Differentiating Human Embryonic Stem Cells in Micropatterns to Study Cell Fate Specification and Morphogenetic Events During Gastrulation

During mammalian embryogenesis, the first major lineage segregation occurs when embryonic epiblast, and extraembryonic trophectoderm and hypoblast arise in the blastocyst. In the next fundamental and conserved phase of animal embryogenesis known as gastrulation, extraembryonic cells provide signals to epiblast to instruct embryonic patterning, and epiblast gives rise to germ layers ectoderm, mesoderm, and endoderm, that will establish all embryonic tissues. Proper specification and morphogenesis of germ layers during gastrulation is vital for correct embryonic development. Due to ethical and legal restrictions limiting human embryo studies, human gastrulation is poorly understood. Our knowledge of human gastrulation has largely been derived from studies in model organisms, including mouse and more recently, cynomolgus monkey. However, interspecies differences underscore the need for alternative human gastrulation models. In this regard, human and mouse embryonic stem cells have been shown to recapitulate aspects of in vivo gastrulation including germ layer specification, and internalization and elongation morphogenesis. These in vitro systems represent powerful models of gastrulation due to the ease of genetic manipulations and the ability to finely control experimental factors. Human embryonic stem cells, treated with BMP4 for 44 hours in spatially confined micro-discs of extracellular matrix, have been shown to differentiate into 2D micro-colonies termed gastruloids. These gastruloids display highly reproducible differentiation of germ layers and extraembryonic cell types in a radial arrangement. We used combinatorial single-cell RNA sequencing and immunofluorescence imaging to characterize these BMP4-treated 2D gastruloids, and showed the formation in gastruloids of seven cell types, including epiblast, prospective ectoderm, two populations of mesoderm, and endoderm, as well as previously undescribed cell types in 2D gastruloids, primordial germ cell-like cells, and extraembryonic cells that are transcriptionally similar to trophectoderm and amnion. Comparative transcriptomic analyses with human, mouse, and cynomolgus monkey gastrulae support the notion that 2D gastruloid differentiation recapitulates formation of cell types relevant to and models an early-mid stage of in vivo gastrulation. Time course scRNA-seq and immunofluorescence analyses of 2D gastruloid differentiation after 12, 24, and 44 hours of BMP4 treatment showed that germ layer emergence in gastruloids follows the temporal sequence of in vivo gastrulation, with epiblast and ectoderm precursors forming at 12 hour, mesendoderm precursors arising from epiblast at 24 hour to give rise to nascent mesoderm and endoderm at 44 hour, when primordial germ cell-like cells also form. Comparison with human gastrula also showed similarity in transcriptomes and differentiation trajectories of gastruloid cells to their in vivo counterparts. Dynamic changes in transcripts encoding components of key signaling pathways support a BMP, WNT and Nodal hierarchy underlying germ layer specification conserved across mammals, with FGF and HIPPO signaling being active throughout the time course of 2D micropattern gastruloid differentiation. To probe morphogenetic properties of gastruloid cells, differentiated gastruloids treated with BMP4 for 44 hours were dissociated and re-seeded onto extracellular matrix micro-discs. The reseeded cells were highly motile and tended to form aggregates with the same but segregate from or mix with distinct cell types, supporting that 2D gastruloid system exhibits evolutionarily conserved sorting behaviors. In particular, ectodermal cells segregated from endodermal and extraembryonic cells but mixed with mesodermal cells. These results demonstrate that 2D gastruloid system models specification of germ layers and extraembryonic cell types, temporal order and differentiation trajectories of germ layer emergence, and signaling interactions found in early-mid in vivo gastrulation. Dissociated and reseeded gastruloid cells also exhibit conserved cell sorting behaviors. Lastly, this work provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation

Umbilical cord blood hematopoietic stem cell transplantation, an alternative to bone marrow

Umbilical cord blood (UCB) is an alternative hematopoietic stem cell (HSC) source that can ameliorate several diseases through transplantation. The purpose of this project is to analyze clinical studies comparing HSCs from a single cord blood unit (CBU) to HSCs from bone marrow, and to explore methods of increasing limited amounts of HSCs. It was found that UCB transplantation in adults is a viable method when a matched bone marrow transplant cannot be identified. Further clinical studies using two CBUs suggest better engraftment and lower risk of relapse. However, double cord blood transplantation has been faced with the challenge of single unit dominance in most studies. Ex vivo expansion of UCB HSCs is another promising method to overcome limited HSC counts

