Human Embryonic Stem Cells

Hepatocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies and toxicology studies. However, before patient-specific iPSCs can be routinely used for these purposes, there is a dire need to critically compare these cells to the golden standard-hESCs. In this study, we aimed at investigating the differences and similarities at the transcriptional level between hepatocyte-like cells (HLCs) derived from both hESCs and iPSCs. Two independent protocols for deriving HLCs from hESCs and iPSCs were adopted and further characterization included immunocytochemistry, real-time (RT)-polymerase chain reaction, and in vitro functional assays. Comparative microarray-based gene expression profiling was conducted on these cells and compared to the transcriptomes of human fetal liver and adult liver progenitors. HLCs derived from hESCs and human iPSCs showed significant functional similarities, similar expression of genes important for liver physiology and common pathways. However, specific differences between the 2 cell types could be observed. For example, among the cytochrome P450 gene family, CYP19A1, CYP1A1, and CYP11A1 were enriched in hESC-derived HLCs, and CYP46A1 and CYP26A1 in iPSC-derived HLCs. HLCs derived from hESCs and human iPSCs exhibited broad similarities but as well meaningful differences. We identified common upregulated transcription factors, which might serve as a source for generating a cocktail of factors able to directly transdifferentiate somatic cells into HLCs. The findings may be vital to the refinement of protocols for the efficient derivation of functional patientspecific HLCs for regenerative and toxicology studies.

Background: Despite their distinct origins, human embryonic stem (hES) and embryonic carcinoma (hEC) cells share a number of similarities such as surface antigen expression, growth characteristics, the ability to either self-renew or differentiate, and control of the undifferentiated state by the same core transcription factors. To obtain further insights into the regulation of selfrenewal, we have silenced hES/hEC cell-specific genes in NCCIT hEC cells and analysed the downstream effects by means of microarrays. Results: RNAi-mediated silencing of OCT4 and SOX2 induced differentiation with mesodermal characteristics. Markers of trophoblast induction were only transiently up-regulated in the OCT4 knock-down. Independent knock-downs of NANOG produced a proliferation rather than a differentiation phenotype, which may be due to high NANOG expression levels in the cell line used. Published ChIP-chip data from hES cells were used to identify putative direct targets. RNAimediated differentiation was accompanied by direct down-regulation of known hES/hEC cell markers. This included all three core transcription factors in the case of the OCT4 and SOX2 knock-downs, confirming previous findings of reciprocal activation in ES cells. Furthermore, large numbers of histone genes as well as epigenetic regulators were differentially expressed, pointing at chromatin remodeling as an additional regulatory level in the differentiation process. Moreover, loss of self-renewal was accompanied by the down-regulation of genes involved in FGF signaling. FGF receptor inhibition for short and prolonged periods of time revealed that the ERK/MAPK cascade is activated by endogenously expressed fibroblast growth factors and that FGF signaling is cruicial for maintaining the undifferentiated state of hEC cells, like in hES cells. Control of self-renewal appears to be very similar in hEC and hES cells. This is supported by large numbers of common transcription factor targets and the requirement for autocrine FGF signaling.

Background: Oct4 is a key factor of an expanded transcriptional network (Oct4-TN) that governs pluripotency and self-renewal in embryonic stem cells (ESCs) and in the inner cell mass from which ESCs are derived. A pending question is whether the establishment of the Oct4-TN initiates during oogenesis or after fertilisation. To this regard, recent evidence has shown that Oct4 controls a poorly known Oct4-TN central to the acquisition of the mouse egg developmental competence. The aim of this study was to investigate the identity and extension of this maternal Oct4-TN, as much as whether its presence is circumscribed to the egg or maintained beyond fertilisation. Results: By comparing the genome-wide transcriptional profile of developmentally competent eggs that express the OCT4 protein to that of developmentally incompetent eggs in which OCT4 is down-regulated, we unveiled a maternal Oct4-TN of 182 genes. Eighty of these transcripts escape post-fertilisation degradation and represent the maternal Oct4-TN inheritance that is passed on to the 2-cell embryo. Most of these 80 genes are expressed in cancer cells and 37 are notable companions of the Oct4 transcriptome in ESCs. Conclusions: These results provide, for the first time, a developmental link between eggs, early preimplantation embryos and ESCs, indicating that the molecular signature that characterises the ESCs identity is rooted in oogenesis. Also, they contribute a useful resource to further study the mechanisms of Oct4 function and regulation during the maternal-to-embryo transition and to explore the link between the regulation of pluripotency and the acquisition of de-differentiation in cancer cells.

