ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

The consequences of recurrent genetic and epigenetic variants in human pluripotent stem cells.

It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.

Diminished AHR Signaling Drives Human Acute Myeloid Leukemia Stem Cell Maintenance.

Eliminating leukemic stem cells (LSC) is a sought after therapeutic paradigm for the treatment of acute myeloid leukemia (AML). While repression of aryl hydrocarbon receptor (AHR) signaling has been shown to promote short-term maintenance of primitive AML cells in culture, no work to date has examined whether altered AHR signaling plays a pathologic role in human AML or whether it contributes at all to endogenous LSC function. Here, we show AHR signaling is repressed in human AML blasts and preferentially downregulated in LSC-enriched populations within leukemias. A core set of AHR targets are uniquely repressed in LSCs across diverse genetic AML subtypes. In vitro and in vivo administration of the specific AHR agonist FICZ significantly impaired leukemic growth, promoted differentiation, and repressed self-renewal. Furthermore, LSCs suppressed a set of FICZ-responsive AHR target genes that function as tumor suppressors and promoters of differentiation. FICZ stimulation did not impair normal hematopoietic stem and progenitor (HSPC) function, and failed to upregulate a prominent LSC-specific AHR target in HSPCs, suggesting that differential mechanisms govern FICZ-induced AHR signaling manifestations in HSCs versus LSCs. Altogether, this work highlights AHR signaling suppression as a key LSC-regulating control mechanism and provides proof of concept in a preclinical model that FICZ-mediated AHR pathway activation enacts unique transcriptional programs in AML that identify it as a novel chemotherapeutic approach to selectively target human LSCs. SIGNIFICANCE: The AHR pathway is suppressed in leukemic stem cells (LSC), therefore activating AHR signaling is a potential therapeutic option to target LSCs and to treat acute myeloid leukemia.

PLAG1 and USF2 Co-regulate Expression of Musashi-2 in Human Hematopoietic Stem and Progenitor Cells.

MSI2, which is expressed predominantly in hematopoietic stem and progenitor cells (HSPCs), enforces HSPC expansion when overexpressed and is upregulated in myeloid leukemias, indicating its regulated transcription is critical to balanced self-renewal and leukemia restraint. Despite this, little is understood of the factors that enforce appropriate physiological levels of MSI2 in the blood system. Here, we define a promoter region that reports on endogenous expression of MSI2 and identify USF2 and PLAG1 as transcription factors whose promoter binding drives reporter activity. We show that these factors co-regulate, and are required for, efficient transactivation of endogenous MSI2. Coincident overexpression of USF2 and PLAG1 in primitive cord blood cells enhanced MSI2 transcription and yielded cellular phenotypes, including expansion of CD34+ cells in vitro, consistent with that achieved by direct MSI2 overexpression. Global chromatin immunoprecipitation sequencing analyses confirm a preferential co-binding of PLAG1 and USF2 at the promoter of MSI2, as well as regulatory regions corresponding to genes with roles in HSPC homeostasis. PLAG1 and USF2 cooperation is thus an important contributor to stem cell-specific expression of MSI2 and HSPC-specific transcriptional circuitry.

Stem Cells in Pulmonary Disease and Regeneration.

The epithelial cells lining the mammalian lung are subjected to constant interaction with the external environment, necessitating robust regeneration strategies to deal with cell loss due to natural turnover or damage arising from inhaled agents or disease. Since lung epithelial function extends beyond respiratory gas exchange to include roles such as immune defense and mucociliary clearance, a diverse complement of epithelial cell types exists that are regionally distributed along the respiratory tree and extensive surface area of the alveolar interface. Although steady-state turnover of the epithelium appears to be relatively low in ideal situations, the vital role of the lung requires stem and progenitor cell populations that can promptly respond to the loss or damage of epithelial tissues. The identity and role of stem cell populations that carry out repair and replacement in the lung has begun to be clarified in recent years, led by cell lineage tracking experiments in the mouse lung, which have revealed a complex interplay of differentiation, transdifferentiation, and dedifferentiation between lung stem cells and functional respiratory cell populations. In this review article, we present the current understanding of the stem cell populations within the pulmonary epithelium and describe ongoing efforts to use these stem cell populations to generate models for exploring lung function and disease.

Functional Enterospheres Derived In Vitro from Human Pluripotent Stem Cells.