Device to dynamically stretch cells during microscopic visualization

Cells in physiological systems are constantly subjected to mechanical forces which affect myriad cellular functions and contribute to pathologies. Current devices to study cellular responses to strain in vitro have drawbacks including the inability to analyze cellular responses in “real-time”, non-uniform strains patterns, and limited operation time (\u3c6 hr). To overcome these limitations, a novel stretch device was developed using four linear motors and a novel silicone culture well. The device is capable of cyclic stretching of cells biaxially up to 30% strain in either or both of two orthogonal axes at 0.01 to 1 Hz frequency for a minimum of 6 hr during “real-time” analysis of cells under a standard inverted microscope. This new system will facilitate controlled mechanobiology studies

Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids

Human embryonic stem cells cultured in 2D micropatterns with BMP4 differentiate into a radial arrangement of germ layers and extraembryonic cells. Single-cell transcriptomes demonstrate generation of cell types transcriptionally similar to their in vivo counterparts in Carnegie stage 7 human gastrula. Time-course analyses indicate sequential differentiation, where the epiblast arises by 12 h between the prospective ectoderm in the center and the cells initiating differentiation toward extraembryonic fates at the edge. Extraembryonic and mesendoderm precursors arise from the epiblast by 24 h, while nascent mesoderm, endoderm, and primordial germ cell-like cells form by 44 h. Dynamic changes in transcripts encoding signaling components support a BMP, WNT, and Nodal hierarchy underlying germ-layer specification conserved across mammals, and FGF and HIPPO pathways being active throughout differentiation. This work also provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation

High rate of virological failure and low rate of switching to second-line treatment among adolescents and adults living with HIV on first-line ART in Myanmar, 2005-2015.

BACKGROUND: The number of people living with HIV on antiretroviral treatment (ART) in Myanmar has been increasing rapidly in recent years. This study aimed to estimate rates of virological failure on first-line ART and switching to second-line ART due to treatment failure at the Integrated HIV Care program (IHC). METHODS: Routinely collected data of all adolescent and adult patients living with HIV who were initiated on first-line ART at IHC between 2005 and 2015 were retrospectively analyzed. The cumulative hazard of virological failure on first-line ART and switching to second-line ART were estimated. Crude and adjusted hazard ratios were calculated using the Cox regression model to identify risk factors associated with the two outcomes. RESULTS: Of 23,248 adults and adolescents, 7,888 (34%) were tested for HIV viral load. The incidence rate of virological failure among those tested was 3.2 per 100 person-years follow-up and the rate of switching to second-line ART among all patients was 1.4 per 100 person-years follow-up. Factors associated with virological failure included: being adolescent; being lost to follow-up at least once; having WHO stage 3 and 4 at ART initiation; and having taken first-line ART elsewhere before coming to IHC. Of the 1032 patients who met virological failure criteria, 762 (74%) switched to second-line ART. CONCLUSIONS: We found high rates of virological failure among one third of patients in the cohort who were tested for viral load. Of those failing virologically on first-line ART, about one quarter were not switched to second-line ART. Routine viral load monitoring, especially for those identified as having a higher risk of treatment failure, should be considered in this setting to detect all patients failing on first-line ART. Strategies also need to be put in place to prevent treatment failure and to treat more of those patients who are actually failing

Long-term outcomes of second-line antiretroviral treatment in an adult and adolescent cohort in Myanmar.