The ability of embryonic stem (ES) cells to self-renew indefinitely and to differentiate into multiple cell lineages hold promise for advances in modeling disease progression, screening drugs and treating diseases. To realize these potentials, it is imperative to study self-assembly in an embryonic microenvironment, as this may increase our understanding of ES cell maintenance and differentiation. In this study, we synthesized an array of one-dimensional alginate gel microstrands and aqueous microstrands through an SU-8 filter device by means of capillary action. Furthermore, we investigated self-assembly behaviors and differentiation potentials of mouse ES cells cultured in microstrands of varying diameters. We found that microstrands with an aqueous interior facilitated high density cell culture and formed compact microtissue structures, while microstrands with gelled interiors promote smaller cell-aggregate structures. In particular, we noticed that ES cells collected from one dimensional aqueous microstrands favored the differentiation towards cell lineages of endoderm and mesoderm, whereas those from gelled microstrands preferred to differentiate into ectoderm and mesoderm lineages. In addition to providing a "liquid-like" tubular microenvironment to understand one dimensional self-assembly process of ES cells, this alginate hydrogel microstrand system also offers an alternative way to manipulate the stem cell fate-decision using bioengineered microenvironments.

The ability to precisely pattern embryonic stem (ES) cells in vitro into predefined arrays/ geometries may allow for the recreation of stem cell niche for better understanding of how cellular microenvironmental factors govern stem cell maintenance and differentiation. In this study, a new gelatin-based laser direct-write (LDW) technique was utilized to deposit mouse ES cells into defined arrays of spots, while maintaining stem cell pluripotency. Results obtained from these studies showed that ES cells were successfully printed into specific patterns and remained viable. Furthermore, ES cells retained the expression of Oct4 in nuclei after LDW, indicating that the laser energy did not affect their maintenance of an undifferentiated state. The differentiation potential of mouse ES cells after LDW was confirmed by their ability to form embryoid bodies (EBs) and to spontaneously become cell lineages representing all three germ layers, revealed by the expression of marker proteins of nestin (ectoderm), Myf-5 (mesoderm) and PDX-1 (endoderm), after 7 days of cultivation. Gelatin-based LDW provides a new avenue for stem cell patterning, with precision and control of the cellular microenvironment.

The recreation of an in vitro microenvironment to understand and manipulate the proliferation and migration of invasive breast cancer cells may allow one to put a halt to their metastasis capacity. Invasive cancer cells have been linked to embryonic stem (ES) cells as they possess certain similar characteristics and gene signatures. Embryonic microenvironments have the potential to reprogram cancer cells into a less invasive phenotype and help elucidate tumorigenesis and metastasis. In this study, we explored the feasibility of reconstructing embryonic microenvironments using mouse ES cells cultured in alginate hydrogel and investigated the interactions of ES cells and highly invasive breast cancer cells in 2D, 2&1/ 2D, and 3D cultures. Results showed that mouse ES cells inhibited the growth and tumor spheroid formation of breast cancer cells. The mouse ES cell microenvironment was further constructed and optimized in 3D alginate hydrogel microbeads, and co-cultured with breast cancer cells. Migration analysis displayed a significant reduction in the average velocity and trajectory of breast cancer cell locomotion compared to control, suggesting that bioengineered mouse ES cell microenvironments inhibited the proliferation and migration of breast cancer cells. This study may act as a platform to open up new options to understand and harness tumor cell plasticity and develop therapeutics for metastatic breast cancer.

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to induced pluripotent stem (iPS) cells that closely resemble embryonic stem (ES) cells. The low efficiency with which iPS cells are derived hinders studies on the molecular mechanism of reprogramming, and integration of viral transgenes, in particular the oncogenes c-Myc and Klf4, may handicap this method for human therapeutic applications. Here we report that valproic acid (VPA), a histone deacetylase inhibitor, enables reprogramming of primary human fibroblasts with only two factors, Oct4 and Sox2, without the need for the oncogenes c-Myc or Klf4. The two factor-induced human iPS cells resemble human ES cells in pluripotency, global gene expression profiles and epigenetic states. These results support the possibility of reprogramming through purely chemical means, which would make therapeutic use of reprogrammed cells safer and more practical.

Directed differentiation of human pluripotent stem cells (hPSCs) into somatic counterparts is a valuable tool for studying disease. However, examination of developmental mechanisms in hPSCs remains challenging given complex multi-factorial actions at different stages. Here, we used TALEN and CRISPR/Cas-mediated gene editing and hPSC-directed differentiation for a systematic analysis of the roles of eight pancreatic transcription factors (PDX1, RFX6, PTF1A, GLIS3, MNX1, NGN3, HES1, and ARX). Our analysis not only verified conserved gene requirements between mice and humans but also revealed a number of previously unsuspected developmental mechanisms with implications for type 2 diabetes. These include a role of RFX6 in regulating the number of pancreatic progenitors, a haploinsufficient requirement for PDX1 in pancreatic β cell differentiation, and a potentially divergent role of NGN3 in humans and mice. Our findings support use of systematic genome editing in hPSCs as a strategy for...

