Sarita Panula

Downregulation of miRNA-200c Links Breast Cancer Stem Cells with Normal Stem Cells

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.

Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos

Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.Summary Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.

Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells

Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications.

Comparison of Biomaterials and Extracellular Matrices as a Culture Platform for Multiple, Independently Derived Human Embryonic Stem Cell Lines

Long-term in vitro culture of undifferentiated human embryonic stem cells (hESCs) traditionally requires a fibroblast feeder cell layer. Using feeder cells in hESC cultures is highly laborious and limits large-scale hESC production for potential application in regenerative medicine. Replacing feeder cells with defined human extracellular matrix (ECM) components or synthetic biomaterials would be ideal for large-scale production of clinical-grade hESCs. We tested and compared different feeder cell-free hESC culture methods based on different human ECM proteins, human and animal sera matrices, and a Matrigel matrix. Also selected biomaterials were tested for feeder cell-free propagation of undifferentiated hESCs. The matrices were tested together with conventional and modified hESC culture media, human foreskin fibroblast-conditioned culture medium, chemically defined medium, TeSR1, and modified TeSR1 media. The results showed the undefined, xenogeneic Matrigel to be a superior matrix for hESC culture compared with the purified human ECM proteins, serum matrices, and the biomaterials tested. A long-term, feeder cell-free culture system was successful on Matrigel in combination with mTeSR1 culture medium, but a xeno-free, fully defined, and reproducible feeder cell-free hESC culture method still remains to be developed.

Adult human and mouse ovaries lack DDX4-expressing functional oogonial stem cells.

The generally accepted viewpoint for more than 50 years has been that the number of oocytes is fixed in fetal or neonatal ovaries, and therefore, oocytes cannot renew themselves in postnatal or adult life. Over the past decade, however, the traditional viewpoint has been challenged by a number of investigators who have presented evidence that postnatal follicular renewal occurs in mammals, and that mitotically active oogonial stem cells (OSCs) exist in postnatal mouse ovaries. Health, Inc. All rights reserved. 30 Obstetrical and Gynecological Survey This letter to the editor presents experimental evidence that disputes the existence of mitotically active OSC in postnatal mouse ovaries. The results presented here are the summary of research conducted independently in 4 laboratories. A previous study (White et al.Nat Med. 2012;18:413–421) reported that OSCs could be purified from adult human and mouse ovaries by use of DEAD box polypeptide 4 (DDX4) antibody–based fluorescence-activated cell sorting (FACS), and that after in vitro manipulation, these isolated OSCs could form oocytes. Based on the well-established cytoplasmic location of DDX4, the use of this protein as a cell surface marker is controversial. Using the same DDX4 antibody–based FACS approach as in the White et al study, the investigators isolated a population of cells from human ovarian cortical tissue biopsied from 16 fertile reproductive-age women who had had at least 1 previous live birth. No DDX4 messenger RNA (mRNA) expression was detected using quantitative polymerase chain reaction in these cells or by a more sensitive single-cell mRNA sequencing analysis that could detect low expression of DDX4. In additional experiments, the sorted human ovarian cells were cultured as described in the previous study. Although no DDX4 expression was detected in the cultured DDX4-positive cells (cultured-POS) or cultured DDX4-negative cells (cultured-NEG) by immunofluorescence staining, the cultured-POS cells and cultured-NEG cells both bound tightly to the DDX4-specific antibody in FACS and became DDX4-positive after culture. The previous study had reported that oocytes enclosed in follicles regenerated 1 week after the DDX4-positive human OSCs were injected into human ovarian cortical tissues that were subsequently xenografted into female severe combined immunodeficient mice. The investigators repeated this experiment and labeled the cultured-POS cells with stable enhanced green fluorescent protein (EGFP) expression. After culturing and expanding this cell population, EGFP-expressing cultured-POS cells were injected into human ovarian cortical tissue biopsies, and these cortical tissues were then xenografted into female severe combined immunodeficient mice for further growth. Grafts were analyzed 1 week, 2 weeks, and 4 weeks after transplantation of the EGFP–cultured-POS cells into the human cortical tissues. The results of this experiment showed that EGFP-positive cells could be observed in the vicinity of the injection sites, but the absence of any EGFP-positive oocytes demonstrated that the DDX4-positive human cells obtained with the DDX4 antibody are not functional stem cells and cannot regenerate oocytes. To confirm these findings that the DDX4-specific antibody–based FACS does not select for a specific cell population expressing DDX4, the same FACS was performed with mouse cells from several organs (including adult liver, spleen, and kidney) that do not express DDX4. DDX4-positive cell populations were obtained from cells of these organs, which provide additional evidence that use of the DDX4-specific antibody using the FACS protocol (critical in purifying the reported OSCs) does not select for DDX4-expressing cells. These findings provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active DDX4-expressing female germline progenitors exist in postnatal mouse ovaries.

Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States

Summary Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.

Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

Summary Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells.

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

Preparation of Human Foreskin Fibroblasts for Human Embryonic Stem Cell Culture

INTRODUCTIONIn order to reduce the risk of immune rejection and zoonosis in human embryonic stem cell (hESC) transplantation recipients, animal-derived components, nonhuman sera, and animal feeder cells need to be removed from hESC culture. To limit the use of animal products, several different human feeder cell types have been tested for hESC culture. These include cells from fetal muscle and skin, as well as adult fallopian tube epithelial cells and adult muscle cells. Human foreskin fibroblasts (hFFs) have been used successfully as feeder cells to support the derivation and undifferentiated growth of hESCs and are commercially available through the American Type Culture Collection (ATCC). This protocol describes a method for preparing hFFs for hESC culture.

Single‐cell RNA sequencing: revealing human pre‐implantation development, pluripotency and germline development

Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single‐cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra‐embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two‐cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single‐cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre‐implantation development, embryonic stem cells and the establishment of the germ line.