Targeting RNA helicase DDX3 in stem cell maintenance and teratoma formation.

DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. Besides the role of DDX3 in transformed cells, there is evidence to indicate that DDX3 expression is at its highest levels during early embryonic development and is also expressed in germ cells of adults. Even though there is a distinct pattern of DDX3 expression during embryonic development and in adults, very little is known regarding its role in embryonic stem cells and pluripotency. In this work, we examined the relationship between DDX3 and human embryonic stem cells and its differentiated lineages. DDX3 expression was analyzed by immunohistochemistry in human embryonic stem cells and embryonal carcinoma cells. From the data obtained, it was evident that DDX3 was overexpressed in undifferentiated stem cells compared to differentiated cells. Moreover, when DDX3 expression was abrogated in multiple stem cells, proliferation was decreased, but differentiation was facilitated. Importantly, this resulted in reduced potency to induce teratoma formation. Taken together, these findings indicate a distinct role for DDX3 in stem cell maintenance.

Primordial germ cells as a potential shared cell of origin for mucinous cystic neoplasms of the pancreas and mucinous ovarian tumors.

Mucinous ovarian tumors (MOTs) morphologically and epidemiologically resemble mucinous cystic neoplasms (MCNs) of the pancreas, sharing a similar stroma and both occurring disproportionately among young females. Additionally, MOTs and MCNs share similar clinical characteristics and immunohistochemical phenotypes. Exome sequencing has revealed frequent recurrent mutations in KRAS and RNF43 in both MOTs and MCNs. The cell of origin for these tumors remains unclear, but MOTs sometimes arise in the context of mature cystic teratomas and other primordial germ cell (PGC) tumors. We undertook the present study to investigate whether non-teratoma-associated MOTs and MCNs share a common cell of origin. Comparisons of the gene expression profiles of MOTs [including both the mucinous borderline ovarian tumors (MBOTs) and invasive mucinous ovarian carcinomas (MOCs)], high-grade serous ovarian carcinomas, ovarian surface epithelium, Fallopian tube epithelium, normal pancreatic tissue, pancreatic duct adenocarcinomas, MCNs, and single-cell RNA-sequencing of PGCs revealed that both MOTs and MCNs are more closely related to PGCs than to either eutopic epithelial tumors or normal epithelia. We hypothesize that MCNs may arise from PGCs that stopped in the dorsal pancreas during their descent to the gonads during early human embryogenesis, while MOTs arise from PGCs in the ovary. Together, these data suggest a common pathway for the development of MCNs and MOTs, and suggest that these tumors may be more properly classified as germ cell tumor variants. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Direct Reprogramming of Human Primordial Germ Cells into Induced Pluripotent Stem Cells: Efficient Generation of Genetically Engineered Germ Cells.

Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SO-derived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.

Prolonged Local Hypothermia Has No Long-Term Adverse Effect on the Spinal Cord.

Hypothermia is known to be neuroprotective and is one of the most effective and promising first-line treatments for central nervous system (CNS) trauma. At present, induction of local hypothermia, as opposed to general hypothermia, is more desired because of its ease of application and safety; fewer side effects and an absence of severe complications have been noted. Local hypothermia involves temperature reduction of a small and specific segment of the spinal cord. Our group has previously shown the neuroprotective effect of short-term, acute moderate general hypothermia through improvements in electrophysiological and motor behavioral assessments, as well as histological examination following contusive spinal cord injury (SCI) in rats. We have also shown the benefit of using short-term local hypothermia versus short-term general hypothermia post-acute SCI. The overall neuroprotective benefit of hypothermia can be categorized into three main components: (1) induction modality, general versus local, (2) invasive, semi-invasive or noninvasive, and (3) duration of hypothermia induction. In this study, a series of experiments were designed to investigate the feasibility, long-term safety, as well as eventual complications and side effects of prolonged, semi-invasive, moderate local hypothermia (30°C±0.5°C for 5 and 8 hours) in rats with uninjured spinal cord while maintaining their core temperature at 37°C±0.5°C. The weekly somatosensory evoked potential and motor behavioral (Basso, Beattie and Bresnahan) assessments of rats that underwent 5 and 8 hours of semi-invasive local hypothermia, which revealed no statistically significant changes in electrical conductivity and behavioral outcomes. In addition, 4 weeks after local hypothermia induction, histological examination showed no anatomical damages or morphological changes in their spinal cord structure and parenchyma. We concluded that this method of prolonged local hypothermia is feasible, safe, and has the potential for clinical translation.

