Survival of Neural Progenitors Derived from Human Embryonic Stem Cells Following Subretinal Transplantation in Rodents.

Purpose: To examine the survival of neural progenitors (NPs) cells derived from human embryonic stem cells (hESCs) following subretinal (SR) transplantation in rodents. Methods: hESCs engineered to express enhanced green fluorescent protein (eGFP) were differentiated in vitro toward an NP fate using a 4-week protocol. State of differentiation was characterized by quantitative-PCR. NPs in suspension (75,000/µl) were transplanted to the SR-space of Royal College of Surgeons (RCS) rats (n = 66), nude-RCS rats (n = 18), and NOD scid gamma (NSG) mice (n = 53). Success of engraftment was determined at 4 weeks post-transplant by in vivo visualization of GFP-expression using a properly filtered rodent fundus camera. Transplanted eyes were examined in vivo at set time points using the fundus camera, and in select cases, by optical coherence tomography imaging, and after enucleation, by retinal histology and immunohistochemistry. Results: In RCS rats, cell rejection was observed in 29% of eyes at 6 weeks, rising to 92% at 8 weeks. In the more immunodeficient nude-RCS rats, the rejection rate was still high reaching 62% of eyes at 6 weeks post-transplant. Following transplantation in highly immunodeficient NSG mice, survival of the hESC-derived NPs was much improved, with 100% survival at 9 weeks and 72% at 20 weeks. A small number of eyes that were followed past 20 weeks showed survival also at 22 weeks. Conclusions: Immune status of recipient animals influences transplant survival. Highly immunodeficient NSG mice provide a better model for studying long-term survival, differentiation, and possible integration of hESC-derived NPs. Clinical Trial Registration numbers: NCT02286089, NCT05626114.

Genome-wide screen for anticancer drug resistance in haploid human embryonic stem cells.

Anticancer drugs are at the frontline of cancer therapy. However, innate resistance to these drugs occurs in one-third to one-half of patients, exposing them to the side effects of these drugs with no meaningful benefit. To identify the genes and pathways that confer resistance to such therapies, we performed a genome-wide screen in haploid human embryonic stem cells (hESCs). These cells possess the advantage of having only one copy of each gene, harbour a normal karyotype, and lack any underlying point mutations. We initially show a close correlation between the potency of anticancer drugs in cancer cell lines to those in hESCs. We then exposed a genome-wide loss-of-function library of mutations in all protein-coding genes to 10 selected anticancer drugs, which represent five different mechanisms of drug therapies. The genetic screening enabled us to identify genes and pathways which can confer resistance to these drugs, demonstrating several common pathways. We validated a few of the resistance-conferring genes, demonstrating a significant shift in the effective drug concentrations to indicate a drug-specific effect to these genes. Strikingly, the p53 signalling pathway seems to induce resistance to a large array of anticancer drugs. The data shows dramatic effects of loss of p53 on resistance to many but not all drugs, calling for clinical evaluation of mutations in this gene prior to anticancer therapy.

The LHX2-OTX2 transcriptional regulatory module controls retinal pigmented epithelium differentiation and underlies genetic risk for age-related macular degeneration.

Tissue-specific transcription factors (TFs) control the transcriptome through an association with noncoding regulatory regions (cistromes). Identifying the combination of TFs that dictate specific cell fate, their specific cistromes and examining their involvement in complex human traits remain a major challenge. Here, we focus on the retinal pigmented epithelium (RPE), an essential lineage for retinal development and function and the primary tissue affected in age-related macular degeneration (AMD), a leading cause of blindness. By combining mechanistic findings in stem-cell-derived human RPE, in vivo functional studies in mice and global transcriptomic and proteomic analyses, we revealed that the key developmental TFs LHX2 and OTX2 function together in transcriptional module containing LDB1 and SWI/SNF (BAF) to regulate the RPE transcriptome. Importantly, the intersection between the identified LHX2-OTX2 cistrome with published expression quantitative trait loci, ATAC-seq data from human RPE, and AMD genome-wide association study (GWAS) data, followed by functional validation using a reporter assay, revealed a causal genetic variant that affects AMD risk by altering TRPM1 expression in the RPE through modulation of LHX2 transcriptional activity on its promoter. Taken together, the reported cistrome of LHX2 and OTX2, the identified downstream genes and interacting co-factors reveal the RPE transcription module and uncover a causal regulatory risk single-nucleotide polymorphism (SNP) in the multifactorial common blinding disease AMD.

Dydrogesterone supplementation in addition to routine micronized progesterone administration for luteal support in cycles triggered with lone GnRH agonist results in an acceptable pregnancy rate and avoids the need to freeze embryos.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is reduced when using antagonist cycle with gonadotrophin releasing hormone (GnRH) agonist trigger before ovum pick up. This trigger induces short luteinizing hormone (LH) and follicle stimulating hormone (FSH) peaks, resulting in an inadequate luteal phase and a reduced implantation rate. We assessed whether the luteal phase can be rescued by supplementing with oral dydrogesterone (duphaston) in antagonist cycles after a lone GnRH agonist trigger. METHODS: A retrospective cohort study. The study group (N.=123) included women who underwent IVF. Patients received a GnRH-antagonist with a lone GnRH-agonist trigger due to imminent OHSS. The control group (N.=374) included patients who underwent a standard antagonist protocol with a dual trigger of a GnRH-agonist and human chorionic gonadotrophin (hCG). All the patients were treated with micronized progesterone (utrogestan) for luteal phase support. Study patients were given duphaston in addition. RESULTS: The fertilization rate was comparable between the two groups. The mean number of embryos transferred, the clinical pregnancy rate and the take-home baby rate were comparable between groups (1.5±0.6 vs. 1.5±0.5 and 46.3% vs. 41.2%, and 66.7% vs. 87.7%, respectively). No OHSS event was reported in either group. CONCLUSIONS: This study was the first to evaluate outcomes of duphaston supplementation for luteal support in an antagonist cycle with lone GnRH agonist trigger. The functionality of the luteal phase of those cycles could be restored by adding duphaston. This approach was found to be safe and prevented the need to postpone embryo transfer in case of pending OHSS.

Modeling sex differences in humans using isogenic induced pluripotent stem cells.

Biological sex is a fundamental trait influencing development, reproduction, pathogenesis, and medical treatment outcomes. Modeling sex differences is challenging because of the masking effect of genetic variability and the hurdle of differentiating chromosomal versus hormonal effects. In this work we developed a cellular model to study sex differences in humans. Somatic cells from a mosaic Klinefelter syndrome patient were reprogrammed to generate isogenic induced pluripotent stem cell (iPSC) lines with different sex chromosome complements: 47,XXY/46,XX/46,XY/45,X0. Transcriptional analysis of the hiPSCs revealed novel and known genes and pathways that are sexually dimorphic in the pluripotent state and during early neural development. Female hiPSCs more closely resembled the naive pluripotent state than their male counterparts. Moreover, the system enabled differentiation between the contributions of X versus Y chromosome to these differences. Taken together, isogenic hiPSCs present a novel platform for studying sex differences in humans and bear potential to promote gender-specific medicine in the future.

Laminin111-based defined culture promoting self-renewing human pluripotent stem cells with properties of the early post-implantation epiblast.

In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-ß signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification.

Mesenchymal stem cell transplantation improves biomechanical properties of vaginal tissue following full-thickness incision in aged rats.

Pelvic organ prolapse (POP) is common among post-menopausal women and is associated with bladder, bowel, and sexual dysfunction. Surgical repair with the patients' native tissues is sub-optimal with high reoperation rates, potentially due to diminished age-related healing. We demonstrate that systemic transplantation of mesenchymal stem cells (MSCs) improves healing of full-thickness vaginal incision in the vaginal wall of old rats, as suggested by both histological and functional analysis. Transplanted MSCs homed and survived at the surgical vaginal site. Attenuation of the injury-induced inflammatory response, increased angiogenesis, and reduced matrix metalloproteinase 9 expression were observed at the surgical site of transplanted rats. Most importantly, the functional biomechanical properties of the healed vagina, at day 30 post-injury, were improved in MSC-transplanted, compared with sham-operated non-transplanted, old rats. These results may pave the way to further translational studies toward clinical transplantation of MSCs adjuvant to POP repair for the improvement of surgical outcome.

