Preservation of ovarian function using human pluripotent stem cell-derived mesenchymal progenitor cells.

Ovarian reserve diminishes with age, and older women experience a corresponding shift in sex hormone levels. These changes contribute to an age-dependent decrease in fertility and a decline in overall health. Furthermore, while survival rates following cancer treatment have improved for young female patients, a reduction in ovarian function due to the side effects of such treatments can be difficult to avoid. To date, no effective therapy has been recommended to preserve ovarian health in these patients. Mesenchymal progenitor cells (MPCs) are considered a promising option for cell therapy aimed at maintaining fertility and fecundity. Although MPCs derived from human adult tissues are recognized for their various protective effects against ovarian senescence, they are limited in quantity. Consequently, human pluripotent stem cell-derived MPCs (hPSC-MPCs), which exhibit high proliferative capacity and retain genetic stability during growth, have been utilized to delay reproductive aging. This review highlights the impact of hPSC-MPCs on preserving the functionality of damaged ovaries in female mouse models subjected to chemotherapy and natural aging. It also proposes their potential as a valuable cell source for fertility preservation in women with a variety of diseases.

Empagliflozin attenuates doxorubicin-induced cardiotoxicity by inhibiting the JNK signaling pathway.

BACKGROUND: Sodium-glucose cotransporter-2 inhibitors, such as empagliflozin, are pivotal therapies for heart failure. However, the effect of empagliflozin on doxorubicin-related cardiac dysfunction remains unclear. METHODS: Human induced pluripotent stem cell- and embryonic stem cell-derived cardiomyocytes were used to investigate the direct effect of empagliflozin on human cardiomyocytes. Then, the c-Jun amino-terminal kinases (JNK) inhibitor SP600125 was administered to the doxorubicin cardiotoxicity model in vitro and in vivo to investigate the role of JNK in empagliflozin. RESULTS: In human stem cell-derived cardiomyocytes, pretreatment with empagliflozin attenuated doxorubicin-induced cleavage of caspase 3 and other apoptosis markers. Empagliflozin significantly attenuated doxorubicin-induced phosphorylation of JNK and p38. Inhibiting the phosphorylation of JNK (SP600125) or STAT3 attenuated doxorubicin-induced apoptosis, but inhibiting the phosphorylation of p38 did not. SP600125 inhibits the phosphorylation of STAT3 (S727), and a STAT3 (Y705) inhibitor also inhibits the phosphorylation of JNK. Empagliflozin and SP600125 attenuated doxorubicin-induced increases in reactive oxygen species (ROS) and decreases in oxidized nicotinamide adenine dinucleotide (NAD+). In animal studies, empagliflozin and SP600125 attenuated doxorubicin-induced cardiac dysfunction and fibrosis. CONCLUSIONS: Empagliflozin attenuated doxorubicin-induced apoptosis by inhibiting the phosphorylation of JNK and its downstream signaling pathways, including ROS and NAD+.

Bio-distribution and toxicity potential of human umbilical cord mesenchymal stem cells in cynomolgus monkeys.

Mesenchymal stem cells (MSCs) have demonstrated promising advantages in the therapies of many diseases, while its multi-directional differentiation potential and immunotoxicity are the major concerns hindered their clinical translation. In this study, human umbilical Mesenchymal stem cell (hUC-MSCs) were labeled with a near-infrared fluorescent dye DiR before infused into cynomolgus monkeys, and the amount of hUC-MSCs in the peripheral blood were dynamically estimated from 5 min to 28 days post a single administration at 3 × 106 cells/kg and 2 × 107 cells/kg intravenously. As results, some hUC-MSCs distributed to the whole body within 5 min, while most of the cells accumulate in the lungs along with the systemic blood circulation, and subsequently released into the blood. The toxicity potentials of hUC-MSCs were investigated in another 30 cynomolgus monkeys, and the cells were repeatedly administrated at doses of 3 × 106 cells/kg and 2 × 107 cells/kg for 5 times on a weekly basis, with a recovery period of 1 months. hUC-MSCs showed no obvious toxic effects in cynomolgus monkeys, except xenogeneic immune rejection to human stem cells. Low levels of the hUC-MSC gene were detected in the peripheral blood of a few animals administered 2 × 107 cells/kg at 30 min subsequent to the first and last administration, and there was no significant difference in the copy number of the hUC-MSC gene in the blood samples compared with the first and last administration, indicating that the hUC-MSC was not significantly amplified in vivo, and it its safe in non-human primates. Our study for the first time verified the safety of long-term use of hUC-MSCs in primates. We have pioneered a technology for the real-time detection of hUC-MSCs in peripheral blood and provide dynamicand rapid monitoring of the distribution characteristics of hUC-MSCs in vivo. Here, we provide data supporting the application of such products for clinical treatment and the application of stem cells in major refractory diseases and regenerative medicine.