Early intervention for spinal cord injury with human induced pluripotent stem cells oligodendrocyte progenitors.

Induced pluripotent stem (iPS) cells are at the forefront of research in regenerative medicine and are envisaged as a source for personalized tissue repair and cell replacement therapy. Here, we demonstrate for the first time that oligodendrocyte progenitors (OPs) can be derived from iPS cells generated using either an episomal, non-integrating plasmid approach or standard integrating retroviruses that survive and differentiate into mature oligodendrocytes after early transplantation into the injured spinal cord. The efficiency of OP differentiation in all 3 lines tested ranged from 40% to 60% of total cells, comparable to those derived from human embryonic stem cells. iPS cell lines derived using episomal vectors or retroviruses generated a similar number of early neural progenitors and glial progenitors while the episomal plasmid-derived iPS line generated more OPs expressing late markers O1 and RIP. Moreover, we discovered that iPS-derived OPs (iPS-OPs) engrafted 24 hours following a moderate contusive spinal cord injury (SCI) in rats survived for approximately two months and that more than 70% of the transplanted cells differentiated into mature oligodendrocytes that expressed myelin associated proteins. Transplanted OPs resulted in a significant increase in the number of myelinated axons in animals that received a transplantation 24 h after injury. In addition, nearly a 5-fold reduction in cavity size and reduced glial scarring was seen in iPS-treated groups compared to the control group, which was injected with heat-killed iPS-OPs. Although further investigation is needed to understand the mechanisms involved, these results provide evidence that patient-specific, iPS-derived OPs can survive for three months and improve behavioral assessment (BBB) after acute transplantation into SCI. This is significant as determining the time in which stem cells are injected after SCI may influence their survival and differentiation capacity.

The effects of local and general hypothermia on temperature profiles of the central nervous system following spinal cord injury in rats.

Local and general hypothermia are used to treat spinal cord injury (SCI), as well as other neurological traumas. While hypothermia is known to provide significant therapeutic benefits due to its neuroprotective nature, it is unclear how the treatment may affect healthy tissues or whether it may cause undesired temperature changes in areas of the body that are not the targets of treatment. We performed 2-hour moderate general hypothermia (32°C core) or local hypothermia (30°C spinal cord) on rats that had received either a moderate contusive SCI or laminectomy (control) while monitoring temperatures at three sites: the core, spinal cord, and cortex. First, we identified that injured rats that received general hypothermia exhibited larger temperature drops at the spinal cord (-3.65°C, 95% confidence intervals [CIs] -3.72, -3.58) and cortex (-3.64°C, CIs -3.73, -3.55) than uninjured rats (spinal cord: -3.17°C, CIs -3.24, -3.10; cortex: -3.26°C, CIs -3.34, -3.17). This was found due to elevated baseline temperatures in the injured group, which could be due to inflammation. Second, both general hypothermia and local hypothermia caused a significant reduction in the cortical temperature (-3.64°C and -1.18°C, respectively), although local hypothermia caused a significantly lower drop in cortical temperature than general hypothermia (p<0.001). Lastly, the rates of rewarming of the cord were not significantly different among the methods or injury groups that were tested; the mean rate of rewarming was 0.13±0.1°C/min. In conclusion, local hypothermia may be more suitable for longer durations of hypothermia treatment for SCI to reduce temperature changes in healthy tissues, including the cortex.

Regulators of prostate cancer stem cells.

PURPOSE OF REVIEW: Significant advances have been made toward identifying prostate cancer stem cells (CSCs). This review will highlight the latest developments in defining this population and the discovery of mechanisms involved in their survival and metastasis. RECENT FINDINGS: Several groups have identified master regulators of stem cells in prostate cancer. These include genetic and epigenetic factors known to control pluripotency in embryonic stem cells and in highly metastatic prostate tumors. For instance, tumors of patients with poor prognosis demonstrate elevated levels of the pluripotent markers OCT4 and SOX2 as well as the polycomb complex protein Bmi-1 and enhancer of zeste homolog 2. Cells that are derived from these patient tumors provide an opportunity to expand our current knowledge regarding how these cells survive and the mechanisms that regulate their proliferation. SUMMARY: Understanding the mechanisms of highly invasive and therapy resistant prostate cancer cells resides in understanding the CSCs, which facilitate cancer recurrence. Some of these factors are just emerging and provide a platform for developing targeted drugs for the future treatment of advanced prostate cancer.

Genome-wide profiling of pluripotent cells reveals a unique molecular signature of human embryonic germ cells.

Human embryonic germ cells (EGCs) provide a powerful model for identifying molecules involved in the pluripotent state when compared to their progenitors, primordial germ cells (PGCs), and other pluripotent stem cells. Microarray and Principal Component Analysis (PCA) reveals for the first time that human EGCs possess a transcription profile distinct from PGCs and other pluripotent stem cells. Validation with qRT-PCR confirms that human EGCs and PGCs express many pluripotency-associated genes but with quantifiable differences compared to pluripotent embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and embryonal carcinoma cells (ECCs). Analyses also identified a number of target genes that may be potentially associated with their unique pluripotent states. These include IPO7, MED7, RBM26, HSPD1, and KRAS which were upregulated in EGCs along with other pluripotent stem cells when compared to PGCs. Other potential target genes were also found which may contribute toward a primed ESC-like state. These genes were exclusively up-regulated in ESCs, IPSCs and ECCs including PARP1, CCNE1, CDK6, AURKA, MAD2L1, CCNG1, and CCNB1 which are involved in cell cycle regulation, cellular metabolism and DNA repair and replication. Gene classification analysis also confirmed that the distinguishing feature of EGCs compared to ESCs, ECCs, and IPSCs lies primarily in their genetic contribution to cellular metabolism, cell cycle, and cell adhesion. In contrast, several genes were found upregulated in PGCs which may help distinguish their unipotent state including HBA1, DMRT1, SPANXA1, and EHD2. Together, these findings provide the first glimpse into a unique genomic signature of human germ cells and pluripotent stem cells and provide genes potentially involved in defining different states of germ-line pluripotency.

Electrophysiological evaluation of sensory and motor pathways after incomplete unilateral spinal cord contusion.

OBJECT: Unilateral contusions represent an increasingly popular model for studying the pathways and recovery mechanisms of spinal cord injury (SCI). Current studies rely heavily on motor behavior scoring and histological evidence to make assessments. Electrophysiology represents one way to reliably quantify the functionality of motor pathways. The authors sought to quantify the functional integrity of the bilateral motor and sensory pathways following unilateral SCI by using measurements of motor and somatosensory evoked potentials (MEPs and SSEPs, respectively). METHODS: Eighteen rats were randomly divided into 3 groups receiving a mild unilateral contusion, a mild midline contusion, or a laminectomy only (control). Contusions were induced at T-8 using a MASCIS impactor. Electrophysiological analysis, motor behavior scoring, and histological quantifications were then performed to identify relationships among pathway conductivity, motor function, and tissue preservation. RESULTS: Hindlimb MEPs ipsilateral to the injury showed recovery by Day 28 after injury and corresponded to approximately 61% of spared corticospinal tract (CST) tissue. In contrast, MEPs of the midline-injured group did not recover, and correspondingly > 90% of the CST tissue was damaged. Somatosensory evoked potentials showed only a moderate reduction in amplitude, with no difference in latency for the pathways ipsilateral to injury. Furthermore, these SSEPs were significantly better than those of the midline-injured rats for the same amount of white matter damage. CONCLUSIONS: Motor evoked potential recovery corresponded to the amount of spared CST in unilateral and midline injuries, but motor behavior consistently recovered independent of MEPs. These data support the idea that spared contralateral pathways aid in reducing the functional deficits of injured ipsilateral pathways and further support the idea of CNS plasticity.