THIS IS AN AGE REGRESSION TECHNOLOGY THAT YOU CAN DO YOURSELF
https://www.academia.edu/129570162/AGE_REGRESSION_VIA_ANTI_PARASITICAL_DRUGS_YOU_MAKE_YOURSELF

"I STILL BELIEVE THAT MACROPHAGES CONVERT TO STEM CELLS"  what leads you to this belief?
GRAINS OF CONSCIOUSNESS = ANUs 

A MACROPHAGE IS A COLLECTION OF WHITE BLOOD CELLS - AN ANU AROUND EACH CELL AND AN ANU AROUND THE WHOLE COLLECTIVE  -  LIKE EVERYTHING ELSE  -  IT'S A MIRROR AND SHAPE SHIFTS  -  SINCE THE ROOT PURPOSE OF THE WHITE BLOOD CELL IS TO BE OF SERVICE TO THE BODY  - THE BODY KNOWS THAT AND CAN DIRECT THAT SEED LEVEL LIFE FORCE ENERGY TO MERGE WITH ANOTHER PART OF THE BODY WHERE IT'S NEEDED - QUITE PLURIPOTENT.

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.

Human stem-cell derivatives are likely to play an important role in the future of regenerative medicine. Evaluation and comparison to their in vivo counterparts is critical for assessment of their therapeutic potential. Transcriptomics was used to compare a new differentiation derivative of human embryonic stem (hES) cells-retinal pigment epithelium (RPE)-to human fetal RPE. Several hES cell lines were differentiated into putative RPE, which expressed RPEspecific molecular markers and was capable of phagocytosis, an important RPE function. Isolated hES cell-derived RPE was able to transdifferentiate into cells of neuronal lineage and redifferentiate into RPE-like cells through multiple passages (Ͼ30 Population doublings). Gene expression profiling demonstrated their higher similarity to primary RPE tissue than of existing human RPE cell lines D407 and ARPE-19, which has been shown to attenuate loss of visual function in animals. This is the first report of the isolation and characterization of putative RPE cells from hES cells, as well as the first application of transcriptomics to assess embryonic stem-cell derivatives and their in vivo counterparts-a "differentiomics" outlook. We describe for the first time, a differentiation system that does not require coculture with animal cells or factors, thus allowing the production of zoonoses-free RPE cells suitable for subretinal transplantation in patients with retinal degenerative diseases. With the further development of therapeutic cloning, or the creation of the banks of homozygous human leucocyte antigen (HLA) hES cells using parthenogenesis, RPE lines could be generated to overcome the problem of immune rejection and could be one of the nearest term applications of stem-cell technology.

Human somatic cells are mortal due in large part to telomere shortening associated with cell division. Limited proliferative capacity may, in turn, limit response to injury and may play an important role in the etiology of age-related pathology. Pluripotent stem cells cultured in vitro appear to maintain long telomere length through relatively high levels of telomerase activity. We propose that the induced reversal of cell aging by transcriptional reprogramming, or alternatively, human embryonic stem cells engineered to escape immune surveillance, are effective platforms for the industrial-scale manufacture of young cells for the treatment of age-related pathologies. Such cell-based regenerative therapies will require newer manufacturing and delivery technologies to insure highly pure, identified and potent pluripotency-based therapeutic formulations.

Methodologies to reprogram somatic cells into patient-specific pluripotent cells, which could potentially be used in personalized drug discovery and cell replacement therapies, are currently under development. Oct4 activation is essential for successful reprogramming and pluripotency of embryonic stem (ES) cells, albeit molecular details of Oct4 activation are not completely understood. Here we report that endogenous SAF-A is involved in regulation of Oct4 expression, binds the Oct4 proximal promoter in ES cells, and dissociates from the promoter upon early differentiation induced by LIF withdrawal. Depletion of SAF-A decreases Oct4 expression even in the presence of LIF, and results in an increase of the mesodermal marker Brachyury. The overexpression of wild-type human SAF-A rescues the mouse knock-down phenotype and results in increased Oct4 level. We also demonstrate that endogenous SAF-A interacts with the C-terminal domain (CTD) of endogenous RNA polymerase II and that the interaction is independent of CTD phosphorylation and mRNA. Moreover, we show that SAF-A exist in complexes with transcription factors Sox2 and Oct4 as well as STAT3 in ES cells. The number of endogenous SAF-A:Oct4 and SAF-A:Sox2 complexes decreases upon LIF depletion. These discoveries allow us to propose a model for activation of Oct4 transcription.

One of the key issues impeding the application of human embryonic stem cells (hESCs) in regenerative medicine is the occurrence of karyotypic instability of hESCs during long term culture. Stem cells grown in culture are exposed to strong selection pressures that often results in genomic alterations, varying in size from point mutations to large chromosomal aberrations, trisomies and monosomies. Different culturing conditions may affect the nature and the frequency of the acquired aberrations; however no culturing technique is immune to genomic instability. The current techniques to evaluate the genomic integrity of stem cells are mostly based on direct cytogenetic and DNA-based analyses; traditional karyotype, spectral karyotyping, array-based molecular analyses of genomic integrity etc. The potential use of aberrant or instable stem cells in cell therapy is obviously hindered as there is increased tumorigenicity risk. It is then important to optimize culturing conditions, cryo-storage and monitoring systems to be applied to the newly derived as well as existing cell lines to decipher any genetic and epigenetic alterations. It was also found that reduced oxygen to physiological levels and additions of antioxidants can be employed as possible strategies to lower oxidative stress and decrease chances of chromosomal transformation. The manual and mechanical dissection are preferentially used during hESCs sub-culturing as being less aggressive and better preserve the genome integrity.

