Cellular Zinc Homeostasis Contributes to Neuronal Differentiation in Human Induced Pluripotent Stem Cells.

Disturbances in neuronal differentiation and function are an underlying factor of many brain disorders. Zinc homeostasis and signaling are important mediators for a normal brain development and function, given that zinc deficiency was shown to result in cognitive and emotional deficits in animal models that might be associated with neurodevelopmental disorders. One underlying mechanism of the observed detrimental effects of zinc deficiency on the brain might be impaired proliferation and differentiation of stem cells participating in neurogenesis. Thus, to examine the molecular mechanisms regulating zinc metabolism and signaling in differentiating neurons, using a protocol for motor neuron differentiation, we characterized the expression of zinc homeostasis genes during neurogenesis using human induced pluripotent stem cells (hiPSCs) and evaluated the influence of altered zinc levels on the expression of zinc homeostasis genes, cell survival, cell fate, and neuronal function. Our results show that zinc transporters are highly regulated genes during neuronal differentiation and that low zinc levels are associated with decreased cell survival, altered neuronal differentiation, and, in particular, synaptic function. We conclude that zinc deficiency in a critical time window during brain development might influence brain function by modulating neuronal differentiation.

The dynactin p150 subunit: cell biology studies of sequence changes found in ALS/MND and Parkinsonian syndromes.

The dynactin p150glued subunit, encoded by the gene DCTN1 is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport. This subunit is a candidate modifier for neurodegenerative diseases, in particular motoneuron and extrapyramidal diseases. Based on an extensive screening effort of all 32 exons in more than 2,500 ALS/MND patients, patients suffering from Parkinsonian Syndromes and controls, we investigated 24 sequence variants of p150 in cell-based studies. We used both non-neuronal cell lines and primary rodent spinal motoneurons and report on cell biological abnormalities in five of these sequence alterations and also briefly report on the clinical features. Our results suggest the presence of biological changes caused by some p150 mutants pointing to a potential pathogenetic significance as modifier of the phenotype of the human disease.

Tubulin-binding cofactor B is a direct interaction partner of the dynactin subunit p150(Glued).

The dynactin p150(Glued) subunit, encoded by the gene DCTN1, is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport in motor neurons. The p150 subunit is a candidate gene for neurodegenerative diseases, in particular motor neuron and extrapyramidal diseases. Tubulin-binding cofactors are believed to be involved in tubulin biogenesis and degradation and therefore to contribute to microtubule functional diversity and regulation. A yeast-two-hybrid screen for putative interacting proteins of dynactin p150(Glued) has revealed tubulin-folding cofactor B (TBCB). We analyzed the interaction of these proteins and investigated the impact of this complex on the microtubule network in cell lines and primary hippocampal neurons in vitro. We especially concentrated on neuronal morphology and synaptogenesis. Overexpression of both proteins or depletion of TBCB alone does not alter the microtubule network and/or neuronal morphology. The demonstration of the interaction of the transport molecule dynactin and the tubulin-regulating factor TBCB is thought to have an impact on several cellular mechanisms. TBCB expression levels have been found to have only a subtle influence on the microtubule network and neuronal morphology. However, overexpression of TBCB leads to the decreased localization of p150 to the microtubule network that might result in a functional modulation of this protein complex.

Major tegument protein pp65 of human cytomegalovirus is required for the incorporation of pUL69 and pUL97 into the virus particle and for viral growth in macrophages.

The tegument protein pp65 of human cytomegalovirus (HCMV) represents the major component of mature virus particles. Nevertheless, deletion of pp65 has been shown to have no effects on virus replication and morphogenesis in fibroblasts in vitro. We have studied the HCMV virion composition in the absence of pp65 and viral growth of a pp65 stop mutant in different cell types, including monocyte-derived macrophages. Two stop codons at amino acids 11 and 12 of pp65 were introduced by bacterial artificial chromosome mutagenesis into the endotheliotropic strain TB40/E. Clear changes of the tegument composition could be observed in purified mutant virus particles, where the amount of tegument protein pUL25 was drastically reduced. In addition, pUL69 and the virally encoded protein kinase UL97 were undetectable in the pp65 stop mutant. Expression of pUL69 in infected cells was unaltered while pUL25 accumulated in the absence of pp65, thus demonstrating that only incorporation into virus particles is dependent on pp65. Coimmunoprecipitation experiments using lysates of infected cells revealed an interaction between pUL69 and pp65. This interaction was verified in pull-down experiments using transfected cells, which showed that pp65 and pUL69 do not require the presence of other viral proteins for their interaction. We conclude that pp65 is required for the incorporation of other viral proteins into the virus particle and thus is involved in the protein-protein interaction network leading to normal tegument formation. When studying growth kinetics of the pp65 stop mutant in different cell types, we found a severe impairment of viral growth in monocyte-derived macrophages, showing for the first time a strong cell-specific role of pp65 in viral growth.