From theory to therapy: a bibliometric and visual study of stem cell advancements in age-related macular degeneration.

BACKGROUND AIMS: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, offer groundbreaking therapeutic potential for degenerative diseases and cellular repair. Despite their significance, a comprehensive bibliometric analysis in this field, particularly in relation to age-related macular degeneration (AMD), is yet to be conducted. This study aims to map the foundational and emerging areas in stem cell and AMD research through bibliometric analysis. METHODS: This study analyzed articles and reviews on stem cells and AMD from 2000 to 2022, sourced from the Web of Science Core Collection. We used VOSviewer and CiteSpace for analysis and visualization of data pertaining to countries, institutions, authors, journals, references and key words. Statistical analyses were conducted using R language and Microsoft Excel 365. RESULTS: In total, 539 publications were included, indicating an increase in global literature on stem cells and AMD from 2000 to 2022. The USA was the leading contributor, with 239 papers and the highest H-index, also the USA had the highest average citation rate per article (59.82). Notably, 50% of the top 10 institutions were from the USA, with the University of California system being the most productive. Key authors included Masayo Takahashi, Michiko Mandai, Dennis Clegg, Pete J. Coffey, Boris Stanzel, and Budd A. Tucker. Investigative Ophthalmology & Visual Science published the majority of relevant papers (n = 27). Key words like "clinical trial," "stem cell therapy," "retinal organoid," and "retinal progenitor cells" were predominant. CONCLUSIONS: Research on stem cells and AMD has grown significantly, highlighting the need for increased global cooperation. Current research prioritizes the relationship between "ipsc," "induced pluripotent stem cell," "cell culture," and "human embryonic stem cell." As stem cell culture and safety have advanced, focus has shifted to prognosis and complications post-transplantation, signifying the movement of stem cell research from labs to clinical settings.

Multiple treatments with human embryonic stem cell-derived mesenchymal progenitor cells preserved the fertility and ovarian function of perimenopausal mice undergoing natural aging.

OBJECTIVES: Currently, no approved stem cell-based therapies for preserving ovarian function during aging. To solve this problem, we developed a long-term treatment for human embryonic stem cell-derived mesenchymal progenitor cells (hESC-MPCs). We investigated whether the cells retained their ability to resist ovarian aging, which leads to delayed reproductive senescence. MATERIALS AND METHODS: In a middle-aged female model undergoing natural aging, we analyzed whether hESC-MPCs benefit the long-term maintenance of reproductive fecundity and ovarian reservoirs and how their transplantation regulates ovarian function. RESULTS: The number of primordial follicles and mice with regular estrous cycles were increased in perimenopausal mice who underwent multiple introductions of hESC-MPCs compared to age-matched controls. The estradiol levels in the hESC-MPCs group were restored to those in the young and adult groups. Embryonic development and live birth rates were higher in the hESC-MPC group than in the control group, suggesting that hESC-MPCs delayed ovarian senescence. In addition to their direct effects on the ovary, multiple-treatments with hESC-MPCs reduced ovarian fibrosis by downregulating inflammation and fibrosis-related genes via the suppression of myeloid-derived suppressor cells (MDSCs) produced in the bone marrow. CONCLUSIONS: Multiple introductions of hESC-MPCs could be a useful approach to prevent female reproductive senescence and that these cells are promising sources for cell therapy to postpone the ovarian aging and retain fecundity in perimenopausal women.

HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells.

Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.

Bright ferritin for long-term MR imaging of human embryonic stem cells.

BACKGROUND: A non-invasive imaging technology that can monitor cell viability, retention, distribution, and interaction with host tissue after transplantation is needed for optimizing and translating stem cell-based therapies. Current cell imaging approaches are limited in sensitivity or specificity, or both, for in vivo cell tracking. The objective of this study was to apply a novel ferritin-based magnetic resonance imaging (MRI) platform to longitudinal tracking of human embryonic stem cells (hESCs) in vivo. METHODS: Human embryonic stem cells (hESCs) were genetically modified to stably overexpress ferritin using the CRISPR-Cas9 system. Cellular toxicity associated with ferritin overexpression and manganese (Mn) supplementation were assessed based on cell viability, proliferation, and metabolic activity. Ferritin-overexpressing hESCs were characterized based on stem cell pluripotency and cardiac-lineage differentiation capability. Cells were supplemented with Mn and imaged in vitro as cell pellets on a preclinical 3 T MR scanner. T1-weighted images and T1 relaxation times were analyzed to assess contrast. For in vivo study, three million cells were injected into the leg muscle of non-obese diabetic severe combined immunodeficiency (NOD SCID) mice. Mn was administrated subcutaneously. T1-weighted sequences and T1 mapping were used to image the animals for longitudinal in vivo cell tracking. Cell survival, proliferation, and teratoma formation were non-invasively monitored by MRI. Histological analysis was used to validate MRI results. RESULTS: Ferritin-overexpressing hESCs labeled with 0.1 mM MnCl2 provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, proliferation, pluripotency, and differentiation into cardiomyocytes. Transplanted hESCs displayed significant bright contrast on MRI 24 h after Mn administration, with contrast persisting for 5 days. Bright contrast was recalled at 4-6 weeks with early teratoma outgrowth. CONCLUSIONS: The bright-ferritin platform provides the first demonstration of longitudinal cell tracking with signal recall, opening a window on the massive cell death that hESCs undergo in the weeks following transplantation before the surviving cell fraction proliferates to form teratomas.

BACH1 regulates the differentiation of vascular smooth muscle cells from human embryonic stem cells via CARM1-mediated methylation of H3R17.

The role of BACH1 in the process of vascular smooth muscle cell (VSMC) differentiation from human embryonic stem cells (hESCs) remains unknown. Here, we find that the loss of BACH1 in hESCs attenuates the expression of VSMC marker genes, whereas overexpression of BACH1 after mesoderm induction increases the expression of VSMC markers during in vitro hESC-VSMC differentiation. Mechanistically, BACH1 binds directly to coactivator-associated arginine methyltransferase 1 (CARM1) during in vitro hESC-VSMC differentiation, and this interaction is mediated by the BACH1 bZIP domain. BACH1 recruits CARM1 to VSMC marker gene promoters and promotes VSMC marker expression by increasing H3R17me2 modification, thus facilitating in vitro VSMC differentiation from hESCs after the mesoderm induction. The increased expression of VSMC marker genes by BACH1 overexpression is partially abolished by inhibition of CARM1 or the H3R17me2 inhibitor TBBD in hESC-derived cells. These findings highlight the critical role of BACH1 in hESC differentiation into VSMCs by CARM1-mediated methylation of H3R17.