Monoclonal antibodies (mAbs) are used as targeted therapies against cancers. These mAbs kill cancer cells via various mechanisms of actions. In this study, human embryonic stem cells (hESCs) was used as the immunogen to generate a panel of antibodies. From this panel of mAbs, A19 was found to bind both hESC and various cancer cell lines. The antigen target of A19 was identified as Erbb-2 and glycan analysis showed that A19 binds to a N-glycan epitope on the antigen. A19 was elucidated to internalize into cancer cells following binding to Erbb-2 and hence developed as an antibody-drug conjugate (ADC). Using ADC as the mechanism of action, A19 was able to kill cancer cells in vitro and delayed the onset of tumour formation in mice xenograft model. When compared to Herceptin, A19 binds to different isoforms of Erbb-2 and does not compete with Herceptin for the same epitope. Hence, A19 has the potential to be developed as an alternative targeted therapeutic agent for cancers expressing ...

Antibody-mediated cell killing has significantly facilitated the elimination of undesired cells in therapeutic applications. Besides the well-known Fc-dependent mechanisms, pathways of antibody-induced apoptosis were also extensively studied. However, with fewer studies reporting the ability of antibodies to evoke an alternative form of programmed cell death, oncosis, the molecular mechanism of antibody-mediated oncosis remains underinvestigated. In this study, a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC) so as to prevent teratoma formation upon transplantation of hESC-derived products. We revealed that A1 induces hESC death via oncosis. Aided with high-resolution scanning electron microscopy (SEM), we uncovered nanoscale morphological changes in A1-induced hESC oncosis, as well as A1 distribution on hESC surface. A1 induces hESC oncosis via binding-initiated signaling cascade, most likely by ligating receptors on surface microvilli. The ability to evoke excess reactive oxygen species (ROS) production via the Nox2 isoform of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical in the cell death pathway. Excess ROS production occurs downstream of microvilli degradation and homotypic adhesion, but upstream of actin reorganization, plasma membrane damage and mitochondrial membrane permeabilization. To our knowledge, this is the first mechanistic model of mAb-induced oncosis on hESC revealing a previously unrecognized role for NAPDH oxidase-derived ROS in mediating oncotic hESC death. These findings in the cell death pathway may potentially be exploited to improve the efficiency of A1 in eliminating undifferentiated hESC and to provide insights into the study of other mAb-induced cell death.

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature.

An automated vision system, Teratom-Eye, was developed for the identification of three representative tissue types: muscle, gut and neural epithelia which are commonly found in teratomas formed from human embryonic stem cells. Muscle tissue, a common structure was identified with an accuracy of 90.3% with high specificity and sensitivity greater than 90%. Gut epithelia were identified with an accuracy of 87.5% with specificity and sensitivity greater than 80%. Neural epithelia which were the most difficult structures to distinguish gave an accuracy of 47.6%. TeratomEye is therefore useful for the automated identification of differentiated tissues in teratoma sections.

Human embryonic stem cells (hESCs) have the potential to offer a virtually unlimited source of chondrogenic cells for use in cartilage repair and regeneration. We have recently shown that expandable chondrogenic cells can be derived from hESCs under selective growth factor-responsive conditions. In this study, we explore the potential of these hESC-derived chondrogenic cells to produce an extracellular matrix (ECM)-enriched cartilaginous tissue construct when cultured in hyaluronic acid (HA)-based hydrogel, and further investigated the long-term reparative ability of the resulting hESC-derived chondrogenic cell-engineered cartilage (HCCEC) in an osteochondral defect model. We hypothesized that HCCEC can provide a functional template capable of undergoing orderly remodeling during the repair of critical-sized osteochondral defects (1.5 mm in diameter, 1 mm depth into the subchondral bone) in a rat model. In the process of repair, we observed an orderly spatial-temporal remodeling of HCCEC over 12 weeks into osteochondral tissue, with characteristic architectural features including a hyaline-like neocartilage layer with good surface regularity and complete integration with the adjacent host cartilage and a regenerated subchondral bone. By 12 weeks, the HCCEC-regenerated osteochondral tissue resembled closely that of age-matched unoperated native control, while only fibrous tissue filled in the control defects which left empty or treated with hydrogel alone. Here we demonstrate that transplanted hESC-derived chondrogenic cells maintain long-term viability with no evidence of tumorigenicity, providing a safe, highly-efficient and practical strategy of applying hESCs for cartilage tissue engineering.

The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. Methods/Findings: Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. Conclusions/Significance: We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases.

