The human embryo undergoes morphogenetic transformations following implantation into the uterus, but our knowledge of this crucial stage is limited by the inability to observe the embryo in vivo. ...Models of the embryo derived from stem cells are important tools for interrogating developmental events and tissue-tissue crosstalk during these stages
. Here we establish a model of the human post-implantation embryo, a human embryoid, comprising embryonic and extraembryonic tissues. We combine two types of extraembryonic-like cell generated by overexpression of transcription factors with wild-type embryonic stem cells and promote their self-organization into structures that mimic several aspects of the post-implantation human embryo. These self-organized aggregates contain a pluripotent epiblast-like domain surrounded by extraembryonic-like tissues. Our functional studies demonstrate that the epiblast-like domain robustly differentiates into amnion, extraembryonic mesenchyme and primordial germ cell-like cells in response to bone morphogenetic protein cues. In addition, we identify an inhibitory role for SOX17 in the specification of anterior hypoblast-like cells
. Modulation of the subpopulations in the hypoblast-like compartment demonstrates that extraembryonic-like cells influence epiblast-like domain differentiation, highlighting functional tissue-tissue crosstalk. In conclusion, we present a modular, tractable, integrated
model of the human embryo that will enable us to probe key questions of human post-implantation development, a critical window during which substantial numbers of pregnancies fail.
Several in vitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the ...interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based in vitro model that reconstitutes the pluripotent ESC lineage and the two extraembryonic lineages of the post-implantation embryo by transcription-factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation; formation of the anterior-posterior axis, brain, and a beating heart structure; and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages but also showed when and where the model diverges from the natural program. Our findings demonstrate an extraordinary plasticity of ESCs to self-organize and generate a whole-embryo-like structure.
Dopaminergic neurons (DAns), generated from human pluripotent stem cells (hPSCs), are capable of functionally integrating following transplantation and have recently advanced to clinical trials for ...Parkinson’s disease (PD). However, pre-clinical studies have highlighted the low proportion of DAns within hPSC-derived grafts and their inferior plasticity compared to fetal tissue. Here, we examined whether delivery of a developmentally critical protein, glial cell line-derived neurotrophic factor (GDNF), could improve graft outcomes. We tracked the response of DAns implanted into either a GDNF-rich environment or after a delay in exposure. Early GDNF promoted survival and plasticity of non-DAns, leading to enhanced motor recovery in PD rats. Delayed exposure to GDNF promoted functional recovery through increases in DAn specification, DAn plasticity, and DA metabolism. Transcriptional profiling revealed a role for mitogen-activated protein kinase (MAPK)-signaling downstream of GDNF. Collectively, these results demonstrate the potential of neurotrophic gene therapy strategies to improve hPSC graft outcomes.
Display omitted
•DAn grafting into a GDNF-rich host aids pan-neuronal survival but impedes plasticity•Delaying graft exposure to GDNF increases DAn maturation, plasticity, and function•GDNF promotes functional integration of DAn grafts via MAPK signaling•Modulating the host environment can improve the integration of human neural grafts
Gantner et al. provide preclinical evidence that the timely overexpression of glial cell-derived neurotrophic factor (GDNF) within the host brain can promote the maturation, plasticity, and functional integration of human stem cell-derived dopamine grafts in a rodent model of Parkinson’s disease.
The development of mouse embryos can be partially recapitulated by combining embryonic stem cells (ESCs), trophoblast stem cells (TS), and extra-embryonic endoderm (XEN) stem cells to generate ...embryo-like structures called ETX embryos. Although ETX embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ESCs transiently expressing transcription factor Gata4, which drives the extra-embryonic endoderm fate, and combined them with ESCs and TS cells to generate induced ETX embryos (iETX embryos). We show that iETX embryos establish a robust anterior signaling center that migrates unilaterally to break embryo symmetry. Furthermore, iETX embryos gastrulate generating embryonic and extra-embryonic mesoderm and definitive endoderm. Our findings reveal that replacement of XEN cells with ESCs transiently expressing Gata4 endows iETX embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system.
Display omitted
•Stem cells generate mouse-embryo-like structures with improved potential•These structures undertake anterior visceral endoderm formation and gastrulation•Single-cell sequencing shows improved resemblance to mouse embryo
Amadei et al. have generated stem-cell-based structures that resemble mouse post-implantation embryos and have the potential to form the anterior-posterior axis and undergo gastrulation in vitro. Single-cell sequencing shows gene-expression patterns similar to those of the natural embryo at a comparable stage of development.
Midbrain dopamine (mDA) neurons can be replaced in patients with Parkinson’s disease (PD) in order to provide long-term improvement in motor functions. The limited capacity for long-distance axonal ...growth in the adult brain means that cells are transplanted ectopically, into the striatal target. As a consequence, several mDA pathways are not re-instated, which may underlie the incomplete restoration of motor function in patients. Here, we show that viral delivery of GDNF to the striatum, in conjunction with homotopic transplantation of human pluripotent stem-cell-derived mDA neurons, recapitulates brain-wide mDA target innervation. The grafts provided re-instatement of striatal dopamine levels and correction of motor function and also connectivity with additional mDA target nuclei not well innervated by ectopic grafts. These results demonstrate the remarkable capacity for achieving functional and anatomically precise reconstruction of long-distance circuitry in the adult brain by matching appropriate growth-factor signaling to grafting of specific cell types.
