The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.

Genetically modified mouse strains derived from embryonic stem (ES) cells have become essential tools for functional genomics and biomedical research. Large scale mutagenesis projects are producing libraries of mutant C57BL/6 (B6) ES cells to enable the functional annotation of every gene of the mouse genome. To realize the utility of these resources, efficient and accessible methods of generating mutant mice from these ES cells are necessary. Here, we describe a combination of ICR morula aggregation and a chemically-defined culture medium with widely available and accessible components for the high efficiency generation of germline transmitting chimeras from C57BL/6N ES cells. Together these methods will ease the access of the broader biomedical research community to the publicly available B6 ES cell resources.

Conversion of EpiSCs to naive ESCs is a rare event that is driven by the reestablishment of the naive transcription factor network. In mice, STAT3 activation is sufficient to drive conversion of EpiSCs to the naive pluripotent stem cell (PSC) state. However, the lack of responsiveness of EpiSCs to LIF presents a bottleneck in this conversion process. Here, we demonstrate that local accumulation of BMP-SMAD1 signaling, in cooperation with GP130 ligands, enhances the recovery of LIF responsiveness by directly controlling transcription of the LIF receptor (Lif-r). Addition of BMP and LIF to EpiSCs increases both LIF responsiveness and conversion frequencies to naive PSCs. Mechanistically, we show that the transcriptional cofactor P300 plays a critical role by mediating complex formation between STAT3 and SMAD1. This demonstration of how the local microenvironment or stem cell niche reactivates dormant signaling responsiveness and developmental potential may be applicable to other stem cell niche-containing systems.

Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor-A (VEGF) biologics, termed VEGF "Sticky-traps," with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky-traps could locally inhibit angiogenesis to at least the same extent as the original VEGF-trap that also gains whole-body access. Sticky-traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky-trap remained localized to various regions of the eye, such as the inner-limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky-trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre-maturity.

The efficient generation of hematopoietic stem cells (HSCs) from human-induced pluripotent stem cells (iPSCs) holds great promise in personalized transplantation therapies. However, the derivation of functional and transplantable HSCs from iPSCs has had very limited success thus far.

Stem cell transplantation represents a promising strategy for the repair of spinal cord injury (SCI). However, the low survival rate of the grafted cells is a major obstacle hindering clinical success because of ongoing secondary injury processes, which includes excitotoxicity, inflammation and oxidative stress. Previous studies have shown that 17b-estradiol (E2) protects several cell types against cytotoxicity. Thus, we examined the effects of E2 on the viability of human eyelid adipose-derived stem cells (hEASCs) in vitro with hydrogen peroxide (H₂O₂)-induced cell model and in vivo within a rat SCI model. Our results showed that E2 protected hEASCs against H₂O₂-induced cell death in vitro, and enhanced the survival of grafted hEASCs in vivo by reducing apoptosis. Additionally, E2 also enhanced the secretion of growth factors by hEASCs, thereby making the local microenvironment more conducive for tissue regeneration. Overall, E2 administration enhanced the therapeutic efficacy of hEASCs transplantation and facilitated motor function recovery after SCI. Hence, E2 administration may be an intervention of choice for enhancing survival of transplanted hEASCs after SCI.

The silver content of the skin regeneration ointments can influence its regeneration process but in the meantime, it can take the benefit of the antibacterial properties of silver by avoiding the bacterial infection of an open wound. In the current study, the skin healing and regeneration capacity of bioactive glass with spherical gold nanocages (BGAuIND) in the Vaseline ointments were evaluated in vivo comparing the bioactive glass (BG)-Vaseline and bioactive glass with spherical gold (BGAuSP)-Vaseline ointments. Spherical gold nanocages are stabilized with silver and as a consequence the BGAuIND exhibits great antibacterial activity. Histological examination of the cutaneous tissue performed on day 8 indicates a more advanced regeneration process in rats treated with BGAuSP-Vaseline. The histopathological examination also confirms the results obtained after 11 days post-intervention, when the skin is completely regenerated at rats treated with BGAuSP-Vaseline compared with the others groups where the healing was incomplete. This result is also confirmed by the macroscopic images of the evolution of wounds healing. As expected, the silver content influences the wound healing process but after two weeks, for all of the post-interventional trials from the groups of rats, the skin healing was completely.