Célunk kiméra-előállítási technikák kidolgozása volt egér, nyúl és szarvasmarha embriókon. Az egér ES-sejtvonalak kiméra alkotó képességét leginkább az euploid sejtek aránya befolyásolta. FISH technikát alkalmazva kimutattuk az X- és Y-kromoszómákat euploid sejtekben, illetve hiányukat az X0 kariotípusú sejtekben. Nyúl 8-sejtes morulák egyetlen sejtjének felhasználásával négy termékeny kiméra utód, két XX/XX nőstény, egy XY/XY és egy XX/XY hím született. Az egyik nőstény és az XY/XY hím germinális kiméra volt. A nyúl ES-like sejt-kolóniákat 6-7 passzázsig fenntartottuk és kidolgoztuk a sejtjeik felhasználásához szükséges kiméra technikát. Sikerült kidolgozni a tetraploid nyúl embrió előállításához szükséges paramétereket. Az A-single spermatogóniumok sejtszuszpenzióban nem azonosíthatók és kis hányadban vannak jelen, így alkalmazásuk kiméra embriók előállítására további kutatást igényel. Hatékony in vitro szarvasmarha-embrió-tenyésztési rendszert dolgoztunk ki és sikerült létrehoznu...

To facilitate understanding of human cardiomyocyte (CM) subtype specification, and the study of ventricular CM biology in particular, we developed a broadly applicable strategy for enrichment of ventricular cardiomyocytes (VCMs) derived from human embryonic stem cells (hESCs). A bacterial artificial chromosome transgenic H9 hESC line in which GFP expression was driven by the human ventricular-specific myosin light chain 2 (MYL2) promoter was generated, and screened to identify cell-surface markers specific for MYL2-GFP-expressing VCMs. A CD77/CD200 cell-surface signature facilitated isolation of >97% cardiac troponin I-positive cells from H9 hESC differentiation cultures, with 65% expressing MYL2-GFP. This study provides a tool for VCM enrichment when using some, but not all, human pluripotent stem cell lines. Tools generated in this study can be utilized toward understanding CM subtype specification, and enriching for VCMs for therapeutic applications.

Since their discovery in the early 1990's, Stem Cell have brought the promise of evolutionary and significant scientific and
medical research with the prospect that possessed the possibility of radically improving treatments for a host of diseases
such as Alzheimer's, Parkinson's disease, various cancers and other diseases that currently render patients and scientists
helpless to combat. With the advent of medical and scientific research, comes the inevitable emergence of the controversy
that has accompanied every major scientific and medical advancement. The use of Stem Cell is no different. Those who
seek to curtail the use of certain stem cell lines, revert to the argument that has defied many medical debates over the
previous decades. The argument, the destruction of human life to create life, is the stalwart philosophical point that all
anti-stem cell advocates attempt to make. The purpose of this discussion is to engage in an analysis of the various aspects
of the ethical debate concerning the use of stem cells in medical research.

Transplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium con...

We have generated mouse null mutants for the cellular retinoic acid (RA) binding protein type I (CRABPI), a protein whose spatio-temporal expression pattern coincides with the target tissues for RA action. Inactivation of the CRABPI gene was accomplished via homologous recombination in embryonic stem cells. Cells carrying the correctly targeted gene were injected into blastocysts and the resulting chimaeras yielded offspring heterozygous for the knockout mutation. Subsequent breeding programs resulted in normal litter sizes containing viable and fertile CRABPI deficient mice. Homozygous mice carrying the knockout mutation were studied in detail to detect possible organ and skeletal anomalies and/or abnormalities of the hematopoietic system. No overt phenotype was evident indicating that a deficiency for CRABPI does not seem to interfere with normal development, growth and reproduction.

Neural cells differentiated in vitro from human embryonic stem cells (hESC) exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. Here, we describe standard conditions for the use and discovery of markers for analysis and cell selection of hESC undergoing neuronal differentiation. To generate betterdefined cell populations, we established a working protocol for sorting heterogeneous hESC-derived neural cell populations by fluorescence-activated cell sorting (FACS). Using genetically labeled synapsin-green fluorescent protein-positive hESC-derived neurons as a proof of principle, we enriched viable differentiated neurons by FACS. Cell sorting methodology using surface markers was developed, and a comprehensive profiling of surface antigens was obtained for immature embryonic stem cell types (such as stage-specific embryonic antigen [SSEA]-3, -4, TRA-1-81, TRA-1-60), neural stem and precursor cells (such as CD133, SSEA-1 [CD15], A2B5, forebrain surface embryonic antigen-1, CD29, CD146, p75 [CD271]), and differentiated neurons (such as CD24 or neural cell adhesion molecule [NCAM; CD56]). At later stages of neural differentiation, NCAM (CD56) was used to isolate hESC-derived neurons by FACS. Such FACS-sorted hESC-derived neurons survived in vivo after transplantation into rodent brain. These results and concepts provide (a) a feasible approach for experimental cell sorting of differentiated neurons, (b) an initial survey of surface antigens present during neural differentiation of hESC, and (c) a framework for developing cell selection strategies for neural cell-based therapies.

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cellbased model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1 -UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state.

Long-range chromosomal interactions bring distal regulatory elements and promoters together to regulate gene expression in biological processes. By performing promoter capture Hi-C (PCHi-C) on human embryonic stem cell-derived cardiomyocytes (hESC-CMs), we show that such promoter interactions are a key mechanism by which enhancers contact their target genes after hESC-CM differentiation from hESCs. We also show that the promoter interactome of hESC-CMs is associated with expression quantitative trait loci (eQTLs) in cardiac left ventricular tissue; captures the dynamic process of genome reorganisation after hESC-CM differentiation; overlaps genome-wide association study (GWAS) regions associated with heart rate; and identifies new candidate genes in such regions. These findings indicate that regulatory elements in hESC-CMs identified by our approach control gene expression involved in ventricular conduction and rhythm of the heart. The study of promoter interactions in other hESC-de...