Potential long-term benefits of acute hypothermia after spinal cord injury: assessments with somatosensory-evoked potentials.

OBJECTIVE: Neuroprotection by hypothermia has been an important research topic over last two decades. In animal models of spinal cord injury, the primary focus has been assessing the effects of hypothermia on behavioral and histologic outcomes. Although a few studies have investigated electrophysiological changes in descending motor pathways with motor-evoked potentials recorded during cooling, we report here hypothermia induced increased electrical conduction in the ascending spinal cord pathways with somatosensory-evoked potentials in injured rats. In our experiments, these effects lasted long after the acute hypothermia and were accompanied by potential long-term improvements in motor movement. DESIGN: Laboratory investigation. SETTING: University medical school. SUBJECTS: Twenty-one female Lewis rats. INTERVENTIONS: Hypothermia. MEASUREMENTS AND MAIN RESULTS: All animals underwent spinal cord contusion with the NYU-Impactor by a 12.5-mm weight drop at thoracic vertebra T8. A group (n = 10) was randomly assigned for a systemic 2-hr hypothermia episode (32 ± 0.5°C) initiated approximately 2.0 hrs postinjury. Eleven rats were controls with postinjury temperature maintained at 37 ± 0.5°C for 2 hrs. The two groups underwent preinjury, weekly postinjury (up to 4 wks) somatosensory-evoked potential recordings and standard motor behavioral tests (BBB). Three randomly selected rats from each group were euthanized for histologic analysis at postinjury day 3 and day 28. Compared with controls, the hypothermia group showed significantly higher postinjury somatosensory-evoked potential amplitudes with longer latencies. The BBB scores were also higher immediately after injury and 4 wks later in the hypothermia group. Importantly, specific changes in the Basso, Beattie, Bresnahan scores in the hypothermia group (not seen in controls) indicated regained functions critical for motor control. Histologic evaluations showed more tissue preservation in the hypothermia group. CONCLUSIONS: After spinal cord injury, early systemic hypothermia provided significant neuroprotection weeks after injury through improved sensory electrophysiological signals in rats. This was accompanied by higher motor behavioral scores and more spared tissue in acute and postacute periods after injury.

Quantitative temporal proteomic analysis of human embryonic stem cell differentiation into oligodendrocyte progenitor cells.

Oligodendrocytes (OLs) are glial cells of the central nervous system, which produce myelin. Cultured OLs provide immense therapeutic opportunities for treating a variety of neurological conditions. One of the most promising sources for such therapies is human embryonic stem cells (ESCs) as well as providing a model to study human OL development. For these purposes, an investigation of proteome level changes is critical for understanding the process of OL differentiation. In this report, an iTRAQ-based quantitative proteomic approach was used to study multiple steps during OL differentiation including neural progenitor cells, glial progenitor cells and oligodendrocyte progenitor cells (OPCs) compared to undifferentiated ESCs. Using a 1% false discovery rate cutoff, ∼3145 proteins were quantitated and several demonstrated progressive stage-specific expression. Proteins such as transferrin, neural cell adhesion molecule 1, apolipoprotein E and wingless-related MMTV integration site 5A showed increased expression from the neural progenitor cell to the OPC stage. Several proteins that have demonstrated evidence or been suspected in OL maturation were also found upregulated in OPCs including fatty acid-binding protein 4, THBS1, bone morphogenetic protein 1, CRYAB, transferrin, tenascin C, COL3A1, TGFBI and EPB41L3. Thus, by providing the first extensive proteomic profiling of human ESC differentiation into OPCs, this study provides many novel proteins that are potentially involved in OL development.

Bone morphogenetic protein 4 mediates human embryonic germ cell derivation.

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and/or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)-polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency.

MicroRNA expression profiling of oligodendrocyte differentiation from human embryonic stem cells.