Epithelial arginase-1 is a key mediator of age-associated delayed healing in vaginal injury.

Pelvic organ prolapse is a disorder that substantially affects the quality of life of millions of women worldwide. The greatest risk factors for prolapse are increased parity and older age, with the largest group requiring surgical intervention being post-menopausal women over 65. Due to ineffective healing in the elderly, prolapse recurrence rates following surgery remain high. Therefore, there is an urgent need to elucidate the cellular and molecular drivers of poor healing in pelvic floor dysfunction to allow effective management and even prevention. Recent studies have uncovered the importance of Arginase 1 for modulating effective healing in the skin. We thus employed novel in vitro and in vivo vaginal injury models to determine the specific role of Arginase 1 in age-related vaginal repair. Here we show, for the first time, that aged rat vaginal wounds have reduced Arginase 1 expression and delayed healing. Moreover, direct inhibition of Arginase 1 in human vaginal epithelial cells also led to delayed scratch-wound closure. By contrast, activation of Arginase 1 significantly accelerated healing in aged vaginal wounds in vivo, to rates comparable to those in young animals. Collectively, these findings reveal a new and important role for Arginase 1 in mediating effective vaginal repair. Targeting age-related Arginase 1 deficiency is a potential viable therapeutic strategy to promote vaginal healing and reduce recurrence rate after surgical repair of pelvic organ prolapse.

Advances in hPSC expansion towards therapeutic entities: A review.

For use in regenerative medicine, large-scale manufacturing of human pluripotent stem cells (hPSCs) under current good manufacturing practice (cGMPs) is required. Much progress has been made since culturing under static two-dimensional (2D) conditions on feeders, including feeder-free cultures, conditioned and xeno-free media, and three-dimensional (3D) dynamic suspension expansion. With the advent of horizontal-blade and vertical-wheel bioreactors, scale-out for large-scale production of differentiated hPSCs became possible; control of aggregate size, shear stress, fluid hydrodynamics, batch-feeding strategies, and other process parameters became a reality. Moving from substantially manipulated processes (i.e., 2D) to more automated ones allows easer compliance to current good manufacturing practices (cGMPs), and thus easier regulatory approval. Here, we review the current advances in the field of hPSC culturing, advantages, and challenges in bioreactor use, and regulatory areas of concern with respect to these advances. Manufacturing trends to reduce risk and streamline large-scale manufacturing will bring about easier, faster regulatory approval for clinical applications.

Establishment of a controlled slow freezing-based approach for experimental clinical cryopreservation of human prepubertal testicular tissues.

Objective: To develop an efficient, clinical-grade, freezing protocol toward experimental clinical cryopreservation of testicular tissues in prepubertal boys suffering from cancer. Design: Experimental cryopreservation of testicular tissue. Setting: University Medical Center. Patients: Adult patients undergoing orchiectomy for various tumors and prepubertal boys scheduled for gonadotoxic treatment. Interventions: None. Main Outcome Measures: Histopathological analysis of tissue architecture, structural integrity, and cellular morphology was performed for control and frozen-thawed cryopreserved tissues.The number of seminiferous tubules per testicular section was calculated. The survival of spermatogonial stem cells (SSCs) and Sertoli cells of the control and frozen-thawed cryopreserved tissues was analyzed by immunofluorescence staining. Results: Uncontrolled Slow Freezing, Controlled slow freezing, and vitrification similarly preserved the integrity of the adult testicular tissues and the survival of SSCs and Sertoli cells. Controlled slow freezing of prepubertal testicular tissues effectively preserved their architecture, the number of tubules, SSCs, and Sertoli cells. In addition, we observed SSC loss after chemotherapy in prepubertal boys, reemphasizing the importance of fertility preservation before gonadotoxic treatment. Conclusions: Future fertility restoration for male survivors of pediatric cancers depends on the development of an optimal prepubertal testicular tissue cryopreservation method. Our findings demonstrate the effectiveness of controlled slow freezing for cryopreservation of human prepubertal testicular tissues and may contribute to more effective banking of these tissues and potential fertility restoration. Clinical Trial Registration Number: NIH research clinical trials number: NCT02529826.

The BNT162b2 mRNA Covid-19 vaccine does not impair sperm parameters.

RESEARCH QUESTION: Does the BNT162b2 COVID-19 vaccine affect sperm parameters of patients with a normal or an abnormal semen analysis? DESIGN: Data were collected from male patients undergoing IVF treatment after completing vaccination between February 2021 and June 2021 (post-vaccine). For comparison, records of the same patients were reviewed before the vaccination (pre-vaccine) back to January 2017. Patients with azoospermia were excluded. Sperm parameters were compared between pre- and post-vaccine groups. Each patient served as self-control. RESULTS: Seventy-two patients were included in the study (median interquartile range [IQR] age 35.7 [33.0-43.0] years), of whom 57 had a normal semen analysis. The time between the first vaccine and the post-vaccine sperm analysis was 71.0 (40.5-104.8) days. The sperm parameters before and after the vaccination were as follows: sperm volume before 3.0 (2.0-4.0) and after 3.0 (1.6-3.9) ml, P = 0.02; sperm concentration before 26.5 (14.0-64.7) and after 31.0 (14.2-80.0) 106/ml, P = 0.35; and total motile sperm count before 33.7 (9.0-66.0) and after 29 (6.0-97.5)106, P = 0.96. Sub-group analyses were conducted for patients with male infertility and patients with a normal semen analysis. Neither of the sub-groups showed significant changes after vaccination. CONCLUSION: Sperm parameters showed no significant changes after vaccination among men with a normal and abnormal semen analysis. Therefore, the BNT162b2 vaccine does not seem to affect sperm parameters. The preliminary results are reassuring for the entire global population, currently undergoing intense vaccination campaigns against COVID-19.

Endometrial thickness following early miscarriage in IVF patients - is there a preferred management approach?

BACKGROUND: Endometrial thickness (ET) has previously been shown to positively correlate with implantation and clinical pregnancy rates. Pregnancies achieved using in-vitro fertilization (IVF) technique are prone to higher rates of early miscarriage. The aim of this study was to compare the effects of expectant management, medical treatment (Misoprostol) and dilation and curettage (D&C) for early miscarriage following IVF cycles on the subsequent cycle outcomes - endometrial thickness and reproductive outcomes. METHODS: A retrospective cohort study of women who underwent embryo transfer, conceived and had first trimester miscarriage with at least one subsequent embryo transfer. ET measurements during fresh or frozen-thawed IVF cycles were assessed for each patient. Comparisons of ET differences between the miscarriage and the subsequent cycles, as well as reproductive outcomes, were performed according to the initial miscarriage management approach. RESULTS: A total of 223 women were included in the study. Seventy-eight women were managed conservatively, 61 were treated with Misoprostol and 84 women underwent D&C. Management by D&C, compared to conservative management and Misoprostol treatment was associated with higher prevalence of a significant (> 2 mm) ET decrease (29.8%% vs. 14.1and 6.6%, respectively; p < .001) and was the only approach associated with a significant increase in the rates of ET under 7 and 8 mm in the following cycle (p = 0.006 and 0.035; respectively). Clinical pregnancy rates were significantly lower following D&C compared with conservative management and Misoprostol (16.7% vs. 38.5 and 27.9%, respectively; p = 0.008) as well as implantation rate (11.1% vs. 30.5.% and 17.7, respectively; p < 0.001). CONCLUSION: Our data suggest that D&C management of a miscarriage is associated with decreased ET and higher rates of thin endometrium in the subsequent IVF cycle, compared with conservative management and Misoprostol treatment. In addition, implantation and pregnancy rates were significantly lower after D&C.

Characterization of C9orf72 haplotypes to evaluate the effects of normal and pathological variations on its expression and splicing.

Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients' cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies.

Vaginal delivery in SARS-CoV-2-infected pregnant women in Israel: a multicenter prospective analysis.

KEY MESSAGE: Among SARS-CoV-2-infected mothers, vaginal delivery rates were high and associated with favorable outcomes with no cases of neonatal COVID-19. PURPOSE: To investigate the mode of delivery and its impact on immediate neonatal outcome in SARS-CoV-2-infected women. METHODS: A prospective study following pregnant women diagnosed with COVID-19 who delivered between March 15th and July 4th in seven university affiliated hospitals in Israel. RESULTS: A total of 52 women with a confirmed diagnosis of COVID-19 delivered in the participating centers during the study period. The median gestational age at the time of delivery was 38 weeks, with 16 (30.8%) cases complicated by spontaneous preterm birth. Forty-three women (82.7%) underwent a trial of labor. The remaining 9 women underwent pre-labor cesarean delivery mostly due to obstetric indications, whereas one woman with a critical COVID-19 course underwent urgent cesarean delivery due to maternal deterioration. Among those who underwent a trial of labor (n = 43), 39 (90.7%) delivered vaginally, whereas 4 (9.3%) cases resulted in cesarean delivery. Neonatal RT-PCR nasopharyngeal swabs tested negative in all cases, and none of the infants developed pneumonia. No maternal and neonatal deaths were encountered. CONCLUSIONS: In this prospective study among SARS-CoV-2-infected mothers, vaginal delivery rates were high and associated with favorable outcomes with no cases of neonatal COVID-19. Our findings underscore that delivery management among SARS-CoV-2-infected mothers should be based on obstetric indications and may potentially reduce the high rates of cesarean delivery previously reported in this setting.

Dual-Targeted Autoimmune Sword in Fatal Epilepsy: Patient's glutamate receptor AMPA GluR3B peptide autoimmune antibodies bind, induce Reactive Oxygen Species (ROS) in, and kill both human neural cells and T cells.

Nodding Syndrome (NS) is a fatal pediatric epilepsy of unknown etiology, accompanied by multiple neurological impairments, and associated with Onchocerca volvulus (Ov), malnutrition, war-induced trauma, and other insults. NS patients have neuroinflammation, and ~50% have cross-reactive Ov/Leiomodin-1 neurotoxic autoimmune antibodies. RESULTS: Studying 30 South Sudanese NS patients and a similar number of healthy subjects from the same geographical region, revealed autoimmune antibodies to 3 extracellular peptides of ionotropic glutamate receptors in NS patients: AMPA-GluR3B peptide antibodies (86%), NMDA-NR1 peptide antibodies (77%) and NMDA-NR2 peptide antibodies (87%) (in either 1:10, 1:100 or 1:1000 serum dilution). In contrast, NS patients did not have 26 other well-known autoantibodies that target the nervous system in several autoimmune-mediated neurological diseases. We demonstrated high expression of both AMPA-GluR3 and NMDA-NR1 in human neural cells, and also in normal human CD3+ T cells of both helper CD4+ and cytotoxic CD8+ types. Patient's GluR3B peptide antibodies were affinity-purified, and by themselves precipitated short 70 kDa neuronal GluR3. NS patient's affinity-purified GluR3B peptide antibodies also bound to, induced Reactive Oxygen Species (ROS) in, and killed both human neural cells and T cells within 1-2 hours only. NS patient's purified IgGs, or serum (1:10 or 1:30), induced similar effects. In vivo video EEG experiments in normal mice, revealed that when NS patient's purified IgGs were released continuously (24/7 for 1 week) in normal mouse brain, they induced all the following: 1.Seizures, 2. Cerebellar Purkinje cell loss, 3. Degeneration in the hippocampus and cerebral cortex, and 4. Elevation of CD3+ T cells, and of activated Mac-2+microglia and GFAP+astrocytes in both the gray and white matter of the cerebral cortex, hippocampus, corpus calossum and cerebellum of mice. NS patient's serum cytokines: IL-1ß, IL-2, IL-6, IL-8, TNFα, IFNγ, are reduced by 85-99% compared to healthy subjects, suggesting severe immunodeficiency in NS patients. This suspected immunodeficiency could be caused by combined effects of the: 1. Chronic Ov infection, 2. Malnutrition, 3. Killing of NS patient's T cells by patient's own GluR3B peptide autoimmune antibodies (alike the killing of normal human T cells by the NS patient's GluR3B peptide antibodies found herein in vitro). CONCLUSIONS: Regardless of NS etiology, NS patients suffer from 'Dual-targeted Autoimmune Sword': autoimmune AMPA GluR3B peptide antibodies that bind, induce ROS in, and kill both neural cells and T cells. These neurotoxic and immunotoxic GluR3B peptide autoimmune antibodies, and also NS patient's NMDA-NR1/NR2A and Ov/Leiomodin-1 autoimmune antibodies, must be silenced or removed. Moreover, the findings of this study are relevant not only to NS, but also to many more patients with other types of epilepsy, which have GluR3B peptide antibodies in serum and/or CSF. This claim is based on the following facts: 1. The GluR3 subunit is expressed in neural cells in crucial brains regions, in motor neurons in the spinal cord, and also in other cells in the body, among them T cells of the immune system, 2. The GluR3 subunit has diverse neurophysiological role, and its deletion or abnormal function can: disrupt oscillatory networks of both sleep and breathing, impair motor coordination and exploratory activity, and increase the susceptibility to generate seizures, 3. GluR3B peptide antibodies were found so far in ~27% of >300 epilepsy patients worldwide, which suffer from various other types of severe, intractable and enigmatic epilepsy, and which turned out to be 'Autoimmune Epilepsy'. Furthermore, the findings of this study could be relevant to different neurological diseases besides epilepsy, since other neurotransmitter-receptors autoantibodies are present in other neurological and psychiatric diseases, e.g. autoimmune antibodies against other GluRs, Dopamine receptors, GABA receptors, Acetylcholine receptors and others. These neurotransmitter-receptors autoimmune autoantibodies might also act as 'Dual-targeted Autoimmune Sword' and damage both neural cells and T cells (as the AMPA-GluR3B peptide antibodies induced in the present study), since T cells, alike neural cells, express most if not all these neurotransmitter receptors, and respond functionally to the respective neurotransmitters - a scientific and clinical topic we coined 'Nerve-Driven Immunity'.

Age-associated differences in macrophage response in a vaginal wound healing rat model.

INTRODUCTION AND HYPOTHESIS: Surgical treatment of pelvic organ prolapse often includes the use of patients' vaginal connective tissue. Wound healing appears to play an important part in the success of such procedures. The aim of this study was to describe the effect of age on inflammatory processes, specifically macrophage response, involved in vaginal wound healing. METHODS: Twenty-five young (12 weeks old) and 25 old (12 months old) virgin female Fischer rats underwent a standardized 9-mm posterior midline vaginal incision. Tissue samples were taken for histological analysis on days 1, 3, 7, 14 and 30 post-injury. Parameters evaluated included wound area, macrophage number and expression of inflammatory markers including tumor necrosis factor alpha (TNFa), inducible nitric oxide synthase (iNOS), CCR7/CD197, arginase I and CD163/M130. RESULTS: Microscopic examination of the vaginal wounds over time demonstrated a clear difference between young and old rats in spontaneous healing capacity. The average wound area in young rats 1 day after injury was significantly smaller than in old rats (16.5 ± 1.7 vs. 23.8 ± 1.5 mm2, P < 0.05). At 3 days post-injury, wounds were closed in young rats but still open in old rats (wound area: 13.5 ± 1.5 mm2). Old rats demonstrated a more excessive and sustained macrophage response compared with young rats. They also demonstrated a disordered pattern of macrophage expression over time, with a prolonged expression of TNFa and iNOS in the tissue and a disordered M2 macrophage response. CONCLUSION: Excessive and prolonged macrophage response in older rats may contribute to poor wound healing in the vagina.