Basic fibroblast growth factor (FGF2) is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiological and pathological processes, including limb development, angiogenesis, wound healing, and tumor growth, but its physiological role is still unclear. To determine the function of FGF2 in vivo , we have generated FGF2 knockout mice, lacking all three FGF2 isoforms, by homologous recombination in embryonic stem cells. FGF2 −/− mice are viable, fertile and phenotypically indistinguishable from FGF2 +/+ littermates by gross examination. However, abnormalities in the cytoarchitecture of the neocortex, most pronounced in the frontal motor-sensory area, can be detected by histological and immunohistochemical methods. A significant reduction in neuronal density is observed in most layers of the motor cortex in the FGF...

Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.

Pluripotent embryonic stem (ES) cells, a potential source of somatic precursors for cell therapies, cause tumors after transplantation. Studies of mammalian carcinogenesis using nuclear magnetic resonance (NMR) spectroscopy have revealed changes in the choline region, particularly increased phosphocholine (PCho) content. High PCho levels in murine ES (mES) cells have recently been attributed to cell pluripotency. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated in tumor-like properties of mES cells. This study aimed to examine a potential link between the metabolic profile associated with choline metabolism of pluripotent mES cells and PI3K/Akt signaling. We used mES (ES-D3) and murine embryonal carcinoma cells (EC-F9) and compared the metabolic profiles of 1) pluripotent mES (ESD0), 2) differentiated mES (ESD14), and 3) pluripotent F9 cells. Involvement of the PI3K/Akt pathway was assessed using LY294002, a selective PI3K inhibitor. Metabolic profiles were characterized in the extracted polar fraction by 1 H NMR spectroscopy. Similarities were found between the levels of choline phospholipid metabolites (PCho/total choline and PCho/glycerophosphocholine [GPCho]) in ESD0 and F9 cell spectra and a greater-than five-fold decrease of the PCho/GPCho ratio associated with mES cell differentiation. LY294002 caused no significant change in relative PCho levels but led to a greater-than two-fold increase in PCho/GPCho ratios. These results suggest that the PCho/GPCho ratio is a metabolic trait shared by pluripotent and malignant cells and that PI3K does not underlie its development. It is likely that the signature identified here in a mouse model may be relevant for safe therapeutic applications of human ES cells.

Human embryonic (hES) stem cells are widely used as an in vitro model to understand global genetic and epigenetic changes that occur during early embryonic development. In-house derived hES cells (KIND1) were subjected to directed differentiation into cardiovascular progenitors (D12) and beating cardiomyocytes (D20). Transcriptome profiling of undifferentiated (D0) and differentiated (D12 and 20) cells was undertaken by microarray analysis. ChIP and sequential ChIP were employed to study role of transcription factor NR2F2 during hES cells differentiation. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20. This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. ChIP sequencing and sequential Ch...

To gain a better understanding of the pathological role of interferon-y (IFN-y) in specific granuloma formation, IFN-y gene-deficient mice (BALB/c and C57BL/6) were produced. The IFN-y gene in embryonic stem (ES) cells was disrupted by inserting the P-galactosidase gene (IacZ) and the neomycin resistance gene ( m u ) at the translation initiation site in exon 1 by homologous recombination. Six-week-old IFN-y-deficient and wild-type mice were inoculated with lo3 -10' bacilli of various strains of Mycobacteriurn tuberculosis (Kurono, H37Rv, H37Ra and BCG Pasteur) through their tail veins. The mice were examined 7 weeks later for granuloma formation. The avirulent BCG Pasteur and H37Ra strains (lo3-lo4 bacilli/ml) induced granulomas in the spleen, liver and lungs of IFN-y-deficient mice. The granulomas consisted of epithelioid macrophages and Langhans multinucleate giant cells, but lacked caseous necrosis. The virulent Kurono and H37Rv strains induced disseminated abscesses but not granulomas in various organs of IFN-y-deficient mice and Mac-3-positive macrophages were not detected in the abscess lesions. These results suggest that IFN-y may be primarily responsible for macrophage activation and that other factor(s) may be involved in the granuloma formation mechanism.

Cartilage tissue can be engineered by starting from a diversity of cell sources, including stem-cell based and primary cell-based platforms. Selecting an appropriate cell source for the process of cartilage tissue engineering or repair is critical and challenging due to the variety of cell options available. In this study, cellular responses of isolated human chondrocytes, human embryonic stem cells and mesenchymal stem cells (MSCs) derived from three sources, human embryonic stem cells, bone marrow and adipose tissue, were assessed for chondrogenic potential in 3D culture. All cell sources were characterized by FACS analysis to compare expression of some surface markers. The cells were differentiated in two different biomaterial matrices, silk and chitosan scaffolds, in the presence and absence of bone morphogenetic protein 6 (BMP-6) along with the standard chondrogenic differentiating factors. Embryonic stem cells derived MSCs showed unique characteristics with preserved chondrogenic phenotype in both scaffolds with regard to chondrogenesis, as determined by real time RT-PCR, histological and microscopic analyses. After 4 weeks of cultivation, embryonic stem cells derived MSCs were promising for chondrogenesis, particularly in the silk scaffolds with BMP-6. The results suggest that cell source differences are important to consider with regard to chondrogenic outcomes and with the variables addressed here, the human embryonic stem cells derived MSCs were the preferred cell source.