Several lines of evidence indicate that the propagation of misfolded α-synuclein (α-syn) plays a central role in the progression and manifestation of Parkinson's disease. Pathogenic α-syn species can be present in the extracellular space. Thus, the identification and modulation of the key enzymes implicated in extracellular α-syn turnover becomes vital. Kallikrein peptidase 6 has been identified as one of the major α-syn degrading enzymes and has been implicated in the clearance of extracellular α-syn. However, the physiological role of this enzyme in regulating α-syn, in vivo, still remains elusive. Here, by utilizing Klk6 knock-out (Klk6-/- ) mice as our experimental model, we provide insight into the physiologic relevance of endogenous KLK6 expression on α-syn processing. Behavioral phenotyping showed that Klk6-/- mice display no gross behavioral abnormalities. Further in vivo characterization of this mouse model, in the context of α-syn accumulation, showed that KLK6 deletion had no impact on the protein levels of intracellular or extracellular α-syn. Upon in vivo administration of α-syn pre-formed fibrils (PFF), α-syn pathologic accumulations were evident both in the brains of Klk6-/- mice and wt mice without significant differences. Intrastriatal delivery of active KLK6, did not affect secreted α-syn levels observed in the A53T α-syn over-expressing mice. These findings suggest that in the in vivo setting of PFF pathology induction, KLK6 alone is not able to modulate pathology transmission. Our study raises implications for the use of recombinant α-syn fibrils in α-syn turnover studies.

The basic helix-loop-helix (bHLH) transcription factor ASCL2 plays essential roles in diploid multipotent trophoblast progenitors, intestinal stem cells, follicular T-helper cells, as well as during epidermal development and myogenesis. During early development, Ascl2 expression is regulated by genomic imprinting and only the maternally inherited allele is transcriptionally active in trophoblast. The paternal allele-specific silencing of Ascl2 requires expression of the long non-coding RNA Kcnq1ot1 in cis and the deposition of repressive histone marks. Here we show that Del7AI, a 280-kb deletion allele neighboring Ascl2, interferes with this process in cis and leads to a partial loss of silencing at Ascl2. Genetic rescue experiments show that the low level of Ascl2 expression from the paternal Del7AI allele can rescue the embryonic lethality associated with maternally inherited Ascl2 mutations, in a level-dependent manner. Despite their ability to support development to term, the rescued placentae have a pronounced phenotype characterized by severe hypoplasia of the junctional zone, expansion of the parietal trophoblast giant cell layer, and complete absence of invasive glycogen trophoblast cells. Transcriptome analysis of ectoplacental cones at E7.5 and differentiation assays of Ascl2 mutant trophoblast stem cells show that ASCL2 is required for the emergence or early maintenance of glycogen trophoblast cells during development. Our work identifies a new cis-acting mutation interfering with Kcnq1ot1 silencing function and establishes a novel critical developmental role for the transcription factor ASCL2.

Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.

T cell immunotherapy is now a mainstay therapy for several blood-borne cancers as well as metastatic melanoma. Unfortunately, many epithelial tumors respond poorly to immunotherapy, and the reasons for this are not well understood. Cancer-associated fibroblasts (CAFs) are the most frequent non-neoplastic cell type in most solid tumors, and they are emerging as a key player in immunotherapy resistance. A range of immortalized CAF lines will be essential tools that will allow us to understand immune responses against cancer and develop novel strategies for cancer immunotherapy. To study the effect of CAFs on T cell proliferation, we created and characterized a number of novel immortalized human CAFs lines (Im-CAFs) from human breast, colon, and pancreatic carcinomas. Im-CAFs shared similar phenotypes, matrix remodeling and contraction capabilities, and growth and migration rates compared to the primary CAFs. Using primary isolates from breast carcinoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma, we report that CAFs across major tumor types are able to potently suppress T cell proliferation in vitro. Im-CAFs retained this property. Im-CAFs are a key tool that will provide important insights into the mechanisms of CAF-mediated T cell suppression through techniques such as CRISPR-Cas9 modification, molecular screens, and pipeline drug testing.

Stroke is a leading cause of death and long-term disability worldwide. Current therapeutic options are limited in terms of their time for implementation and efficacy in promoting recovery. Cell transplantation has been shown to have promise in several animal models however significant challenges remain, including the optimal source of cells to promote neural repair. Here, we report on the use of a population of human ESC derived, cortically specified, neuroepithelial precursor cells (cNEPs) that are neurally restricted in their lineage potential. CNEPs have the potential to give rise to mature neural cell types following transplantation, including neurons, astrocytes and oligodendrocytes. With a view towards translation, we sought to determine whether this human cell source was effective in promoting improved functional outcomes following stroke. Undifferentiated cNEPs were transplanted in a pre-clinical endothelin-1 (ET-1) model of ischemic motor cortical stroke in immunocompromised SCID-beige mice and cellular and functional outcomes were assessed. We demonstrate that cNEP transplantation in the acute phase (4 days post-stroke) improves motor function as early as 20 days post-stroke, compared to stroke-injured, non-transplanted mice. At the time of recovery, a small fraction (<6%) of the transplanted cNEPs are observed within the stroke injury site. The surviving cells expressed the immature neuronal marker, doublecortin, with no differentiation into mature neural phenotypes. At longer survival times (40 days), the majority of recovered, transplanted mice had a complete absence of surviving cNEPS. Hence, human cNEPs grafted at early times post-stroke support the observed functional recovery following ET-1 stroke but their persistence is not required, thereby supporting a by-stander effect rather than cell replacement.