Zinc deficiency and low enterocyte zinc transporter expression in human patients with autism related mutations in SHANK3.

Phelan McDermid Syndrome (PMDS) is a genetic disorder characterized by features of Autism spectrum disorders. Similar to reports of Zn deficiency in autistic children, we have previously reported high incidence of Zn deficiency in PMDS. However, the underlying mechanisms are currently not well understood. Here, using inductively coupled plasma mass-spectrometry to measure the concentration of Zinc (Zn) and Copper (Cu) in hair samples from individuals with PMDS with 22q13.3 deletion including SHANK3 (SH3 and multiple ankyrin repeat domains 3), we report a high rate of abnormally low Zn/Cu ratios. To investigate possible underlying mechanisms, we generated enterocytes from PMDS patient-derived induced pluripotent stem cells and used Caco-2 cells with knockdown of SHANK3. We detected decreased expression of Zn uptake transporters ZIP2 and ZIP4 on mRNA and protein level correlating with SHANK3 expression levels, and found reduced levels of ZIP4 protein co-localizing with SHANK3 at the plasma membrane. We demonstrated that especially ZIP4 exists in a complex with SHANK3. Furthermore, we performed immunohistochemistry on gut sections from Shank3αß knockout mice and confirmed a link between enterocytic SHANK3, ZIP2 and ZIP4. We conclude that apart from its well-known role in the CNS, SHANK3 might play a specific role in the GI tract.

Reprogramming to pluripotency does not require transition through a primitive streak-like state.

Pluripotency can be induced in vitro from adult somatic mammalian cells by enforced expression of defined transcription factors regulating and initiating the pluripotency network. Despite the substantial advances over the last decade to improve the efficiency of direct reprogramming, exact mechanisms underlying the conversion into the pluripotent stem cell state are still vaguely understood. Several studies suggested that induced pluripotency follows reversed embryonic development. For somatic cells of mesodermal and endodermal origin that would require the transition through a Primitive streak-like state, which would necessarily require an Eomesodermin (Eomes) expressing intermediate. We analyzed reprogramming in human and mouse cells of mesodermal as well as ectodermal origin by thorough marker gene analyses in combination with genetic reporters, conditional loss of function and stable fate-labeling for the broad primitive streak marker Eomes. We unambiguously demonstrate that induced pluripotency is not dependent on a transient primitive streak-like stage and thus does not represent reversal of mesendodermal development in vivo.

TBX3 Knockdown Decreases Reprogramming Efficiency of Human Cells.

TBX3 is a member of the T-box transcription factor family and is involved in the core pluripotency network. Despite this role in the pluripotency network, its contribution to the reprogramming process during the generation of human induced pluripotent stem cells remains elusive. In this respect, we performed reprogramming experiments applying TBX3 knockdown in human fibroblasts and keratinocytes. Knockdown of TBX3 in both somatic cell types decreased the reprogramming efficiencies in comparison to control cells but with unchanged reprogramming kinetics. The resulting iPSCs were indistinguishable from control cells and displayed a normal in vitro differentiation capacity by generating cells of all three germ layers comparable to the controls.

A Fresh Look on T-Box Factor Action in Early Embryogenesis (T-Box Factors in Early Development).

T-Box transcription factors are expressed throughout the gestational period and coordinate a variety of embryonic events that enable proper development, from the first differentiation of embryonic and extraembryonic tissues until final organogenesis. Although the T-Box gene family comprises essential roles in early cellular differentiation, in adult tissues it has also been associated with cancer development. In spite of their common T-Box regulatory binding domain, T-Box family members utilize different cofactors and different spatiotemporal expression patterns to confer their specificity in diverse developmental processes. The earliest expression note of T-Box factors can be observed even before fertilization in primordial germ cells and just after zygotic gene activation (around the eight-cell blastomere stage). Thus, particularly the early stages of development are highly influenced by these key regulators in line with the notion that T-Box mutations lead to developmental disorders and even lethality. In this review, we summarize recently acquired findings on T-Box factors to provide a comprehensive overview on their role during early embryogenesis.

A Dynamic Role of TBX3 in the Pluripotency Circuitry.