In August 2000, I received a phone call from Michael West, President and Chief Scientific Officer of Advanced Cell Technology (ACT), a small Massachusetts biotech company. West told me that ACT was about to embark on a program of research involving the use of somatic cell nuclear transfer (SCNT), also called therapeutic or research cloning, for the purpose of creating immunologically compatible embryonic stem cell lines. He wanted to know whether I was interested in serving as Chair of an Ethics Advisory Board (EAB) that ACT was setting up to provide guidance for its research. Thus began an adventure that continues to occupy my time seven years later. Like all adventures, it has had its emotional highs and lows: moments of exhilarating accomplishment and others of seeming failure. Among them: the year-long collaborative effort of setting up the world's first SCNT research egg donor program and watching the first eggs arrive for cloning experiments; 1 the November 2001 announcement of the creation of the world's first cloned human embryo; 2 and then days later, a bitter controversy over whether the announcement was premature in terms of its

As regulatory bodies encourage alternatives to animal testing, there is renewed interest in engineering disease models, particularly for cardiac tissues. The aligned organization of cells in the mammalian heart controls the electrical and ionic currents and its ability to efficiently circulate blood to the body. Although the development of engineered cardiac systems is rising, insights into the topographical aspects, in particular, the necessity to design in vitro cardiac models incorporating cues for unidirectional cell growth, is lacking. This review first summarizes the widely used methods to organize cardiomyocytes (CMs) unidirectionally and the ways to quantify the resulting cellular alignment. The behavior of CMs in response to alignment is described, with emphasis on their functions and underlying mechanisms. Lastly, the limitations of state-of-the-art techniques to modulate CM alignment in vitro and opportunities for further development in the future to improve the cardiac tissue models that more faithfully mimic the pathophysiological hallmarks are outlined. This review serves as a call to action for bioengineers to delve deeper into the in vivo role of cellular organization in cardiac muscle tissue and draw inspiration to effectively mimic in vitro for engineering reliable disease models.

The essay questions on the development of the statement of the Court of Justice, according to which: «... Article 6 , paragraph 2, letter c) of Directive 98/44 , must be interpreted as meaning that an unfertilized human egg which through parthenogenesis, has been induced to divide and grow is not a "human embryo" within the meaning of that provision if , in the light of current knowledge of science, it is free, as such , the inherent capacity to develop into a human being…». Consequently, it reflects the possibility that, in future, the uses of such a body for industrial or commercial purposes are, in principle, subject to patent.

Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation, but they often clump in culture, which has always represented a challenge for neurodifferentiation. In this study, we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM) with 10% CO 2 , which doubled the expression of the NESTIN, PAX6 and FOXG1 genes compared with those cultured with 5% CO 2 . Furthermore, an additional step (AdSTEP) was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the "neurosphederm". The large neural tube-type rosette (NTTR) structure formed from the neurosphederm, and the NTTR expressed higher levels of the PAX6, SOX2 and NESTIN genes compared with the neuroectoderm-derived neuroprogenitors. Different layers of cortical, pyramidal, GABAergic, glutamatergic, cholinergic neurons appeared within 27 days using the neurosphederm, which is a shorter period than in traditional neurodifferentiation-protocols (42-60 days). With additional supplements and timeline dopaminergic and Purkinje neurons were also generated in culture too. Furthermore, our in vivo results indicated that the fragmented neurospheres facilitated significantly better neurogenesis in severe combined immunodeficiency (SCID) mouse brains compared with the non-fragmented neurospheres. Therefore, this neurospherebased neurodifferentiation protocol is a valuable tool for studies of neurodifferentiation, neuronal transplantation and high throughput screening assays.

Cigarette smoke is a mixture of over 7000 toxic components with at least 69 smoke chemicals producing cancers, suggesting that smoking is harmful to smokers. More importantly, the World Health Organization has estimated that 10% of the world's population is involuntarily exposed to environmental tobacco smoke, a number that does not include developing embryos. However, tobacco exposure during embryonic development is one of the risk factors for developing a congenital birth defect. An understudied, yet important abnormality caused by maternal smoking is the improper development of the embryonic skeleton, which can result in long-term burdens on the affected child and their family, creating a need for investigation that is addressed in this thesis. Due to the difficulty in quitting smoking while pregnant, women often look to use harm reduction tobacco products (HRTPs) because they are perceived as a safer alternative in comparison to conventional cigarettes. These products include ultra-filtered cigarettes with reduced carcinogen content, and chewing tobacco, which omits exposure to harmful vii combustion products. However, there is a lack of investigation to reveal the molecular mechanisms involved in skeletal embryotoxicity induced by conventional and harmreduction tobacco, the unraveling of which is another objective of this thesis. In the process of this investigation, an in vitro model based on human embryonic stem cells was developed. These cells are the only currently available cell culture system, which mimics the development of the human embryo. As this thesis shows, their differentiation into osteoblasts in vitro can be used to study chemical toxicity and the molecular mechanisms thereof.