BACKGROUND: Cells of the oligodendrocyte (OL) lineage play a vital role in the production and maintenance of myelin, a multilamellar membrane which allows for saltatory conduction along axons. These cells may provide immense therapeutic potential for lost sensory and motor function in demyelinating conditions, such as spinal cord injury, multiple sclerosis, and transverse myelitis. However, the molecular mechanisms controlling OL differentiation are largely unknown. MicroRNAs (miRNAs) are considered the "micromanagers" of gene expression with suggestive roles in cellular differentiation and maintenance. Although unique patterns of miRNA expression in various cell lineages have been characterized, this is the first report documenting their expression during oligodendrocyte maturation from human embryonic stem (hES) cells. Here, we performed a global miRNA analysis to reveal and identify characteristic patterns in the multiple stages leading to OL maturation from hES cells including those targeting factors involved in myelin production. METHODOLOGY/PRINCIPAL FINDINGS: We isolated cells from 8 stages of OL differentiation. Total RNA was subjected to miRNA profiling and validations preformed using real-time qRT-PCR. A comparison of miRNAs from our cultured OLs and OL progenitors showed significant similarities with published results from equivalent cells found in the rat and mouse central nervous system. Principal component analysis revealed four main clusters of miRNA expression corresponding to early, mid, and late progenitors, and mature OLs. These results were supported by correlation analyses between adjacent stages. Interestingly, the highest differentially-expressed miRNAs demonstrated a similar pattern of expression throughout all stages of differentiation, suggesting that they potentially regulate a common target or set of targets in this process. The predicted targets of these miRNAs include those with known or suspected roles in oligodendrocyte development and myelination including C11Orf9, CLDN11, MYTL1, MBOP, MPZL2, and DDR1. CONCLUSIONS/SIGNIFICANCE: We demonstrate miRNA profiles during distinct stages in oligodendroglial differentiation that may provide key markers of OL maturation. Our results reveal pronounced trends in miRNA expression and their potential mRNA target interactions that could provide valuable insight into the molecular mechanisms of differentiation.

Efficient differentiation of human embryonic stem cells into oligodendrocyte progenitors for application in a rat contusion model of spinal cord injury.

This study utilized a contusion model of spinal cord injury (SCI) in rats using the standardized NYU-MASCIS impactor, after which oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem cell (ESC) were transplanted into the spinal cord to study their survival and migration route toward the areas of injury. One critical aspect of successful cell-based SCI therapy is the time of injection following injury. OPCs were injected at two clinically relevant times when most damage occurs to the surrounding tissue, 3 and 24 hours following injury. Migration and survivability after eight days was measured postmortem. In-vitro immunofluorescence revealed that most ESC-derived OPCs expressed oligodendrocyte markers, including CNPase, GalC, Olig1, O4, and O1. Results showed that OPCs survived when injected at the center of injury and migrated away from the injection sites after one week. Histological sections revealed integration of ESC-derived OPCs into the spinal cord with contusion injury without disruption to the parenchyma. Cells survived for a minimum of eight days after injury, without tumor or cyst formation. The extent of injury and effect of early cell transplant was measured using behavioral and electrophysiological assessments which demonstrated increased neurological responses in rats transplanted with OPCs compared to controls.

Comparative proteomics of human embryonic stem cells and embryonal carcinoma cells.

Pluripotent human embryonic stem cells (ESCs) can be differentiated in vitro into a variety of cells which hold promise for transplantation therapy. Human embryonal carcinoma cells (ECCs), stem cells of human teratocarcinomas, are considered a close but malignant counterpart to human ESCs. In this study, a comprehensive quantitative proteomic analysis of ESCs and ECCs was carried out using the iTRAQ method. Using two-dimensional LC and MS/MS analyses, we identified and quantitated approximately 1800 proteins. Among these are proteins associated with pluripotency and development as well as tight junction signaling and TGFbeta receptor pathway. Nearly approximately 200 proteins exhibit more than twofold difference in abundance between ESCs and ECCs. Examples of early developmental markers high in ESCs include beta-galactoside-binding lectin, undifferentiated embryonic cell transcription factor-1, DNA cytosine methyltransferase 3beta isoform-B, melanoma antigen family-A4, and interferon-induced transmembrane protein-1. In contrast, CD99-antigen (CD99), growth differentiation factor-3, cellular retinoic acid binding protein-2, and developmental pluripotency associated-4 were among the highly expressed proteins in ECCs. Several proteins that were highly expressed in ECCs such as heat shock 27 kDa protein-1, mitogen-activated protein kinase kinase-1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor like-2, and S100 calcium-binding protein-A4 have also been attributed to malignancy in other systems. Importantly, immunocytochemistry was used to validate the proteomic analyses for a subset of the proteins. In summary, this is the first large-scale quantitative proteomic study of human ESCs and ECCs, which provides critical information about the regulators of these two closely related, but developmentally distinct, stem cells.