Display omitted
•A PITX3-eGFP iPSC line reveals patterns of mDA neuron growth from stem cell grafts•mDA neurons in homotopic grafts target nuclei not well innervated by ectopic grafts•Forebrain GDNF facilitates robust striatal innervation by homotopic mDA grafts
Moriarty et al. show that dopamine neurons in homotopic grafts generated from pluripotent stem cells can provide long-distance, anatomically precise innervation of appropriate targets throughout the brain, including robust striatal innervation when combined with forebrain GDNF delivery, in order to restore motor function in a rodent model of Parkinson’s disease.
Abstract
Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are ...established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development.
Clinical studies have provided evidence for dopamine (DA) cell replacement therapy in Parkinson's Disease. However, grafts derived from foetal tissue or pluripotent stem cells (PSCs) remain ...heterogeneous, with a high proportion of non-dopaminergic cells, and display subthreshold reinnervation of target tissues, thereby highlighting the need to identify new strategies to improve graft outcomes. In recent work, Stromal Cell-Derived Factor-1 (SDF1), secreted from meninges, has been shown to exert many roles during ventral midbrain DA development and DA-directed differentiation of PSCs. Related, co-implantation of meningeal cells has been shown to improve neural graft outcomes, however, no direct evidence for the role of SDF1 in neural grafting has been shown. Due to the rapid degradation of SDF1 protein, here, we utilised a hydrogel to entrap the protein and sustain its delivery at the transplant site to assess the impact on DA progenitor differentiation, survival and plasticity. Hydrogels were fabricated from self-assembling peptides (SAP), presenting an epitope for laminin, the brain's main extracellular matrix protein, thereby providing cell adhesive support for the grafts and additional laminin-integrin signalling to influence cell fate. We show that SDF1 functionalised SAP hydrogels resulted in larger grafts, containing more DA neurons, increased A9 DA specification (the subpopulation of DA neurons responsible for motor function) and enhanced innervation. These findings demonstrate the capacity for functionalised, tissue-specific hydrogels to improve the composition of grafts targeted for neural repair.
Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in ...protocols for the
generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP
, and GFP
cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP
, or PITX3-eGFP
cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP
cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP
grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP
progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP
cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function.
Clinical trials have shown functional integration of transplanted fetal-derived dopamine progenitors in Parkinson's disease. Human pluripotent stem cell (hPSC)-derived midbrain progenitors are now being tested as an alternative cell source; however, despite current differentiation protocols generating >80% correctly specified cells for implantation, resultant grafts contain a small fraction of dopamine neurons. Cell-sorting approaches, to select for correctly patterned cells before implantation, are being explored yet have been suboptimal to date. This study provides the first evidence of using 2 hPSC reporter lines (LMX1A-GFP and PITX3-GFP) to isolate correctly specified cells for transplantation. We show LMX1A-GFP
, but not PITX3-GFP
, cell grafts are more predictable, with smaller grafts, enriched in dopamine neurons, showing appropriate integration and accelerated functional recovery in Parkinsonian rats.
The survival and synaptic integration of transplanted dopaminergic (DA) progenitors are essential for ameliorating motor symptoms in Parkinson's disease (PD). Human pluripotent stem cell ...(hPSC)‐derived DA progenitors are, however, exposed to numerous stressors prior to, and during, implantation that result in poor survival. Additionally, hPSC‐derived grafts show inferior plasticity compared to fetal tissue grafts. These observations suggest that a more conducive host environment may improve graft outcomes. Here, tissue‐specific support to DA progenitor grafts is provided with a fully characterized self‐assembling peptide hydrogel. This biomimetic hydrogel matrix is programmed to support DA progenitors by i) including a laminin epitope within the matrix; and ii) shear encapsulating glial cell line‐derived neurotrophic factor (GDNF) to ensure its sustained delivery. The biocompatible hydrogel biased a 51% increase in A9 neuron specification—a subpopulation of DA neurons critical for motor function. The sustained delivery of GDNF induced a 2.7‐fold increase in DA neurons and enhanced graft plasticity, resulting in significant improvements in motor deficits at 6 months. These findings highlight the therapeutic benefit of stepwise customization of tissue‐specific hydrogels to improve the physical and trophic support of human PSC‐derived neural transplants, resulting in improved standardization, predictability and functional efficacy of grafts for PD.
The benefits of a functionalized tissue‐specific hydrogel are demonstrated to support human stem cell‐derived neural transplants in a Parkinson's disease model. The laminin epitope‐presenting hydrogel selectively supports A9 dopamine neurons, critical for motor function, while sustained glial cell line‐derived neurotrophic factor delivery enhances graft survival and plasticity—collectively resulting in improved recovery of motor symptoms.
PDF