Pluripotency represents a cell state comprising a fine-tuned pattern of transcription factor activity required for embryonic stem cell (ESC) self-renewal. TBX3 is the earliest expressed member of the T-box transcription factor family and is involved in maintenance and induction of pluripotency. Hence, TBX3 is believed to be a key member of the pluripotency circuitry, with loss of TBX3 coinciding with loss of pluripotency. We report a dynamic expression of TBX3 in vitro and in vivo using genetic reporter tools tracking TBX3 expression in mouse ESCs (mESCs). Low TBX3 levels are associated with reduced pluripotency, resembling the more mature epiblast. Notably, TBX3-low cells maintain the intrinsic capability to switch to a TBX3-high state and vice versa. Additionally, we show TBX3 to be dispensable for induction and maintenance of naive pluripotency as well as for germ cell development. These data highlight novel facets of TBX3 action in mESCs.

A Comparative View on Human Somatic Cell Sources for iPSC Generation.

The breakthrough of reprogramming human somatic cells was achieved in 2006 by the work of Yamanaka and Takahashi. From this point, fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells (iPSCs). Various characteristics of fibroblasts supported their utilization for the groundbreaking experiments of iPSC generation. One major advantage is the high availability of fibroblasts which can be easily isolated from skin biopsies. Furthermore, their cultivation, propagation, and cryoconservation properties are uncomplicated with respect to nutritional requirements and viability in culture. However, the required skin biopsy remains an invasive approach, representing a major drawback for using fibroblasts as the starting material. More and more studies appeared over the last years, describing the reprogramming of other human somatic cell types. Cells isolated from blood samples or urine, as well as more unexpected cell types, like pancreatic islet beta cells, synovial cells, or mesenchymal stromal cells from wisdom teeth, show promising characteristics for a reprogramming strategy. Here, we want to highlight the advantages of keratinocytes from human plucked hair as a widely usable, noninvasive harvesting method for primary material in comparison with other commonly used cell types.

Calcium activated potassium channel expression during human iPS cell-derived neurogenesis.

The family of calcium activated potassium channels of low and intermediate conductance, known as SK channels, consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes, such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date, the role of SK channels in developmental processes has been merely a marginal focus of investigation, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein, we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells, neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly, we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis.

Definitive Endoderm Formation from Plucked Human Hair-Derived Induced Pluripotent Stem Cells and SK Channel Regulation.

Pluripotent stem cells present an extraordinary powerful tool to investigate embryonic development in humans. Essentially, they provide a unique platform for dissecting the distinct mechanisms underlying pluripotency and subsequent lineage commitment. Modest information currently exists about the expression and the role of ion channels during human embryogenesis, organ development, and cell fate determination. Of note, small and intermediate conductance, calcium-activated potassium channels have been reported to modify stem cell behaviour and differentiation. These channels are broadly expressed throughout human tissues and are involved in various cellular processes, such as the after-hyperpolarization in excitable cells, and also in differentiation processes. To this end, human induced pluripotent stem cells (hiPSCs) generated from plucked human hair keratinocytes have been exploited in vitro to recapitulate endoderm formation and, concomitantly, used to map the expression of the SK channel (SKCa) subtypes over time. Thus, we report the successful generation of definitive endoderm from hiPSCs of ectodermal origin using a highly reproducible and robust differentiation system. Furthermore, we provide the first evidence that SKCas subtypes are dynamically regulated in the transition from a pluripotent stem cell to a more lineage restricted, endodermal progeny.

Microarray-Based Comparisons of Ion Channel Expression Patterns: Human Keratinocytes to Reprogrammed hiPSCs to Differentiated Neuronal and Cardiac Progeny.

Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of, for example, ion selectivity, gating mechanism, composition, or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source, keratinocytes from plucked human hair. This comparison revealed that 26% of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6% were downregulated. Additionally, iPSCs express a much higher number of ion channels compared to keratinocytes. Further, to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny, namely, neurons and cardiomyocytes derived from iPS cells. To conclude, hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms.

The potential of iPS cells in synucleinopathy research.

α-synuclein is a protein involved in the pathogenesis of several so-called synucleinopathies including Parkinson's disease. A variety of models have been so far assessed. Human induced pluripotent stem cells provide a patient- and disease-specific model for in vitro studies, pharmacotoxicological screens, and hope for future cell-based therapies. Initial experimental procedures include the harvest of patients' material for the reprogramming process, the investigation of the patients genetic background in the cultured cells, and the evaluation of disease-relevant factors/proteins under various cell culture conditions.

Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells.

An SK3 channel/nWASP/Abi-1 complex is involved in early neurogenesis.

BACKGROUND: The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell. PRINCIPAL FINDINGS: We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners. CONCLUSIONS: Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins.

An inducible expression system of the calcium-activated potassium channel 4 to study the differential impact on embryonic stem cells.

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells. Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation. Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus. Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression.