SMC5 Plays Independent Roles in Congenital Heart Disease and Neurodevelopmental Disability.

Up to 50% of patients with severe congenital heart disease (CHD) develop life-altering neurodevelopmental disability (NDD). It has been presumed that NDD arises in CHD cases because of hypoxia before, during, or after cardiac surgery. Recent studies detected an enrichment in de novo mutations in CHD and NDD, as well as significant overlap between CHD and NDD candidate genes. However, there is limited evidence demonstrating that genes causing CHD can produce NDD independent of hypoxia. A patient with hypoplastic left heart syndrome and gross motor delay presented with a de novo mutation in SMC5. Modeling mutation of smc5 in Xenopus tropicalis embryos resulted in reduced heart size, decreased brain length, and disrupted pax6 patterning. To evaluate the cardiac development, we induced the conditional knockout (cKO) of Smc5 in mouse cardiomyocytes, which led to the depletion of mature cardiomyocytes and abnormal contractility. To test a role for Smc5 specifically in the brain, we induced cKO in the mouse central nervous system, which resulted in decreased brain volume, and diminished connectivity between areas related to motor function but did not affect vascular or brain ventricular volume. We propose that genetic factors, rather than hypoxia alone, can contribute when NDD and CHD cases occur concurrently.

Modeling Human Gonad Development in Organoids.

BACKGROUND: Our learning about human reproductive development is greatly hampered due to the absence of an adequate model. Animal studies cannot truthfully recapitulate human developmental processes, and studies of human fetal tissues are limited by their availability and ethical restrictions. Innovative three-dimensional (3D) organoid technology utilizing human pluripotent stem cells (hPSCs) offered a new approach to study tissue and organ development in vitro. However, a system for modeling human gonad development has not been established, thus, limiting our ability to study causes of infertility. METHODS: In our study we utilized the 3D hPSC organoid culture in mini-spin bioreactors. Relying on intrinsic self-organizing and differentiation capabilities of stem cells, we explored whether organoids could mimic the development of human embryonic and fetal gonad. RESULTS: We have developed a simple, bioreactor-based organoid system for modeling early human gonad development. Male hPSC-derived organoids follow the embryonic gonad developmental trajectory and differentiate into multipotent progenitors, which further specialize into testicular supporting and interstitial cells. We demonstrated functional activity of the generated cell types by analyzing the expression of cell type-specific markers. Furthermore, the specification of gonadal progenitors in organoid culture was accompanied by the characteristic architectural tissue organization. CONCLUSION: This organoid system opens the opportunity for detailed studies of human gonad and germ cell development that can advance our understanding of sex development disorders. Implementation of human gonad organoid technology could be extended to modeling causes of infertility and regenerative medicine applications.

SF3B1 homeostasis is critical for survival and therapeutic response in T cell leukemia.

The production of noncanonical mRNA transcripts is associated with cell transformation. Driven by our previous findings on the sensitivity of T cell acute lymphoblastic leukemia (T-ALL) cells to SF3B1 inhibitors, we identified that SF3B1 inhibition blocks T-ALL growth in vivo with no notable associated toxicity. We also revealed protein stabilization of the U2 complex component SF3B1 via deubiquitination. Our studies showed that SF3B1 inhibition perturbs exon skipping, leading to nonsense-mediated decay and diminished levels of DNA damage response-related transcripts, such as the serine/threonine kinase CHEK2, and impaired DNA damage response. We also identified that SF3B1 inhibition leads to a general decrease in R-loop formation. We further demonstrate that clinically used SF3B1 inhibitors synergize with CHEK2 inhibitors and chemotherapeutic drugs to block leukemia growth. Our study provides the proof of principle for posttranslational regulation of splicing components and associated roles and therapeutic implications for the U2 complex in T cell leukemia.

SMC5/6 is required for replication fork stability and faithful chromosome segregation during neurogenesis.

Mutations of SMC5/6 components cause developmental defects, including primary microcephaly. To model neurodevelopmental defects, we engineered a mouse wherein Smc5 is conditionally knocked out (cKO) in the developing neocortex. Smc5 cKO mice exhibited neurodevelopmental defects due to neural progenitor cell (NPC) apoptosis, which led to reduction in cortical layer neurons. Smc5 cKO NPCs formed DNA bridges during mitosis and underwent chromosome missegregation. SMC5/6 depletion triggers a CHEK2-p53 DNA damage response, as concomitant deletion of the Trp53 tumor suppressor or Chek2 DNA damage checkpoint kinase rescued Smc5 cKO neurodevelopmental defects. Further assessment using Smc5 cKO and auxin-inducible degron systems demonstrated that absence of SMC5/6 leads to DNA replication stress at late-replicating regions such as pericentromeric heterochromatin. In summary, SMC5/6 is important for completion of DNA replication prior to entering mitosis, which ensures accurate chromosome segregation. Thus, SMC5/6 functions are critical in highly proliferative stem cells during organism development.

Adaptation of the AID system for stem cell and transgenic mouse research.

The auxin-inducible degron (AID) system is becoming a widely used method for rapid and reversible degradation of target proteins. This system has been successfully used to study gene and protein functions in eukaryotic cells and common model organisms, such as nematode and fruit fly. To date, applications of the AID system in mammalian stem cell research are limited. Furthermore, standard mouse models harboring the AID system have not been established. Here we have explored the utility of the H11 safe-harbor locus for integration of the TIR1 transgene, an essential component of auxin-based protein degradation system. We have shown that the H11 locus can support constitutive and conditional TIR1 expression in mouse and human embryonic stem cells, as well as in mice. We demonstrate that the AID system can be successfully employed for rapid degradation of stable proteins in embryonic stem cells, which is crucial for investigation of protein functions in quickly changing environments, such as stem cell proliferation and differentiation. As embryonic stem cells possess unlimited proliferative capacity, differentiation potential, and can mimic organ development, we believe that these research tools will be an applicable resource to a broad scientific audience.

Adaptation of Human Testicular Niche Cells for Pluripotent Stem Cell and Testis Development Research.

BACKGROUND: Human testicular cells are greatly valuable to the research community as tools for studying testicular physiology and the effects of environmental pollutants. Because adult testicular cells have a limited self-organization capacity and life span, we investigated whether human pluripotent stem cells (hPSCs) can be used together with testicular cells to move a step closer toward making an optimal model of the human testis. METHODS: We used in vitro culture of donor testicular cells under serum-containing and chemically defined conditions. CRISPR-Cas9 technology was applied to introduce fluorescent transgenes (mCherry2 and EGFP) into hPSCs and testicular cells. hPSC-derived spheroids were co-cultured with human testicular cells in mini-spin bioreactors. RESULTS: Traditional cell culture conditions used for maintenance of testicular somatic cells generally contain serum and pose limitations on evaluating the role of active molecules on cell functions. We established that chemically defined culture conditions can be used to maintain testicular cells without the loss of proliferative activity. These cultures demonstrate marker expression which is characteristic of common testicular cell types: Sertoli, Leydig, endothelial, myoid cells, and macrophages. In order to model testicular physiology, it is important to be able to perform live cell microscopy. Thus, we generated fluorescent protein-expressing human testicular cells and hPSCs and demonstrated that these cell types can be successfully co-cultured for prolonged periods of time in a three-dimensional microenvironment. CONCLUSION: Our research extends the possible applications of human testis-derived somatic cells and shows that they can be used together with hPSCs for further studies of human male reproductive biology.

Conditional Mutation of SMC5 in Mouse Embryonic Fibroblasts.

The structural maintenance of chromosomes (SMC) complex, SMC5/6, is important for genome maintenance in all model eukaryotes. To date, the most extensive studies have focused on the roles of Smc5/6 in lower eukaryotes, such as yeast and fly. In the handful of studies that have used mammalian cells, siRNA was used by most to knockdown SMC5/6 components. RNAi methods have been very important for scientific progression, but they are hindered by incomplete silencing of protein expression and off-target effects. This chapter outlines the use of a conditional knockout approach in mouse embryonic fibroblasts to study the function of the SMC5/6 complex. These cell lines provide an alternative method to study the function and properties of the SMC5/6 complex in mammals.

Conditional mutation of Smc5 in mouse embryonic stem cells perturbs condensin localization and mitotic progression.

Correct duplication of stem cell genetic material and its appropriate segregation into daughter cells are requisites for tissue, organ and organism homeostasis. Disruption of stem cell genomic integrity can lead to developmental abnormalities and cancer. Roles of the Smc5/6 structural maintenance of chromosomes complex in pluripotent stem cell genome maintenance have not been investigated, despite its important roles in DNA synthesis, DNA repair and chromosome segregation as evaluated in other model systems. Using mouse embryonic stem cells (mESCs) with a conditional knockout allele of Smc5, we showed that Smc5 protein depletion resulted in destabilization of the Smc5/6 complex, accumulation of cells in G2 phase of the cell cycle and apoptosis. Detailed assessment of mitotic mESCs revealed abnormal condensin distribution and perturbed chromosome segregation, accompanied by irregular spindle morphology, lagging chromosomes and DNA bridges. Mutation of Smc5 resulted in retention of Aurora B kinase and enrichment of condensin on chromosome arms. Furthermore, we observed reduced levels of Polo-like kinase 1 at kinetochores during mitosis. Our study reveals crucial requirements of the Smc5/6 complex during cell cycle progression and for stem cell genome maintenance.

Carbon nanotube-based substrates for modulation of human pluripotent stem cell fate.

We investigated the biological response of human pluripotent stem cells (hPSCs) cultured on a carbon nanotube (CNT) array-based substrate with the long term goal to direct hPSC germ layer specification for a wide variety of tissue engineering applications. CNT arrays were fabricated using a chemical vapor deposition system allowing for control over surface roughness and mechanical stiffness. Our results demonstrated that hPSCs readily attach to hydrophilized and extracellular matrix coated CNT arrays. hPSCs cultured as colonies in conditions supporting self-renewal demonstrated the morphology and marker expression of undifferentiated hPSCs. Conditions inducing spontaneous differentiation lead to hPSC commitment to all three embryonic germ layers as assessed by immunostaining and RT-PCR analysis. Strikingly, the physical characteristics of CNT arrays favored mesodermal specification of hPSCs. This is contradictory to the behavior of hPSCs on traditional tissue culture plastic which promotes the development of ectoderm. Altogether, these results demonstrate the potential of CNT arrays to be used in the generation of new platforms that allow for precise control of hPSC differentiation by tuning the characteristics of their physical microenvironment.

Concise review: carbon nanotechnology: perspectives in stem cell research.

Carbon nanotechnology has developed rapidly during the last decade, and carbon allotropes, especially graphene and carbon nanotubes, have already found a wide variety of applications in industry, high-tech fields, biomedicine, and basic science. Electroconductive nanomaterials have attracted great attention from tissue engineers in the design of remotely controlled cell-substrate interfaces. Carbon nanoconstructs are also under extensive investigation by clinical scientists as potential agents in anticancer therapies. Despite the recent progress in human pluripotent stem cell research, only a few attempts to use carbon nanotechnology in the stem cell field have been reported. However, acquired experience with and knowledge of carbon nanomaterials may be efficiently used in the development of future personalized medicine and in tissue engineering.

Patterning Pluripotent Stem Cells at a Single Cell Level.

Studies of cell-extracellular matrix (ECM) interactions at a single cell level have drawn interest from scientists around the world. Subcellular ECM micropatterning techniques allow researchers to control cell shape, migration, and spindle orientation during mitosis potentially influencing the stem cell fate. Generally these studies have been limited to somatic cells rather than human pluripotent stem cells (hPSCs) which are capable of enormous differentiation potential. hPSCs require a defined ECM for attachment and express characteristic integrins mediating cell-substrate interactions. hPSCs also rely on cell-cell contacts for survival and to maintain self-renewal properties, but these circumstances also significantly limit hPSC observation at a single cell level. In addition, currently available methods for ECM micropatterning generally require a facility with trained personnel and intricate equipment to produce protein micropatterns. To overcome this problem, we have developed a protocol for vitronectin micropatterning using simple UV/ozone modification of polystyrene. Single hPSCs were able to attach and form characteristic stress fibers and focal adhesions similar to somatic cell types which demonstrate hPSC responsiveness to extracellular adhesive cues. Micropatterned hPSCs were able to be cultured for up to 48 hours while maintaining expression of pluripotency-associated transcription factor OCT4. Although further studies are necessary, the results of our investigation will potentially have a large impact on cell regenerative medicine and tissue engineering.

Erythropoietic differentiation of a human embryonic stem cell line harbouring the sickle cell anaemia mutation.

Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and in-situ immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research.

Challenges and strategies for generating therapeutic patient-specific hemangioblasts and hematopoietic stem cells from human pluripotent stem cells.

Recent characterization of hemangioblasts differentiated from human embryonic stem cells (hESC) has further confirmed evidence from murine, zebrafish and avian experimental systems that hematopoietic and endothelial lineages arise from a common progenitor. Such progenitors may provide a valuable resource for delineating the initial developmental steps of human hemato-endotheliogenesis, which is a process normally difficult to study due to the very limited accessibility of early human embryonic/fetal tissues. Moreover, efficient hemangioblast and hematopoietic stem cell (HSC) generation from patient-specific pluripotent stem cells has enormous potential for regenerative medicine, since it could lead to strategies for treating a multitude of hematologic and vascular disorders. However, significant scientific challenges remain in achieving these goals, and the generation of transplantable hemangioblasts and HSC derived from hESC currently remains elusive. Our previous work has suggested that the failure to derive engraftable HSC from hESC is due to the fact that current methodologies for differentiating hESC produce hematopoietic progenitors developmentally similar to those found in the human yolk sac, and are therefore too immature to provide adult-type hematopoietic reconstitution. Herein, we outline the nature of this challenge and propose targeted strategies for generating engraftable human pluripotent stem cell-derived HSC from primitive hemangioblasts using a developmental approach. We also focus on methods by which reprogrammed somatic cells could be used to derive autologous pluripotent stem cells, which in turn could provide unlimited sources of patient-specific hemangioblasts and HSC.