Carol B. Ware

Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response

Embryonic stem (ES) cells are promising for cardiac repair’ but directing their differentiation toward cardiomyocytes remains challenging. We investigated whether the heart guides ES cells toward cardiomyocytes in vivo and whether allogeneic ES cells were immunologically tolerated. Undifferentiated mouse ES cells consistently formed cardiac teratomas in nude or immunocompetent syngeneic mice. Cardiac teratomas contained no more cardiomyocytes than hind‐limb teratomas’ suggesting lack of guided differentiation. ES cells also formed teratomas in infarcted hearts’ indicating injury‐related signals did not direct cardiac differentiation. Allogeneic ES cells also caused cardiac teratomas’ but these were immunologically rejected after several weeks’ in association with increased inflammation and up‐regulation of class I and II histocompatibility antigens. Fusion between ES cells and cardiomyocytes occurred in vivo’ but was rare. Infarct autofluorescence was identified as an artifact that might be mistaken for enhanced GFP expression and true regeneration. Hence’ undifferentiated ES cells were not guided toward a cardiomyocyte fate in either normal or infarcted hearts’ and there was no evidence for allogeneic immune tolerance of ES cell derivatives. Successful cardiac repair strategies involving ES cells will need to control cardiac differentiation’ avoid introducing undifferentiated cells’ and will likely require immune modulation to avoid rejection.—Nussbaum, J., Minami, E., Laflamme, M. A., Virag, J. A. I., Ware, C. B., Masino, A., Muskheli, V., Pabon, L., Reinecke, H., Murry, C. E. Transplantation of undifferentiated mu‐rine embryonic stem cells in the heart: teratoma formation and immune response. FASEB J. 21, 1345–1357 (2007)

Efficient generation of retinal progenitor cells from human embryonic stem cells.

The retina is subject to degenerative conditions, leading to blindness. Although retinal regeneration is robust in lower vertebrates, regeneration does not occur in the adult mammalian retina. Thus, we have developed efficient methods for deriving retinal neurons from human embryonic stem (hES) cells. Under appropriate culture conditions, up to 80% of the H1 line can be directed to the retinal progenitor fate, and express a gene expression profile similar to progenitors derived from human fetal retina. The hES cell-derived progenitors differentiate primarily into inner retinal neurons (ganglion and amacrine cells), with functional glutamate receptors. Upon coculture with retinas derived from a mouse model of retinal degeneration, the hES cell derived retinal progenitors integrate with the degenerated mouse retina and increase in their expression of photoreceptor-specific markers. These results demonstrate that human ES cells can be selectively directed to a neural retinal cell fate and thus may be useful in the treatment of retinal degenerations.

Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock-in mice.

Excitotoxicity, a form of neuronal injury in which excessive activation of glutamate receptors results in cellular calcium overload, has been implicated in the pathogenesis of Alzheimer disease (AD), although direct evidence is lacking. Mutations in the presenilin-1 (PS1) gene on chromosome 14 are causally linked to many cases of early-onset inherited AD (refs. 5,6). We generated PS1 mutant mice (PS1M146VKI) that express the PS1 M146V targeted allele at normal physiological levels. Although PS1M146VKI mice have no overt mutant phenotype, they are hypersensitive to seizure-induced synaptic degeneration and necrotic neuronal death in the hippocampus. Cultured hippocampal neurons from PS1M146VKI mice have increased vulnerability to death induced by glutamate, which is correlated with perturbed calcium homeostasis, increased oxidative stress and mitochondrial dysfunction. Agents that suppress calcium influx or release and antioxidants protect neurons against the excitotoxic action of the PS1 mutation. These findings establish a direct link between a genetic defect that causes AD and excitotoxic neuronal degeneration, and indicate new avenues for therapeutic intervention in AD patients.

HIF induces human embryonic stem cell markers in cancer cells.

Low oxygen levels have been shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have been shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia-inducible factor (HIF), can induce an hESC-like transcriptional program, including the induced pluripotent stem cell (iPSC) inducers, OCT4, NANOG, SOX2, KLF4, cMYC, and microRNA-302 in 11 cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver, and breast tumors). Furthermore, nondegradable forms of HIFα, combined with the traditional iPSC inducers, are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4-, and HIF1α-positive regions. Furthermore, NANOG and OCT4 expressions positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells, hypoxia was able to induce neurospheres and hESC markers. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathologic conditions.

MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries.

We used massively parallel pyrosequencing to discover and characterize microRNAs (miRNAs) expressed in human embryonic stem cells (hESC). Sequencing of small RNA cDNA libraries derived from undifferentiated hESC and from isogenic differentiating cultures yielded a total of 425,505 high‐quality sequence reads. A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 13 novel miRNAs, and 56 candidate miRNAs. Further characterization of a subset of the novel miRNAs in Dicer‐knockdown hESC demonstrated Dicer‐dependent expression, providing additional validation of our results. A set of 14 miRNAs (9 known and 5 novel) was noted to be expressed in undifferentiated hESC and then strongly downregulated with differentiation. Functional annotation analysis of predicted targets of these miRNAs and comparison with a null model using non‐hESC‐expressed miRNAs identified statistically enriched functional categories, including chromatin remodeling and lineage‐specific differentiation annotations. Finally, integration of our data with genome‐wide chromatin immunoprecipitation data on OCT4, SOX2, and NANOG binding sites implicates these transcription factors in the regulation of nine of the novel/candidate miRNAs identified here. Comparison of our results with those of recent deep sequencing studies in mouse and human ESC shows that most of the novel/candidate miRNAs found here were not identified in the other studies. The data indicate that hESC express a larger complement of miRNAs than previously appreciated, and they provide a resource for additional studies of miRNA regulation of hESC physiology.