Continuous Immune-Modulatory Effects of Human Olig2+ Precursor Cells Attenuating a Chronic-Active Model of Multiple Sclerosis.

Neuroglial precursor cells (NPC) possess immune-modulatory properties by which they prevent immune-mediated injury in experimental autoimmune encephalomyelitis (EAE). It is unclear whether cell transplantation in a clinical-relevant setup induces ongoing therapeutic effects in a chronic-active model of progressive multiple sclerosis (MS). We examined whether human embryonic stem cell (hESC)-derived NPCs inhibit progressive EAE in Biozzi AB/H mice, manifesting with chronic-active neuroinflammation and demyelinated plaques. hESC-derived NPCs were propagated for 6-8 weeks as spheres enriched for Olig2+ cells to switch from neuronal to glial commitment and to enrich for oligodendrocyte progenitor cells. NPC were transplanted intracerebroventricularly at 30 days post-EAE induction, after the acute relapse. We evaluated effects of cell transplantation on clinical parameters, neuroinflammation, myelination, and axonal loss. Transplanted animals exhibited a significantly milder disease, reduced neuroinflammation, reduced demyelination, and reduced axonal loss as compared to control EAE mice. Toluidine-blue semi-thin staining showed a bystander neuroprotective effect of human precursor cells preventing the loss of myelinated fibers in superficial layer of the cervical dorsal funiculus. Human Olig2+ cells were detected along spinal cord meninges after 65 days of follow-up. In co-cultures in vitro, Olig2+ human precursors inhibited Concanavalin A-induced murine T cell activation and proliferation. To conclude, glial-committed human NPC induce ongoing immune-regulatory and neuroprotective effects, following transplantation into mice with a clinical-relevant model of chronic-active MS and during established disease, entering the chronic phase. These properties highlight the therapeutic potential of human NPC transplantation in chronic MS and their delivery via the cerebrospinal fluid.

Hadassah, provider of "Regulatory-Ready" pluripotent clinical-grade stem cell banks.

The Hadassah hESC Research Center's aim is to be a supplier of clinical and research-grade human embryonic stem cell (hESC) lines. In 2012, we derived the first three entirely GMP-compliant and xeno-free, fully-characterised, feeder-dependent (human umbilical cord) hESC lines developed under cleanroom conditions. In 2018, we established four new GMP and xeno-free, feeder-independent MCB hESCs under GMP conditions using commercially available reagents, media and matrix. All cell lines were derived under Israeli Ministry of Health's National Ethics Committee for Genetic Research in Humans and the ethical considerations that guided the development of the hESCs strictly followed Israeli law. Hadassah has provided its clinical-grade hESC lines to commercial entities of which two are already in clinical trials, establishing Hadassah as a key provider of clinical-grade hESC lines.

Human iPSC-derived astrocytes from ALS patients with mutated C9ORF72 show increased oxidative stress and neurotoxicity.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons (MNs). It was shown that human astrocytes with mutations in genes associated with ALS, like C9orf72 (C9) or SOD1, reduce survival of MNs. Astrocyte toxicity may be related to their dysfunction or the release of neurotoxic factors. METHODS: We used human induced pluripotent stem cell-derived astrocytes from ALS patients carrying C9orf72 mutations and non-affected donors. We utilized these cells to investigate astrocytic induced neuronal toxicity, changes in astrocyte transcription profile as well as changes in secretome profiles. FINDINGS: We report that C9-mutated astrocytes are toxic to MNs via soluble factors. The toxic effects of astrocytes are positively correlated with the length of astrocyte propagation in culture, consistent with the age-related nature of ALS. We show that C9-mutated astrocytes downregulate the secretion of several antioxidant proteins. In line with these findings, we show increased astrocytic oxidative stress and senescence. Importantly, media conditioned by C9-astrocytes increased oxidative stress in wild type MNs. INTERPRETATION: Our results suggest that dysfunction of C9-astrocytes leads to oxidative stress of themselves and MNs, which probably contributes to neurodegeneration. Our findings suggest that therapeutic strategies in familial ALS must not only target MNs but also focus on astrocytes to abrogate nervous system injury.