Exosomal miR-17-5p from human embryonic stem cells prevents pulmonary fibrosis by targeting thrombospondin-2.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible lung disease characterized by pulmonary fibrosis and lung dysfunction, ultimately leading to respiratory failure. Many preclinical studies have investigated the therapeutic potential of stem cell-derived exosomes in this disease, particularly mesenchymal stem cell-derived exosomes. However, the effects of embryonic stem cell-derived exosomes in IPF remain unclear. METHODS: We established a bleomycin (BLM)-induced pulmonary fibrosis mice model and administered human embryonic stem cell exosomes (hESC-exo) from the first day after BLM treatment. The effects of hESC-exo were assessed by pulmonary function tests, biochemical analysis, histochemistry, quantitative real-time polymerase chain reaction (qPCR), and western blot (WB). RNA-seq was used to screen for the potential therapeutic targets of hESC-exo in fibrotic lungs; the identified signaling axis was characterized using a luciferase assay, qPCR, and WB. RESULTS: Results indicated hESC-exo administration notably alleviated inflammation, removed deposited collagen, and rescued alveolar architecture in the lungs of BLM-induced mice. In vivo and in vitro tests revealed that hESC-exo-derived miR-17-5p directly bound thrombospondin-2 (Thbs2) to regulate inflammation and fibrosis; thus, hESC-exo protected against BLM toxicity in the lungs via the miR-17-5p/Thbs2 axis. CONCLUSION: These results suggest a promising new treatment for fibrosis-associated diseases.

Functional and long-lived melanocytes from human pluripotent stem cells with transient ectopic expression of JMJD3.

BACKGROUND: Melanocytes are an essential part of the epidermis, and their regeneration has received much attention because propagation of human adult melanocytes in vitro is too slow for clinical use. Differentiation from human pluripotent stem cells to melanocytes has been reported, but the protocols to produce them require multiple and complex differentiation steps. METHOD: We differentiated human embryonic stem cells (hESCs) that transiently express JMJD3 to pigmented cells. We investigated whether the pigmented cells have melanocytic characteristics and functions by qRT-PCR, immunocytochemical analysis and flow cytometry. We also investigated their biocompatibility by injecting the cells into immunodeficient mice for clinical use. RESULT: We successfully differentiated and established a pure culture of melanocytes. The melanocytes maintained their growth rate for a long time, approximately 200 days, and were functional. They exhibited melanogenesis and transfer of melanin to peripheral keratinocytes. Moreover, melanocytes simulated the developmental processes from melanoblasts to melanocytes. The melanocytes had high engraftability and biocompatibility in the immunodeficient mice. CONCLUSION: The robust generation of functional and long-lived melanocytes are key to developing clinical applications for the treatment of pigmentary skin disorders.

Huntingtin CAG-expansion mutation results in a dominant negative effect.

Introduction: Huntington's disease (HD) remains an incurable and fatal neurodegenerative disease long after CAG-expansion mutation in the huntingtin gene (HTT) was identified as the cause. The underlying pathological mechanism, whether HTT loss of function or gain of toxicity results from mutation, remains a matter of debate. Methods: In this study, we genetically modulated wild-type or mutant HTT expression levels in isogenic human embryonic stem cells to systematically investigate their contribution to HD-specific phenotypes. Results: Using highly reproducible and quantifiable in vitro micropattern-based assays, we observed comparable phenotypes with HD mutation and HTT depletion. However, halving endogenous wild-type HTT levels did not strongly recapitulate the HD phenotypes, arguing against a classical loss of function mechanism. Remarkably, expression of CAG-expanded HTT in non-HD cells induced HD like phenotypes akin to HTT depletion. Discussion: By corollary, these results indicate a dominant negative effect of mutated HTT on its wild-type counterpart. Complementation with additional copies of wild-type HTT ameliorated the HD-associated phenotypes, strongly supporting a classical dominant negative mechanism. Understanding the molecular basis of this dominant negative effect will guide the development of efficient clinical strategies to counteract the deleterious impact of mutant HTT on the wild-type HTT function.

Enhancing Viability of Human Embryonic Stem Cells during Cryopreservation via RGD-REP-Mediated Activation of FAK/AKT/FoxO3a Signaling Pathway.