The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However, the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis, the noncanonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a two-day window showed significantly enhanced osteogenic yield. Mechanistically, rWNT5a supplementation up-regulated PKC, CamKII and JNK activity while antagonizing the key effector of canonical Wnt signaling: beta-catenin. Conversely, when recombinant WNT3a (rWNT3a) or other positive regulators of catenin were employed during this same time-window there was a decrease in osteogenic marker expression. However, if rWNT3a was supplemented during a time-window following rWNT5a treatment, osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine.

Objective Sexual dimorphism in mammals can be described as subsequent transcriptional differences from their distinct sex chromosome complements. Following X inactivation in females, the Y chromosome is the major genetic difference between sexes. In this study, we used a male embryonic stem cell line (Royan H6) to identify the potential role of the male-specific region of the Y chromosome (MSY) during spontaneous differentiation into embryoid bodies (EBs) as a model of early embryonic development. Materials and Methods In this experimental study, RH6 cells were cultured on inactivated feeder layers and Matrigel. In a dynamic suspension system, aggregates were generated in the same size and were spontaneously differentiated into EBs. During differentiation, expression patterns of specific markers for three germ layers were compared with MSY genes. Results Spontaneous differentiation was determined by downregulation of pluripotent markers and upregulation of fourteen differentiation m...

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.

Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene (neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1 +/- mice produced no Pcsk1 -/-offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1 +/-. These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT-PCR analysis on RNA from endocrine tissues from Pcsk1 +/-mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1 +/-and Pcsk1 +/+ mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele.

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.

Embryogenesis, the process of human development from fertilization to
birth, involves several stages such as the formation of bones, muscles, and facial features, which modern science has detailed. Remarkably, the Quran described many of these stages over 1,400 years ago. The Quran accurately identifies the male sperm as determining the baby’s sex, describes the development of the embryo in stages, and mentions the fetus being protected in “a triple darkness,” referring to the layers surrounding it. The Quran also touches on the changes in the womb lining, the protective amniotic fluid, and the formation of bones and muscles, all of which align with modern scientific findings. This shows a striking harmony between ancient scripture and contemporary embryology.

Background: Next Generation Sequencing has proven to be an exceptionally powerful tool in the field of genomics and transcriptomics. With recent development it is nowadays possible to analyze ultra-low input sample material down to single cells. Nevertheless, investigating such sample material often limits the analysis to either the genome or transcriptome. We describe here a combined analysis of both types of nucleic acids from the same sample material. Methods: The method described enables the combined preparation of amplified cDNA as well as amplified wholegenome DNA from an ultra-low input sample material derived from a sub-colony of in-vitro cultivated human embryonic stem cells. cDNA is prepared by the application of oligo-dT coupled magnetic beads for mRNA capture, first strand synthesis and 3'-tailing followed by PCR. Whole-genome amplified DNA is prepared by Phi29 mediated amplification. Illumina sequencing is applied to short fragment libraries prepared from the amplified samples. Results: We developed a protocol which enables the combined analysis of the genome as well as the transcriptome by Next Generation Sequencing from ultra-low input samples. The protocol was evaluated by sequencing sub-colony structures from human embryonic stem cells containing 150 to 200 cells. The method can be adapted to any available sequencing system. Conclusions: To our knowledge, this is the first report where sub-colonies of human embryonic stem cells have been analyzed both at the genomic as well as transcriptome level. The method of this proof of concept study may find useful practical applications for cases where only a limited number of cells are available, e.g. for tissues samples from biopsies, tumor spheres, circulating tumor cells and cells from early embryonic development. The results we present demonstrate that a combined analysis of genomic DNA and messenger RNA from ultra-low input samples is feasible and can readily be applied to other cellular systems with limited material available.

Embryonic stem (ES) cells have generated enormous interest due to their capacity to self-renew and the potential for growing many different cell types in vitro. Leukemia inhibitory factor (LIF), bone morphogenetic proteins, octamer-binding protein 3 or 4, and Nanog are important factors in the maintenance of pluripotency in mouse ES cells. However, the mechanisms by which these factors regulate the pluripotency remain poorly understood. To identify other proteins involved in this process, we did a proteomic analysis of mouse ES cells that were cultured in the presence or absence of LIF. More than 100 proteins were found to be involved specifically in either the differentiation process or the maintenance of undifferentiated state. Among these, chromatin-related proteins were identified as the major proteins in nuclear extracts of undifferentiated cells. Analysis with real-time RT-PCR revealed that enrichment of these proteins in pluripotent ES cells was regulated at the transcriptional levels. These results suggest that specific chromatin-related proteins may be involved in maintaining the unique properties of pluripotent ES cells.