The dazzle in germ cell differentiation.

Embryonic stem cells have demonstrated the capacity to differentiate into germ cells in vitro. Until recently, the molecular basis of early post-meiotic germ cell development was largely unknown. Now, two reports including one published here recently, have demonstrated the significant contribution of Dazl in the differentiation of embryonic stem cells into pre- and post-meiotic germ cells. Although factors that Dazl influences during this process have been identified, the underlying mechanisms warrant future studies.

Temporal analysis of neural differentiation using quantitative proteomics.

The ability to derive neural progenitors, differentiated neurons and glial cells from human embryonic stem cells (hESCs) with high efficiency holds promise for a number of clinical applications. However, investigating the temporal events is crucial for defining the underlying mechanisms that drive this process of differentiation along different lineages. We carried out quantitative proteomic profiling using a multiplexed approach capable of analyzing eight different samples simultaneously to monitor the temporal dynamics of protein abundance as human embryonic stem cells differentiate into motor neurons or astrocytes. With this approach, a catalog of approximately 1200 proteins along with their relative quantitative expression patterns was generated. The differential expression of the large majority of these proteins has not previously been reported or studied in the context of neural differentiation. As expected, two of the widely used markers of pluripotency, alkaline phosphatase (ALPL) and LIN28, were found to be downregulated during differentiation, while S-100 and tenascin C were upregulated in astrocytes. Neurofilament 3 protein, doublecortin and CAM kinase-like 1 and nestin proteins were upregulated during motor neuron differentiation. We identified a number of proteins whose expression was largely confined to specific cell types, embryonic stem cells, embryoid bodies and differentiating motor neurons. For example, glycogen phosphorylase (PYGL) and fatty acid binding protein 5 (FABP5) were enriched in ESCs, while beta spectrin (SPTBN5) was highly expressed in embryoid bodies. Karyopherin, heat shock 27 kDa protein 1 and cellular retinoic acid binding protein 2 (CRABP2) were upregulated in differentiating motor neurons but were downregulated in mature motor neurons. We validated some of the novel markers of the differentiation process using immunoblotting and immunocytochemical labeling. To our knowledge, this is the first large-scale temporal proteomic profiling of human stem cell differentiation into neural cell types highlighting proteins with limited or undefined roles in neural fate.

Current protocols in the generation of pluripotent stem cells: theoretical, methodological and clinical considerations.

Pluripotent stem cells have been derived from various embryonic, fetal and adult sources. Embryonic stem cells (ESCs) and parthenogenic ESCs (pESCs) are derived from the embryo proper while embryonic germ cells (EGCs), embryonal carcinoma cells (ECCs), and germ-line stem cells (GSC) are produced from germ cells. ECCs were the first pluripotent stem cell lines established from adult testicular tumors while EGCs are generated in vitro from primordial germ cells (PGCs) isolated in late embryonic development. More recently, studies have also demonstrated the ability to produce GSCs from adult germ cells, known as spermatogonial stem cells. Unlike ECCs, the source of GSCs are normal, non-cancerous adult tissue. The study of these unique cell lines has provided information that has led to the ability to reprogram somatic cells into an ESC-like state. These cells, called induced pluripotent stem cells (iPSCs), have been derived from a number of human fetal and adult origins. With the promises pluripotent stem cells bring to cell-based therapies there remain several considerations that need to be carefully studied prior to their clinical use. Many of these issues involve understanding key factors regulating their generation, including those which define pluripotency. In this regard, the following article discusses critical aspects of pluripotent stem cell derivation and current issues about their therapeutic potential.