James A. Byrne

LRRK2 Mutant iPSC-Derived DA Neurons Demonstrate Increased Susceptibility to Oxidative Stress

Studies of Parkinsons disease (PD) have been hindered by lack of access to affected human dopaminergic (DA) neurons. Here, we report generation of induced pluripotent stem cells that carry the p.G2019S mutation (G2019S-iPSCs) in the Leucine-Rich Repeat Kinase-2 (LRRK2) gene, the most common PD-related mutation, and their differentiation into DA neurons. The high penetrance of the LRRK2 mutation and its clinical resemblance to sporadic PD suggest that these cells could provide a valuable platform for disease analysis and drug development. We found that DA neurons derived from G2019S-iPSCs showed increased expression of key oxidative stress-response genes and α-synuclein protein. The mutant neurons were also more sensitive to caspase-3 activation and cell death caused by exposure to stress agents, such as hydrogen peroxide, MG-132, and 6-hydroxydopamine, than control DA neurons. This enhanced stress sensitivity is consistent with existing understanding of early PD phenotypes and represents a potential therapeutic target.

Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts.

BACKGROUND The derivation of induced pluripotent stem cells (iPSCs) provides new possibilities for basic research and novel cell-based therapies. Limitations, however, include our current lack of understanding regarding the underlying mechanisms and the inefficiency of reprogramming. METHODOLOGY/PRINCIPAL FINDINGS Here, we report identification and isolation of a subpopulation of human dermal fibroblasts that express the pluripotency marker stage specific embryonic antigen 3 (SSEA3). Fibroblasts that expressed SSEA3 demonstrated an enhanced iPSC generation efficiency, while no iPSC derivation was obtained from the fibroblasts that did not express SSEA3. Transcriptional analysis revealed NANOG expression was significantly increased in the SSEA3 expressing fibroblasts, suggesting a possible mechanistic explanation for the differential reprogramming. CONCLUSIONS/SIGNIFICANCE To our knowledge, this study is the first to identify a pluripotency marker in a heterogeneous population of human dermal fibroblasts, to isolate a subpopulation of cells that have a significantly increased propensity to reprogram to pluripotency and to identify a possible mechanism to explain this differential reprogramming. This discovery provides a method to significantly increase the efficiency of reprogramming, enhancing the feasibility of the potential applications based on this technology, and a tool for basic research studies to understand the underlying reprogramming mechanisms.

Germline stem cells: toward the regeneration of spermatogenesis

Improved therapies for cancer and other conditions have resulted in a growing population of long-term survivors. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or prereproductive years. Some of these patients have the opportunity to preserve their fertility using standard technologies that include sperm, egg, or embryo banking, followed by IVF and/or ET. However, these options are not available to all patients, especially the prepubertal patients who are not yet producing mature gametes. For these patients, there are several stem cell technologies in the research pipeline that may give rise to new fertility options and allow infertile patients to have their own biological children. We will review the role of stem cells in normal spermatogenesis as well as experimental stem cell-based techniques that may have potential to generate or regenerate spermatogenesis and sperm. We will present these technologies in the context of the fertility preservation paradigm, but we anticipate that they will have broad implications for the assisted reproduction field.

Generation of isogenic pluripotent stem cells

The ability to reprogram somatic cell nuclei back into a pluripotent epigenetic state provides exciting new possibilities for in vitro research and cell transplantation therapy. There has been a significant quantity of recent research studies demonstrating that this epigenetic reprogramming process is possible with human and non-human primate somatic cells. In this review, various methodologies for reprogramming primate somatic cells into pluripotent stem cells are examined, epigenetic reprogramming following somatic cell nuclear transfer and normal primate embryonic development is compared, and future potential methods to induce direct reprogramming without using genetic modification are discussed.

