Gist F. Croft

Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patients disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS.

Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines

The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

A functionally characterized test set of human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.

Self-organization of the in vitro attached human embryo

Implantation of the blastocyst is a developmental milestone in mammalian embryonic development. At this time, a coordinated program of lineage diversification, cell-fate specification, and morphogenetic movements establishes the generation of extra-embryonic tissues and the embryo proper, and determines the conditions for successful pregnancy and gastrulation. Despite its basic and clinical importance, this process remains mysterious in humans. Here we report the use of a novel in vitro system to study the post-implantation development of the human embryo. We unveil the self-organizing abilities and autonomy of in vitro attached human embryos. We find human-specific molecular signatures of early cell lineage, timing, and architecture. Embryos display key landmarks of normal development, including epiblast expansion, lineage segregation, bi-laminar disc formation, amniotic and yolk sac cavitation, and trophoblast diversification. Our findings highlight the species-specificity of these developmental events and provide a new understanding of early human embryonic development beyond the blastocyst stage. In addition, our study establishes a new model system relevant to early human pregnancy loss. Finally, our work will also assist in the rational design of differentiation protocols of human embryonic stem cells to specific cell types for disease modelling and cell replacement therapy.

Accelerated High-Yield Generation of Limb-Innervating Motor Neurons from Human Stem Cells

Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1, and column-specific markers that mirror those observed in vivo in human embryonic spinal cord. They also exhibited spontaneous and induced activity, and projected axons toward muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3−). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays.

Maturation of Spinal Motor Neurons Derived from Human Embryonic Stem Cells

Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.

Metabolism of dehydroepiandrosterone by rodent brain cell lines: relationship between 7-hydroxylation and aromatization.

The rate of aromatization of 4-androstenedione (AD) and 7-hydroxylation of dehydroepiandrosterone (DHEA) by different neuronal cell lines from fetal rat and mouse brain was compared to that of embryonic rat hippocampal cells in primary culture. The (3)H-labeled steroids were incubated with the cells and the metabolites extracted and separated by thin layer chromatography (TLC), as well as analyzed by high-performance liquid chromatography (HPLC) for further identification. All cell types produced estrone (E(1)) and estradiol (E(2)) from [(3)H]AD but the rate of aromatization was lowest with the rat hippocampal cells in primary culture. With [(3)H]DHEA, BHc.2 mouse hippocampal cells and E(t)C.1 neurons behaved like the mixed cells from rat hippocampus, forming 7-hydroxy DHEA as the almost exclusive product. In contrast, mouse brain BV2 microglia were virtually unable to hydroxylate DHEA at C-7 and yielded estrogen and more testosterone (T) than other cell types tested. These experiments highlight the pivotal role of 3beta-hydroxysteroid dehydrogenase/ketoisomerase in the control of AD formation for its subsequent aromatization to estrogen. It raises the possibility that differences in metabolism of DHEA by certain brain cells could account for differences in their immunomodulatory and neuroprotective functions. Some could exert their effects by converting DHEA to its 7-hydroxylated form while others, like BV2 microglia, by converting DHEA primarily to other C-19 steroids and to estrogen by way of AD.

Dehydroepiandrosterone (DHEA) metabolism in the brain: identification by liquid chromatography/mass spectrometry of the delta-4-isomer of DHEA and related steroids formed from androstenedione by mouse BV2 microglia.

Studies to elucidate the role of dehydroepiandrosterone (DHEA) metabolism in neuroprotection have compared its relative 7-hydroxylation against estrogen formation by way of 4-androstenedione (AD) in various rodent brain cell lines. In all cases, the 7alpha- and 7beta-hydroxy epimers of DHEA were found to be the dominant products with one notable exception. BV2 mouse microglia were virtually unable to hydroxylate DHEA at C-7 and converted AD to a major unknown metabolite not observed with mouse BHc hippocampal cells. In this paper, we describe the identification of this compound based on its physical properties and analysis by TLC and HPLC. Its identity as 3beta-hydroxy-4-androstene-17-one, the Delta(4)-isomer of DHEA, was confirmed by mass spectrometry (LC/MS), as well as by reverse isotope dilution analysis involving co-crystallization with the synthetic steroid. Possible mechanisms for the formation of this isomer of DHEA by BV2 microglia are proposed, together with that of other C-19 steroids detected which include testosterone (T), 5alpha-dihydrotestosterone and 5alpha-androstanedione.

A single trophectoderm biopsy at blastocyst stage is mathematically unable to determine embryo ploidy accurately enough for clinical use

BackgroundIt has become increasingly apparent that the trophectoderm (TE) at blastocyst stage is much more mosaic than has been appreciated. Whether preimplantation genetic screening (PGS), utilizing a single TE biopsy (TEB), can reliably determine embryo ploidy has, therefore, increasingly been questioned in parallel.MethodsWe for that reason here established 2 mathematical models to assess probabilities of false-negative and false-positive results of an on average 6-cell biopsy from an approximately 300-cell TE. This study was a collaborative effort between investigators at The Center for Human Reproduction in New York City and the Center for Studies in Physics and Biology and the Brivanlou Laboratory of Stem Cell Biology and Molecular Embryology, the latter two both at Rockefeller University in New York City.ResultsBoth models revealed that even under best case scenario, assuming even distribution of mosaicism in TE (since mosaicism is usually clonal, a highly unlikely scenario), a biopsy of at least 27 TE cells would be required to reach minimal diagnostic predictability from a single TEB.ConclusionsAs currently performed, a single TEB is, therefore, mathematically incapable of reliably determining whether an embryo can be transferred or should be discarded. Since a single TEB, as currently performed, apparently is not representative of the complete TE, this study, thus, raises additional concern about the clinical utilization of PGS.

Transcriptional activity of estrogen receptors ERα, and ERβ in the EtC.1 cerebellar granule cell line

Abstract Estrogen receptors alpha and beta (ERα and ERβ) are expressed in the cerebellum throughout development and in the adult suggesting an important role of 17-β-estradiol (E2) in this brain structure. In the present study, we have characterized the functionality of estrogen receptors (ERs) expressed in the immature cerebellar granule cell line EtC.1 by transfecting such cells with a luciferase reporter gene (ERE-Luc) coupled to an estrogen response element promoter. The induction of luciferase activity in EtC.1 cells by E2 and ER-subtype selective agonists was compared in normal cells and in cells overexpressing human ERα or ERβ (hERα or hERβ). E2-mediated transcription of the reporter gene was blocked by the ER antagonist ICI 182,780 (ICI), demonstrating the presence of functional native ERs. The selective agonist for ERα (PPT) showed a reduced response in luciferase induction compared to E2. Moreover, the ERβ agonist (DPN) was unable to induce luciferase activity. E2-induced ERE-Luc transcription was not increased by overexpression of hERα. In contrast, hERβ overexpression reduced the efficacy of E2 and abolished ERα-selective agonist activity. The ERβ-specific agonist did not induce gene reporter activity unless hERβ was overexpressed in the cells, suggesting that the endogenous ERβ in EtC.1 cells is transcriptionally inactive. ICI inhibition of E2 responses was not affected by overexpression of the human ERs. The data suggest that ERα plays a predominant role in E2-mediated transcription in EtC.1 cells. Our data are discussed in view of other reports alluding to the complexity and cell-type specificity of E2-mediated transcription.