BACKGROUND: Cryopreservation is a crucial method for long-term storage and stable allocation of human pluripotent stem cells (hPSCs), which are increasingly being used in various applications. However, preserving hPSCs in cryogenic conditions is challenging due to reduced recovery rates. METHODS: To address this issue, the Arginine-Glycine-Aspartate (RGD) motif was incorporated into a recombinant elastin-like peptide (REP). Human embryonic stem cells (hESCs) were treated with REP containing RGD motif (RGD-REP) during suspension and cryopreservation, and the survival rate was analyzed. The underlying mechanisms were also investigated. RESULTS: The addition of RGD-REP to the cryopreservation solution improved cell survival and pluripotency marker expression. The improvement was confirmed to be due to the activation of the FAK-AKT cascade by RGD-REP binding to hESC surface interin protein, and consequent inhibition of FoxO3a. The inactivation of FoxO3a reduced the expression of apoptosis-related genes, such as BIM, leading to increased survival of PSCs in a suspension state. CONCLUSION: RGD-REP, as a ligand for integrin protein, improves the survival and maintenance of hPSCs during cryopreservation by activating survival signals via the RGD motif. These results have potential implications for improving the efficiency of stem cell usage in both research and therapeutic applications.

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

IGF-1 boosts mitochondrial function by a Ca2+ uptake-dependent mechanism in cultured human and rat cardiomyocytes.

A physiological increase in cardiac workload results in adaptive cardiac remodeling, characterized by increased oxidative metabolism and improvements in cardiac performance. Insulin-like growth factor-1 (IGF-1) has been identified as a critical regulator of physiological cardiac growth, but its precise role in cardiometabolic adaptations to physiological stress remains unresolved. Mitochondrial calcium (Ca2+) handling has been proposed to be required for sustaining key mitochondrial dehydrogenase activity and energy production during increased workload conditions, thus ensuring the adaptive cardiac response. We hypothesized that IGF-1 enhances mitochondrial energy production through a Ca2+-dependent mechanism to ensure adaptive cardiomyocyte growth. We found that stimulation with IGF-1 resulted in increased mitochondrial Ca2+ uptake in neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes, estimated by fluorescence microscopy and indirectly by a reduction in the pyruvate dehydrogenase phosphorylation. We showed that IGF-1 modulated the expression of mitochondrial Ca2+ uniporter (MCU) complex subunits and increased the mitochondrial membrane potential; consistent with higher MCU-mediated Ca2+ transport. Finally, we showed that IGF-1 improved mitochondrial respiration through a mechanism dependent on MCU-mediated Ca2+ transport. In conclusion, IGF-1-induced mitochondrial Ca2+ uptake is required to boost oxidative metabolism during cardiomyocyte adaptive growth.

Functional assessment of donated human embryos for the generation of pluripotent embryonic stem cell lines.

RESEARCH QUESTION: Can discarded embryos at blastocyst stage, donated to research because of genetic abnormalities and poor morphological quality, become a reliable source of human embryonic stem cell (HESC) lines? DESIGN: This study was consecutively conducted with 23 discarded embryos that were donated to research between February 2020 and April 2021. All embryos, except one, were morphologically evaluated and underwent trophectoderm biopsy for preimplantation genetic testing using next-generation sequencing (NGS), and then vitrified. After warming, the embryos were placed in appropriate culture conditions for the generation of HESCs, which was functionally assessed with immunofluorescence and flow cytometry for pluripotency capacity and spontaneous in-vitro differentiation. Cytogenetic assessment of the HESC was conducted with multiplex ligation-dependent probe amplification, and micro array comparative genomic hybridization. RESULTS: From the 23 embryos initially included, 17 survived warming, and 16 of them presented viability. Overall, the embryos presented poor morphological quality after warming. Only the previously untested embryo was capable of generating a new HESC line. Further characterization of this line revealed fully functional, euploid HESCs with preserved pluripotency, becoming a useful resource for research into human development and therapeutic investigation. CONCLUSIONS: None of the donated blastocysts with poor morphological quality in association with genetic abnormalities detected by NGS had the capacity for further in-vitro expansion to originate pluripotent HESC lines. This finding seems to provide extra support to genetic counselling on the suitability of this type of embryo for clinical use.

The Essential Function of miR-5739 in Embryonic Muscle Development.