Intestinal organoids derived from human pluripotent stem cells (hPSCs) are valuable in vitro research models that enable simplified access to human gastrointestinal tissues. Here, we report the in vitro generation of enterospheres (hEnS) from hPSC-derived gastrointestinal epithelial precursors. hEnS are cystic spheroids with a simple uniform structure composed entirely of intestinal epithelium. hEnS express markers of mature brush border cells and share a transcriptome profile similar to that of more mature intestinal organoids. Modulation of signaling cues enables control of hEnS growth and differentiation, including long-term propagation. We show that hEnS can be exploited for functional studies: hEnS display an innate immune response when treated with enteric pathogens, and transgenic modification of hEnS with a fluorescence cell-cycle reporter enables hEnS-forming stem cell enrichment. Our work establishes hEnS as an accessible and tractable in vitro modeling system for studying human gastrointestinal biology.

Organoids as a model system for studying human lung development and disease.

The lung is a complex organ comprising multiple cell types that perform a variety of vital processes, including immune defense and gas exchange. Diseases of the lung, such as chronic obstructive pulmonary disease, asthma and lung cancer, together represent one of the largest causes of patient suffering and mortality. Logistical barriers that hamper access to embryonic, normal adult or diseased lung tissue currently hinder the study of lung disease. In vitro lung modeling represents an attractive and accessible avenue for investigating lung development, function and disease pathology, but accurately modeling the lung in vitro requires a system that recapitulates the structural features of the native lung. Organoids are stem cell-derived three-dimensional structures that are supported by an extracellular matrix and contain multiple cell types whose spatial arrangement and interactions mimic those of the native organ. Recently, organoids representative of the respiratory system have been generated from adult lung stem cells and human pluripotent stem cells. Ongoing studies are showing that organoids may be used to model human lung development, and can serve as a platform for interrogating the function of lung-related genes and signalling pathways. In a therapeutic context, organoids may be used for modeling lung diseases, and as a platform for screening for drugs that alleviate respiratory disease. Here, we summarize the organoid-forming capacity of respiratory cells, current lung organoid technologies and their potential use in future therapeutic applications.

Treatment-induced cell cycle kinetics dictate tumor response to chemotherapy.

Chemotherapy fails to provide durable cure for the majority of cancer patients. To identify mechanisms associated with chemotherapy resistance, we identified genes differentially expressed before and after chemotherapeutic treatment of breast cancer patients. Treatment response resulted in either increased or decreased cell cycle gene expression. Tumors in which cell cycle gene expression was increased by chemotherapy were likely to be chemotherapy sensitive, whereas tumors in which cell cycle gene transcripts were decreased by chemotherapy were resistant to these agents. A gene expression signature that predicted these changes proved to be a robust and novel index that predicted the response of patients with breast, ovarian, and colon tumors to chemotherapy. Investigations in tumor cell lines supported these findings, and linked treatment induced cell cycle changes with p53 signaling and G1/G0 arrest. Hence, chemotherapy resistance, which can be predicted based on dynamics in cell cycle gene expression, is associated with TP53 integrity.

Demarcation of stable subpopulations within the pluripotent hESC compartment.

Heterogeneity is a feature of stem cell populations, resulting from innate cellular hierarchies that govern differentiation capability. How heterogeneity impacts human pluripotent stem cell populations is directly relevant to their efficacious use in regenerative medicine applications. The control of pluripotency is asserted by a core transcription factor network, of which Oct4 is a necessary member. In mouse embryonic stem cells (ESCs), the zinc finger transcription factor Rex1 (Zfp42) closely tracks the undifferentiated state and is capable of segregating Oct4 positive mESCs into metastable populations expressing or lacking Rex1 that are inter-convertible. However, little is currently understood about the extent or function of heterogeneous populations in the human pluripotent compartment. Human ESCs express REX1 transcripts but the distribution and properties of REX1 expressing cells have yet to be described. To address these questions, we used gene targeting in human ESCs to insert the fluorescent protein Venus and an antibiotic selection marker under the control of the endogenous REX1 transcription regulatory elements, generating a sensitive, selectable reporter of pluripotency. REX1 is co-expressed in OCT4 and TRA-1-60 positive hESCs and rapidly lost upon differentiation. Importantly, REX1 expression reveals significant heterogeneity within seemingly homogenous populations of OCT4 and TRA-1-60 hESCs. REX1 expression is extinguished before OCT4 during differentiation, but, in contrast to the mouse, loss of REX1 expression demarcates a stable, OCT4 positive lineage-primed state in pluripotent hESCs that does not revert back to REX1 positivity under normal conditions. We show that loss of REX1 expression correlates with altered patterns of DNA methylation at the REX1 locus, implying that epigenetic mechanisms may interfere with the metastable phenotype commonly found in murine pluripotency.

Lengthened G1 phase indicates differentiation status in human embryonic stem cells.

