Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche
ABSTRACT: In this work, we generated early human amnion-like tissues by culturing human pluripotent stem cells (hPSCs) in a bioengineered implantation-like niche in vitro. To explore the gene expression profile of hPSC-derived amnion-like cells (hPSC-amnion), we performed mRNA-sequencing for both undifferentiated hPSCs and hPSC-amnion. Here we show that hPSC-amnion differs from hPSCs by actively regulating a comprehensive set of transcriptional regulation network and developmental signaling pathways such as BMP-SMAD signaling. Overall design: hPSCs cultured in conventional maintenance condition (mTeSR1-based feeder-free culture system; three biological replicates) as well as in the engineered implantation-like niche (the Gel-3D system composed of mTeSR1 and Geltrex; three biological replicates) were harvested and subjected to RNA-seq.
Project description:Amniogenesis-the development of amnion-is a critical developmental milestone for early human embryogenesis and successful pregnancy. However, human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development, thereby helping advance human embryology and reproductive medicine.
Project description:Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are capable of differentiating into any cell type in the human body and thus can be used in studies of early human development, as cell models for different diseases and eventually also in regenerative medicine applications. Since the first derivation of hESCs in 1998, a variety of culture conditions have been described for the undifferentiated growth of hPSCs. In this study, we cultured both hESCs and hiPSCs in three different culture conditions: on mouse embryonic fibroblast (MEF) and SNL feeder cell layers together with conventional stem cell culture medium containing knockout serum replacement and basic fibroblast growth factor (bFGF), as well as on a Matrigel matrix in mTeSR1 medium. hPSC lines were subjected to cardiac differentiation in mouse visceral endodermal-like (END-2) co-cultures and the cardiac differentiation efficiency was determined by counting both the beating areas and Troponin T positive cells, as well as studying the expression of OCT-3/4, mesodermal Brachyury T and NKX2.5 and endodermal SOX-17 at various time points during END-2 differentiation by q-RT-PCR analysis. The most efficient cardiac differentiation was observed with hPSCs cultured on MEF or SNL feeder cell layers in stem cell culture medium and the least efficient cardiac differentiation was observed on a Matrigel matrix in mTeSR1 medium. Further, hPSCs cultured on a Matrigel matrix in mTeSR1 medium were found to be more committed to neural lineage than hPSCs cultured on MEF or SNL feeder cell layers. In conclusion, culture conditions have a major impact on the propensity of the hPSCs to differentiate into a cardiac lineage.
Project description:Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%-0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process.
Project description:Development of the asymmetric amniotic sac-with the embryonic disc and amniotic ectoderm occupying opposite poles-is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology.Early in human embryonic development, it is unclear how amniotic sac formation is regulated. Here, the authors use a human pluripotent stem cell-based model, termed the post-implantation amniotic sac embryoid, to recapitulate early embryogenic events of human amniotic sac development.
Project description:The success of tissue regenerative therapies is contingent on functional and multicellular vasculature within the redeveloping tissue. Although endothelial cells (ECs), which compose the vasculature's inner lining, are intrinsically able to form nascent networks, these structures regress without the recruitment of pericytes, supporting cells that surround microvessel endothelium. Reconstruction of typical in vivo microvascular architecture traditionally has been done using distinct cell sources of ECs and pericytes within naturally occurring matrices; however, the limited sources of clinically relevant human cells and the inherent chemical and physical properties of natural materials hamper the translational potential of these approaches. Here we derived a bicellular vascular population from human pluripotent stem cells (hPSCs) that undergoes morphogenesis and assembly in a synthetic matrix. We found that hPSCs can be induced to codifferentiate into early vascular cells (EVCs) in a clinically relevant strategy amenable to multiple hPSC lines. These EVCs can mature into ECs and pericytes, and can self-organize to form microvascular networks in an engineered matrix. These engineered human vascular networks survive implantation, integrate with the host vasculature, and establish blood flow. This integrated approach, in which a derived bicellular population is exploited for its intrinsic self-assembly capability to create microvasculature in a deliverable matrix, has vast ramifications for vascular construction and regenerative medicine.
Project description:The utilization of human pluripotent stem cells (hPSCs) offers new avenues in the generation of organs and opportunities to understand development and diseases. The hPSC-derived human intestinal organoids (HIOs) provide a new tool to gain insights in small intestinal development, physiology, and associated diseases. Herein, we provide a method for orthotropic transplantation of HIOs in immunocompromised mice. This method highlights the specific steps to successful engraftment and provides insight into the study of bioengineered human small intestine.
Project description:Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFR?), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies.
Project description:In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.
Project description:Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.
Project description:Recently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.