Background and Objectives: Embryologically, mesodermal development is closely related to the development of various organs such as muscles, blood vessels, and hearts, which are the main organs that make up the body. However, treatment for mesoderm developmental disorders caused by congenital or acquired factors has so far relied on surgery and drug treatment for symptom relief, and more fundamentally, treatment for mesoderm developmental disorders is needed. Methods and Results: In our study, microRNA (miRNA), which plays an important role in the mesoderm development process, was identified and the developmental function was evaluated. miRNAs consist of small nucleotides, which act as transcription factors that bind to the 3' untranslated region and suppressed target gene expression. We constructed the human embryonic stem cell (hESC) knockout cell line and analyzed the function and characteristics of miR-5739, which plays an important role in mesoderm lineage. miR-5739 acts as a transcription factor targeting SMA, Brachyury T, Hand1, which controls muscle proliferation and differentiation, and KDR gene, which regulates vessel formation in vitro. In vivo results suggest a role in regulating muscle proliferation and differentiation. Gene ontology analysis confirmed that the miR-5739 is closely related to genes that regulate muscle and vessel proliferation and differentiation. Importantly, abnormal expression of miR-5739 was detected in somatic cells derived from patients with congenital muscle disease. Conclusions: Our study demonstrate that miR-5739 gene function significantly affects transcriptional circuits that regulate muscle and vascular differentiation during embryonic development.

Proliferation toxicity and mechanism of novel mixed bromine/chlorine transformation products of tetrabromobisphenol A on human embryonic stem cell.

Mixed bromine/chlorine transformation products of tetrabromobisphenol A (ClyBrxBPAs) are mixed halogenated-type compounds recently identified in electronic waste dismantling sites. There are a lack of toxicity data on these compounds. To study their development toxicity, the proliferation toxicity was investigated using human embryonic stem cells (hESC) exposed to the lowest effective dose of two ClyBrxBPA analogues (2-chloro-2',6-dibromobisphenol A and 2,2'-dichloro-6-monobromobisphenol A). For comparison, tetrabromobisphenol A, 2,2',6-tribromobisphenol A, and bisphenol A were also assessed. It was observed that ClyBrxBPAs inhibited hESCs proliferation in a concentration-dependent manner. The cell bioaccumulation efficiency of ClyBrxBPAs was higher than that of tetrabromobisphenol A. Also, ClyBrxBPAs were more toxic than tetrabromobisphenol A, with 2,2'-dichloro-6-monobromobisphenol A exhibiting the most potent toxicity. Furthermore, flow cytometry and oxidative stress results showed that increased reactive oxygen species raised the degree of apoptosis and reduced DNA synthesis. Metabolomics analysis on the effect of ClyBrxBPAs on metabolic pathway alteration showed that ClyBrxBPAs mainly interfered with four metabolic pathways related to amino acid metabolism and biosynthesis. These results provide an initial perspective on the proliferation toxicity of ClyBrxBPAs, indicating development toxicity in children.

Distribution of Embryonic Stem Cell-Derived Mesenchymal Stem Cells after Intravenous Infusion in Hypoxic-Ischemic Encephalopathy.

Systemic administration of mesenchymal stem cells (MSCs) has been reported to improve neurological function in brain damage, including hypoxic-ischemic encephalopathy (HIE), though the action mechanisms have not been fully elucidated. In this study, the cells were tracked live using a Pearl Trilogy Small Animal fluorescence imaging system after human embryonic stem Cell-Derived MSCs (ES-MSCs) infusion for an HIE mouse model. ES-MSC-treated HIE mice showed neurobehavioral improvement. In vivo imaging showed similar sequential migration of ES-MSCs from lungs, liver, and spleen within 7 days in both HIE and normal mice with the exception of lungs, where there was higher entrapment in the HIE 1 h after infusion. In addition, ex vivo experiments confirmed time-dependent infiltration of ES-MSCs into the organs, with similar findings in vivo, although lungs and brain revealed small differences. ES-MSCs seemed to remain in the brain only in the case of HIE on day 14 after the cell infusion. The homing effect in the host brain was confirmed with immunofluorescence staining, which showed that grafted cells remained in the brain tissue at the lesion area with neurorestorative findings. Further research should be carried out to elucidate the role of each host organ's therapeutic effects when stem cells are systemically introduced.