The cell cycle in pluripotent stem cells is notable for the brevity of the G1 phase, permitting rapid proliferation and reducing the duration of differentiation signal sensitivity associated with the G1 phase. Changes in the length of G1 phase are understood to accompany the differentiation of human embryonic stem cells (hESCs), but the timing and extent of such changes are poorly defined. Understanding the early steps governing the differentiation of hESCs will facilitate better control over differentiation for regenerative medicine and drug discovery applications. Here we report the first use of real-time cell cycle reporters in hESCs. We coexpressed the chromatin-decorating H2B-GFP fusion protein and the fluorescence ubiquitination cell cycle indicator (FUCCI)-G1 fusion protein, a G1 phase-specific reporter, in hESCs to measure the cell cycle status in live cells. We found that FUCCI-G1 expression is weakly detected in undifferentiated hESCs, but rapidly increases upon differentiation. hESCs in the G1 phase display a reduction in undifferentiated colony-initiating cell function, underscoring the relationship between G1 phase residence and differentiation. Importantly, we demonstrate inter- and intracolony variation in response to chemicals that induce differentiation, implying extensive cell-cell variation in the threshold necessary to alter the G1 phase length. Finally, gain of differentiation markers appears to be coincident with G1 phase lengthening, with distinct G1 phase profiles associated with different markers of early hESC differentiation. Our data demonstrate the tight coupling of cell cycle changes to hESC differentiation, and highlight the cell cycle reporter system and assays we have implemented as a novel avenue for investigating pluripotency and differentiation.

Cell fate potential of human pluripotent stem cells is encoded by histone modifications.

Human embryonic stem cells (hESCs) expressing pluripotency markers are assumed to possess equipotent developmental potential. However, disparate responses to differentiation stimuli functionally illustrate that hESCs generate a spectrum of differentiated cell types, suggestive of lineage bias. Here, we reveal specific cell surface markers that allow subfractionation of hESCs expressing hallmark markers of pluripotency. By direct de novo isolation of these subsets, we demonstrate that propensities for lineage differentiation are balanced with reduced clonogenic self-renewal. Histone modification marks of gene loci associated with pluripotency versus lineage specificity predicted cell fate potential of these subfractions, thereby supporting the absence of uniform bivalency as a molecular paradigm to describe cell fate determination of pluripotent cells. Our study reveals that cell fate potential is encoded within cells comprising hESC cultures, highlighting them as a means to understand the mechanisms of lineage specification of pluripotent cells.

Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2.

The mouse blastocyst and stem cells derived from its tissue lineages provide a unique genetic system for examining the establishment and loss of pluripotency. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4, and promoting extraembryonic trophoblast fate at the blastocyst stage. However, genetic evidence has suggested that Cdx2 does not work alone in the trophoblast lineage. We have used bioinformatic and functional genomic strategies to identify the transcription factor Gata3 as a trophoblast factor. We show Gata3 to be capable of inducing trophoblast fate in embryonic stem cells and driving trophoblast differentiation in trophoblast stem cells. In addition, Cdx2 is not required for Gata3-induced expression of a subset of trophoblast genes in embryonic stem cells. We show that Gata3 is coexpressed with Cdx2 in the blastocyst, but this does not depend on Cdx2. In the embryo, expression of Gata3, like that of Cdx2, depends on Tead4, and the expression of both factors becomes restricted to trophoblast by a mechanism that does not initially rely on Oct4. These observations suggest that Gata3 and Cdx2 can act in parallel pathways downstream of Tead4 to induce the expression of common and independent targets in the trophoblast lineage, whereas Oct4 is required for continued repression of trophoblast fate in the embryonic lineage.

Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency.

Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome-specific mouse and human iPS cell lines with known mutations in MECP2.

Establishment of endoderm progenitors by SOX transcription factor expression in human embryonic stem cells.

In this study, we explore endoderm cell fate regulation through the expression of lineage-determining transcription factors. We demonstrate that stable endoderm progenitors can be established from human ES cells by constitutive expression of SOX7 or SOX17, producing extraembryonic endoderm and definitive endoderm progenitors, respectively. In teratoma assays and growth factor-mediated differentiation, SOX7 cells appear restricted to the extraembryonic endoderm, and SOX17 cells demonstrate a mesendodermal phenotype in teratomas and the ability to undergo endoderm maturation in vitro in the absence of cytokine-mediated endoderm induction. These endoderm progenitor cells maintain a stable phenotype through many passages in culture, thereby providing new tools to explore the pathways of endoderm differentiation.

Transient and stable transgene expression in human embryonic stem cells.