BACKGROUND: Myanmar has a high burden of Human Immunodeficiency Virus (HIV) and second-line antiretroviral treatment (ART) has been available since 2008 in the public health sector. However, there have been no published data about the outcomes of such patients until now. OBJECTIVE: To assess the treatment and programmatic outcomes and factors associated with unfavorable outcomes (treatment failure, death and loss to follow-up from care) among people living with HIV (aged ≥ 10 years) receiving protease inhibitor-based second-line ART under the Integrated HIV Care Program in Myanmar between October 2008 and June 2015. DESIGN: Retrospective cohort study using routinely collected program data. RESULTS: Of 824 adults and adolescents on second-line ART, 52 patients received viral load testing and 19 patients were diagnosed with virological failure. However, their treatment was not modified. At the end of a total follow-up duration of 7 years, 88 (11%) patients died, 35 (4%) were lost to follow-up, 21 (2%) were transferred out to other health facilities and 680 (83%) were still under care. The incidence rate of unfavorable outcomes was 7.9 patients per 100 person years follow-up. Patients with a history of injecting drug use, with a history of lost to follow-up, with a higher baseline viral load and who had received didanosine and abacavir had a higher risk of unfavorable outcomes. Patients with higher baseline C4 counts, those having taken first-line ART at a private clinic, receiving ART at decentralized sites and taking zidovudine and lamivudine had a lower risk of unfavorable outcomes. CONCLUSIONS: Long-term outcomes of patients on second-line ART were relatively good in this cohort. Virological failure was relatively low, possibly because of lack of viral load testing. No patient who failed on second-line ART was switched to third-line treatment. The National HIV/AIDS Program should consider making routine viral load monitoring and third-line ART drugs available after a careful cost-benefit analysis

Early Success With Retention in Care Among People Living With HIV at Decentralized ART Satellite Sites in Yangon, Myanmar, 2015–2016

Introduction: Myanmar is one of the countries in the Asia-Pacific region hit hardest by the HIV epidemic that is concentrated among urban areas and key populations. In 2014, the National AIDS Programme (NAP) launched a new model of decentralized service delivery with the establishment ART satellite sites with care delivered by HIV peer workers.Methods: ART satellite sites are implemented by non-government organizations to service high burden HIV areas and populations that suffer stigma or find access to public sector services difficult. They provide continuity of HIV care from outreach testing, counseling, linkage to care, and retention in care. Anti-retroviral (ART) initiation occurs at health facilities by specialist physicians. We conducted a retrospective cohort study of people living with HIV (PLHIV) who were initiated on ART from 2015 to 2016 at five ART satellite sites in Yangon, Myanmar to assess outcomes and time from enrolment to ART initiation.Results: Of 1,339 PLHIV on ART treatment in 2015–16, 1,157 (89%) were retained, and 5% were lost from care and 5% reported dead, at the end of March 2018. Attrition rates (death and lost-to-follow-up) were found to be significantly associated with a CD4 count ≤ 50 cells/mm3 and having baseline weight ≤ 50 kg. Median time taken from enrolment to ART initiation was 1.9 months (interquartile range: 1.4–2.5).Conclusion: We report high rates of retention in care of PLHIV in a new model of ART satellite sties in Yangon, Myanmar after 3 years of follow-up. The delays identified in time taken from enrolment to ART initiation need to be explored further and addressed. This initial study supports continuation of plans to scale-up ART satellite sites in Myanmar. To optimize outcomes for patients and the program and accelerate progress to reduce HIV transmission and end the HIV epidemic, operational research needs to be embedded within the response

High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human ESC gastruloid cultures

During mammalian gastrulation, germ layers arise and are shaped into the body plan while extraembryonic layers sustain the embryo. Human embryonic stem cells, cultured with BMP4 on extracellular matrix micro-discs, reproducibly differentiate into gastruloids, expressing markers of germ layers and extraembryonic cells in radial arrangement. Using single-cell RNA sequencing and cross-species comparisons with mouse, cynomolgus monkey gastrulae, and post-implantation human embryos, we reveal that gastruloids contain cells transcriptionally similar to epiblast, ectoderm, mesoderm, endoderm, primordial germ cells, trophectoderm, and amnion. Upon gastruloid dissociation, single cells reseeded onto micro-discs were motile and aggregated with the same but segregated from distinct cell types. Ectodermal cells segregated from endodermal and extraembryonic but mixed with mesodermal cells. Our work demonstrates that the gastruloid system models primate-specific features of embryogenesis, and that gastruloid cells exhibit evolutionarily conserved sorting behaviors. This work generates a resource for transcriptomes of human extraembryonic and embryonic germ layers differentiated in a stereotyped arrangement