Functional reconstruction of the basal ganglia neural circuit by human striatal neurons in hypoxic-ischaemic injured brain.

Perinatal hypoxic-ischaemic encephalopathy is the leading cause of neonatal death and permanent neurological deficits, while the basal ganglia is one of the major nuclei that is selectively and greatly affected in the brains of hypoxic-ischaemic encephalopathy patients, especially in severe cases. Human embryonic stem cell-derived neurons have shown great potential in different types of brain disorders in adults. However, it remains unknown whether and how grafted human embryonic stem cell-derived neurons can repair immature brains with hypoxic-ischaemic encephalopathy. Here, by administrating genetically labelled human embryonic stem cell-derived striatal neural progenitors into the ipsilateral striatum of hypoxic-ischaemic encephalopathy-injured mice, we found that the grafted cells gradually matured into GABA spiny projection neurons morphologically and electrophysiologically, and significantly rescued the area loss of hypoxic-ischaemic encephalopathy-injured brains. Intriguingly, using immunohistochemical staining combined with enhanced ascorbate peroxidase-based immunoelectron microscopy and rabies virus-mediated trans-synaptic tracing, we show that the grafts start to extend axonal projections to the endogenous target areas (globus pallidus externa, globus pallidus internus, substantia nigra), form synapses with host striatal, globus pallidus and nigra neurons, and receive extensive and stable synaptic inputs as early as 2 months post-transplantation. Importantly, we further demonstrated functional neural circuits re-established between the grafted neurons and host cortical, striatal and substantial nigra neurons at 3-6 months post-transplantation in the hypoxic-ischaemic encephalopathy-injured brain by optogenetics combined with electrophysiological recording. Finally, the transplanted striatal spiny projection neurons but not spinal GABA neurons restored the motor defects of hypoxic-ischaemic encephalopathy, which were reversed by clozapine-N-oxide-based inhibition of graft function. These findings demonstrate anatomical and functional reconstruction of the basal ganglia neural circuit including multiple loops by striatal spiny projection neurons in hypoxic-ischaemic encephalopathy-injured immature brains, which raises the possibility of such a cell replacement therapeutic strategy for hypoxic-ischaemic encephalopathy in neonates.

HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway.

Objectives: Normal commitment of the endoderm of the third pharyngeal pouch (3PP) is essential for the development and differentiation of the thymus. The aim of this study was to investigate the role of transcription factor HOXA3 in the development and differentiation of 3PP endoderm (3PPE) from human embryonic stem cells (hESCs). Methods: The 3PPE was differentiated from hESC-derived definitive endoderm (DE) by mimicking developmental queues with Activin A, WNT3A, retinoic acid and BMP4. The function of 3PPE was assessed by further differentiating into functional thymic epithelial cells (TECs). The effect of HOXA3 inhibition on cells of 3PPE was subsequently investigated. Results: A highly efficient approach for differentiating 3PPE cells was developed and these cells expressed 3PPE related genes HOXA3, SIX1, PAX9 as well as EpCAM. 3PPE cells had a strong potential to develop into TECs which expressed both cortical TEC markers K8 and CD205, and medullary TEC markers K5 and AIRE, and also promoted the development and maturation of T cells. More importantly, transcription factor HOXA3 not only regulated the differentiation of 3PPE, but also had a crucial role for the proliferation and migration of 3PPE cells. Our further investigation revealed that HOXA3 controlled the commitment and function of 3PPE through the regulation of Wnt signaling pathway by activating EPHB2. Conclusion: Our results demonstrated that HOXA3 functioned as the on-off switch to regulate the development of hESC-derived 3PPE through EPHB2-mediated Wnt pathway, and our findings will provide new insights into studying the development of 3PP and thymic organ in vitro and in vivo.