conditions, the quality and quantity of MSCs, have an influence on the growth factors, cytokines, extracellular vesicles, and secrete bioactive factors of the regenerative milieu, thus influencing the clinical result. Thus, optimal source selection should harmonize this complex setting and ensure a well-personalized and effective treatment. Mesenchymal stem cells (MSCs) can be obtained from several sources, including bone marrow and adipose tissue, already used in orthopedic regenerative applications. In this sense, for bone, dental, and oral injuries, MSCs could provide an innovative and effective therapy. The present review aims to compare the properties (proliferation, migration, clonogenicity, angiogenic capacity, differentiation potential, and secretome) of MSCs derived from bone marrow, adipose tissue, and dental tissue to enable clinicians to select the best source of MSCs for their clinical application in bone and oral tissue regeneration to delineate new translational perspectives. A review of the literature was conducted using the search engines Web of Science, Pubmed, Scopus, and Google Scholar. An analysis of different publications showed that all sources compared (bone marrow mesenchymal stem cells (BM-MSCs), adipose tissue mesenchymal stem cells (AT-MSCs), and dental tissue mesenchymal stem cells (DT-MSCs)) are good options to promote proper migration and angiogenesis, and they turn out to be useful for gingival, dental pulp, bone, and periodontal regeneration. In particular, DT-MSCs have better proliferation rates and AT and G-MSC sources showed higher clonogenicity. MSCs from bone marrow, widely used in orthopedic regenerative medicine, are preferable for their differentiation ability. Considering all the properties among sources, BM-MSCs, AT-MSCs, and DT-MSCs present as potential candidates for oral and dental regeneration. Full article