Plasmid vectors remain a valuable yet capricious tool for the genetic manipulation of human embryonic stem (hES) cells. We have compared the efficacy of four promoters to mediate transient and stable transfection in hES and human embryonal carcinoma cell lines with the reporter enhanced green fluorescent protein (eGFP). In transient assays, the two mammalian promoters, UbiquitinC and Rosa26 (pUbiC and pR26), the human cytomegalovirus major immediate early promoter (HCMV-MIE; pCMV), and the HCMV-MIE/chicken beta-actin/rabbit beta-globin hybrid promoter (pCAGG) gave variable results that depended upon the cell line transfected but in an unpredictable way: each promoter supported strong transient expression in at least one cell line. The results for stable transfection were generally at variance with the transient assays. In each case, transgene silencing was quite marked, most notably with the pCMV, with which no eGFP-positive clones were obtained. An exception was the pCAG vector, in which the CAGG composite promoter is linked to the polyoma virus mutant enhancer PyF101; stable eGFP-positive transfectants were obtained, and these clones retained eGFP expression for over 120 passages, even in the absence of selection. However, if the PyF101 elements were removed, the resulting transfectants were also subjected to progressive gene silencing. Thus, the choice of promoter is critical for determining the desired effect of transgene expression in hES cells. Our data also demonstrate that pUbiC, pR26, pCAGG, and pCAG are more superior to the pCMV for generation of stable transfectants in hES cells. Disclosure of potential conflicts of interest is found at the end of this article.

Culture and characterization of human embryonic stem cells.

Human embryonic stem (ES) cells offer substantial opportunities for providing well-defined differentiated cells for drug discovery, toxicology, and regenerative medicine, but the development of efficient techniques for their large-scale culture under defined conditions, and for controlling and directing their differentiation, presents a substantial challenge. Markers for defining the undifferentiated cells are well established, based upon previous studies of embryonal carcinoma (EC) cells, their malignant counterparts from teratocarcinomas. These provide valuable tools for monitoring human ES cultures and their state of differentiation. However, current culture techniques are suboptimal and involve the use of poorly defined culture media and the use of feeder cells. Over time, the cells may also acquire karyotypic changes, reflecting genetic selection and adaptation to in vitro culture conditions. Nevertheless, progress is being made. Originally, human ES cells were derived and maintained in medium containing fetal calf serum. They are now widely cultured in a proprietary serum-free formulation (serum replacement from Invitrogen Corp., Carlsbad, CA), and recently we have derived a new human ES line in this medium without fetal calf serum. Human fibroblasts can also be used to replace mouse embryo fibroblasts as feeder cells. We have now found it possible to culture a subline of human ES cells on Matrigel, or purified collagen type IV, laminin, and fibronectin, without feeders or feeder-conditioned medium. These cells nevertheless retain the features of undifferentiated human ES cells, including a capacity for differentiation. Although these cells also carried karyotypic changes, further research focused upon understanding the mechanisms that control self-renewal, apoptosis, and commitment to differentiation will facilitate the development of defined culture conditions that minimize genetic change and optimize the maintenance of the undifferentiated stem cells.

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.

We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.

Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors.

Remarkably little is known about the transcriptional profiles of human embryonic stem (ES) cells or the molecular mechanisms that underlie their pluripotency. To identify commonalties among the transcriptional profiles of different human pluripotent cells and to search for clues into the genesis of human germ cell tumors, we compared the expression profiles of human ES cell lines, human germ cell tumor cell lines and tumor samples, somatic cell lines, and testicular tissue samples by using cDNA microarray analysis. Hierarchical cluster analysis of gene expression profiles showed that the five independent human ES cell lines clustered tightly together, reflecting highly similar expression profiles. The gene expression patterns of human ES cell lines showed many similarities with the human embryonal carcinoma cell samples and more distantly with the seminoma samples. We identified 895 genes that were expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples. These genes are candidates for involvement in the maintenance of a pluripotent, undifferentiated phenotype.

Human embryonic stem cells: multilineage differentiation and mechanisms of self-renewal.

Human ES (hES) cells are pluripotent stem cells isolated from the inner cell mass (ICM) of blastocysts, with the theoretical capacity to differentiate in vitro to produce all somatic and germ cell types. The diverse differentiation repertoire of hES cells makes them ideal candidates for the generation of tissues for transplantation therapies and drug discovery. However, to realize the full potential of hES cells it will be necessary to characterize the mechanisms that control self-renewal and differentiation into specific cell types. We review here the recent developments to differentiate human ES cell into lineages including neural and cardiac. Further, by reference to the self-renewal system established in murine ES we will discuss the possible mechanisms of self-renewal in hES cells.