Mesenchymal to Epithelial Conversion in Rat Metanephros Is Induced by LIF

Inductive signals cause conversion of mesenchyme into epithelia during the formation of many organs. Yet a century of study has not revealed the inducing molecules. Using a standard model of induction, we found that ureteric bud cells secrete factors that convert kidney mesenchyme to epithelia that, remarkably, then form nephrons. Purification and sequencing of one such factor identified it as leukemia inhibitory factor (LIF). LIF acted on epithelial precursors that we identified by the expression of Pax2 and Wnt4. Other IL-6 type cytokines acted like LIF, and deletion of their shared receptor reduced nephron development. In situ, the ureteric bud expressed LIF, and metanephric mesenchyme expressed its receptors. The data suggest that IL-6 cytokines are candidate regulators of mesenchymal to epithelial conversion during kidney development.

HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition

The function of metabolic state in stemness is poorly understood. Mouse embryonicstem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent statesrepresenting the inner cell mass (ICM) and epiblast embryos. Human embryonic stemcells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic differencebetween these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalentin their energy production, dynamically switching from glycolysis to mitochondrialrespiration on demand. Despite having a more developed and expanding mitochondrialcontent, EpiSC/hESC have low mitochondrial respiratory capacity due to lowcytochrome c oxidase (COX) expression. Similarly, in vivo epiblastssuppress COX levels. These data reveal EpiSC/hESC functional similarity to theglycolytic phenotype in cancer (Warburg effect). We further show thathypoxia‐inducible factor 1α (HIF1α) is sufficient to drive ESC to aglycolytic Activin/Nodal‐dependent EpiSC‐like stage. This metabolic switch duringearly stem‐cell development may be deterministic.

Facioscapulohumeral Dystrophy: Incomplete Suppression of a Retrotransposed Gene

Each unit of the D4Z4 macrosatellite repeat contains a retrotransposed gene encoding the DUX4 double-homeobox transcription factor. Facioscapulohumeral dystrophy (FSHD) is caused by deletion of a subset of the D4Z4 units in the subtelomeric region of chromosome 4. Although it has been reported that the deletion of D4Z4 units induces the pathological expression of DUX4 mRNA, the association of DUX4 mRNA expression with FSHD has not been rigorously investigated, nor has any human tissue been identified that normally expresses DUX4 mRNA or protein. We show that FSHD muscle expresses a different splice form of DUX4 mRNA compared to control muscle. Control muscle produces low amounts of a splice form of DUX4 encoding only the amino-terminal portion of DUX4. FSHD muscle produces low amounts of a DUX4 mRNA that encodes the full-length DUX4 protein. The low abundance of full-length DUX4 mRNA in FSHD muscle cells represents a small subset of nuclei producing a relatively high abundance of DUX4 mRNA and protein. In contrast to control skeletal muscle and most other somatic tissues, full-length DUX4 transcript and protein is expressed at relatively abundant levels in human testis, most likely in the germ-line cells. Induced pluripotent (iPS) cells also express full-length DUX4 and differentiation of control iPS cells to embryoid bodies suppresses expression of full-length DUX4, whereas expression of full-length DUX4 persists in differentiated FSHD iPS cells. Together, these findings indicate that full-length DUX4 is normally expressed at specific developmental stages and is suppressed in most somatic tissues. The contraction of the D4Z4 repeat in FSHD results in a less efficient suppression of the full-length DUX4 mRNA in skeletal muscle cells. Therefore, FSHD represents the first human disease to be associated with the incomplete developmental silencing of a retrogene array normally expressed early in development.

Derivation of naïve human embryonic stem cells

Significance We report on generation of nontransgenic, naïve human pluripotent cells that represent the developmentally earliest state described for human established cells. Existing human ES cell lines in the later primed state can be toggled in reverse to naïve by exposure to histone deacetylase inhibitors prior to naïve culture. A new line was established directly from an eight-cell embryo under naïve culture conditions. We describe the naïve state in humans and show that naïve human ES cells have expanded endoderm developmental capacity. The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222–9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid β-peptide toxicity : Central roles of superoxide production and caspase activation

Abstract : Many cases of early‐onset inherited Alzheimers disease (AD) are caused by mutations in the presenilin‐1 (PS1) gene. Overexpression of PS1 mutations in cultured PC12 cells increases their vulnerability to apoptosis‐induced trophic factor withdrawal and oxidative insults. We now report that primary hippocampal neurons from PS1 mutant knock‐in mice, which express the human PS1M146V mutation at normal levels, exhibit increased vulnerability to amyloid β‐peptide toxicity. The endangering action of mutant PS1 was associated with increased superoxide production, mitochondrial membrane depolarization, and caspase activation. The peroxynitrite‐scavenging antioxidant uric acid and the caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone protected hippocampal neurons expressing mutant PS1 against cell death induced by amyloid β‐peptide. Increase oxidative stress may contribute to the pathogenic action of PS1 mutations, and antioxidants may counteract the adverse property of such AD‐linked mutations.