Human trophoblast stem cells (hTSCs) provide a valuable model to study placental development and function. While primary hTSCs have been derived from embryos/early placenta, and transdifferentiated hTSCs from naïve human pluripotent stem cells (hPSCs), the generation of hTSCs from primed PSCs is problematic. We report the successful generation of TSCs from primed hPSCs and show that BMP4 substantially enhances this process. TSCs derived from primed hPSCs are similar to blastocyst-derived hTSCs in terms of morphology, proliferation, differentiation potential, and gene expression. We define the chromatin accessibility dynamics and histone modifications (H3K4me3/H3K27me3) that specify hPSC-derived TSCs. Consistent with low density of H3K27me3 in primed hPSC-derived hTSCs, we show that knockout of H3K27 methyltransferases (EZH1/2) increases the efficiency of hTSC derivation from primed hPSCs. Efficient derivation of hTSCs from primed hPSCs provides a simple and powerful model to understand human trophoblast development, including the pathogenesis of trophoblast-related disorders, by generating disease-specific hTSCs.

USP7, a ubiquitin-specific peptidase (USP), plays an important role in many cellular processes through its catalytic deubiquitination of various substrates. However, its nuclear function that shapes the transcriptional network in mouse embryonic stem cells (mESCs) remains poorly understood. We report that USP7 maintains mESC identity through both catalytic activity-dependent and -independent repression of lineage differentiation genes. Usp7 depletion attenuates SOX2 levels and derepresses lineage differentiation genes thereby compromising mESC pluripotency. Mechanistically, USP7 deubiquitinates and stabilizes SOX2 to repress mesoendodermal (ME) lineage genes. Moreover, USP7 assembles into RYBP-variant Polycomb repressive complex 1 and contributes to Polycomb chromatin-mediated repression of ME lineage genes in a catalytic activity-dependent manner. USP7 deficiency in its deubiquitination function is able to maintain RYBP binding to chromatin for repressing primitive endoderm-associated genes. Our study demonstrates that USP7 harbors both catalytic and noncatalytic activities to repress different lineage differentiation genes, thereby revealing a previously unrecognized role in controlling gene expression for maintaining mESC identity.

Many autoimmune and infectious diseases are characterized by the formation of granulomas which are inflammatory lesions that consist of spatially organized immune cells. These sites protect the host and control pathogens like Mycobacterium tuberculosis (Mtb), but are highly inflammatory and cause pathology. Using bacille Calmette-Guerin (BCG) and Mtb infection in mice that induce sarcoid or caseating granulomas, we show that a subpopulation of granuloma macrophages produces vascular endothelial growth factor (VEGF-A), which recruits immune cells to the granuloma by a non-angiogenic pathway. Selective blockade of VEGF-A in myeloid cells, combined with granuloma transplantation, shows that granuloma VEGF-A regulates granulomatous inflammation. The severity of granuloma-related inflammation can be ameliorated by pharmaceutical or genetic inhibition of VEGF-A, which improves survival of mice infected with virulent Mtb without altering host protection. These data show that VEGF-A inhibitors could be used as a host-directed therapy against granulomatous diseases like tuberculosis and sarcoidosis, thereby expanding the value of already existing and approved anti-VEGF-A drugs.

The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using oncogenic transcription factors. However, this method leads to genetic aberrations in iPSCs via unknown mechanisms, which may limit their clinical use. Here, we demonstrate that the supplementation of growth media with antioxidants reduces the genome instability of cells transduced with the reprogramming factors. Antioxidant supplementation did not affect transgene expression level or silencing kinetics. Importantly, iPSCs made with antioxidants had significantly fewer de novo copy number variations, but not fewer coding point mutations, than iPSCs made without antioxidants. Our results suggest that the quality and safety of human iPSCs might be enhanced by using antioxidants in the growth media during the generation and maintenance of iPSCs.

Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ~E6.0-E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ~E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ~E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Ischemic stroke results in a loss of neurons for which there are no available clinical strategies to stimulate regeneration. While preclinical studies have demonstrated that functional recovery can be obtained by transplanting an exogenous source of neural progenitors into the brain, it remains unknown at which stage of neuronal maturity cells will provide the most benefit. We investigated the role of neuronal maturity on cell survival, differentiation, and long-term sensorimotor recovery in stroke-injured rats using a population of human cortically-specified neuroepithelial progenitor cells (cNEPs) delivered in a biocompatible, bioresorbable hyaluronan/methylcellulose hydrogel. We demonstrate that transplantation of immature cNEPs result in the greatest tissue and functional repair, relative to transplantation of more mature neurons. The transplantation process itself resulted in the least cell death and phenotypic changes in the immature cNEPs, and the greatest acute cell death in the mature cells. The latter negatively impacted host tissue and negated any potential positive effects associated with cell maturity and the hydrogel vehicle, which itself showed some functional and tissue benefit. Moreover, we show that more mature cell populations are drastically altered during the transplantation process itself. The phenotype of the cells before and after transplantation had an enormous impact on their survival and the consequent tissue and behavioral response, emphasizing the importance of characterizing injected cells in transplantation studies more broadly.