From Skin Biopsy to Neurons through a Pluripotent Intermediate Under Good Manufacturing Practice Protocols

The clinical application of human‐induced pluripotent stem cells (hiPSCs) requires not only the production of Good Manufacturing Practice‐grade (GMP‐grade) hiPSCs but also the derivation of specified cell types for transplantation under GMP conditions. Previous reports have suggested that hiPSCs can be produced in the absence of animal‐derived reagents (xenobiotics) to ease the transition to production under GMP standards. However, to facilitate the use of hiPSCs in cell‐based therapeutics, their progeny should be produced not only in the absence of xenobiotics but also under GMP conditions requiring extensive standardization of protocols, documentation, and reproducibility of methods and product. Here, we present a successful framework to produce GMP‐grade derivatives of hiPSCs that are free of xenobiotic exposure from the collection of patient fibroblasts, through reprogramming, maintenance of hiPSCs, identification of reprogramming vector integration sites (nrLAM‐PCR), and finally specification and terminal differentiation of clinically relevant cells. Furthermore, we developed a primary set of Standard Operating Procedures for the GMP‐grade derivation and differentiation of these cells as a resource to facilitate widespread adoption of these practices.

Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status

IntroductionThe reprogramming of a patient’s somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known.MethodsIn this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions.ResultsWe found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status.ConclusionFor the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.

Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

Background Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. Methodology/Principal Finding Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. Conclusions/Significance Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer.

Lethal phenotype in conditional late-onset arginase 1 deficiency in the mouse

Human arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia, which lead to neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation; uncommonly, patients suffer early death from this disorder. In a murine targeted knockout model, onset of the phenotypic abnormality is heralded by weight loss at around day 15, and death occurs typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. This discrepancy between the more attenuated juvenile-onset human disease and the lethal neonatal murine model has remained suboptimal for studying and developing therapy for the more common presentation of arginase deficiency. These investigations aimed to address this issue by creating an adult conditional knockout mouse to determine whether later onset of arginase deficiency also resulted in lethality. Animal survival and ammonia levels, body weight, circulating amino acids, and tissue arginase levels were examined as outcome parameters after widespread Cre-recombinase activation in a conditional knockout model of arginase 1 deficiency. One hundred percent of adult female and 70% of adult male mice died an average of 21.0 and 21.6 days, respectively, after the initiation of tamoxifen administration. Animals demonstrated elevated circulating ammonia and arginine at the onset of phenotypic abnormalities. In addition, brain and liver amino acids demonstrated abnormalities. These studies demonstrate that (a) the absence of arginase in adult animals results in a disease profile (leading to death) similar to that of the targeted knockout and (b) the phenotypic abnormalities seen in the juvenile-onset model are not exclusive to the age of the animal but instead to the biochemistry of the disorder. This adult model will be useful for developing gene- and cell-based therapies for this disorder that will not be limited by the small animal size of neonatal therapy and for developing a better understanding of the characteristics of hyperargininemia.

Human Skin Cells That Express Stage-Specific Embryonic Antigen 3 Associate with Dermal Tissue Regeneration

Abstract Stage-specific embryonic antigen 3 (SSEA3) is a glycosphingolipid that has previously been used to identify cells with stem cell-like, multipotent, and pluripotent characteristics. A rare subpopulation of SSEA3-expressing cells exists in the dermis of adult human skin. These SSEA3-expressing cells undergo a significant increase in cell number in response to injury, suggesting a possible role in regeneration. These SSEA3-expressing regeneration-associated (SERA) cells were derived through primary cell culture, purified by fluorescence-activated cell sorting (FACS), and characterized. Longer in vitro culture of the primary skin cells led to lower SSEA3 expression stability after FACS-based purification, suggesting that the current culture conditions may need to be optimized to permit the large-scale expansion of SERA cells. The SERA cells demonstrated a global transcriptional state that was most similar to bone marrow- and fat-derived mesenchymal stem cells (MSCs), and the highest expressing SSEA3-expressing cells co-expressed CD105 (clone 35). However, while a rare population of MSCs was observed in primary human skin cell cultures that could differentiate into adipocytes, osteoblasts, or chondrocytes, SERA cells did not possess this differentiation capacity, suggesting that there are at least two different rare subpopulations in adult human skin primary cultures. The identification, efficient purification, and large-scale expansion of these rare subpopulations (SERA cells and MSCs) from heterogeneous adult human skin primary cell cultures may have applications for future patient-specific cellular therapies.