Bhlhe40 Regulates Proliferation and Angiogenesis in Mouse Embryoid Bodies under Hypoxia.

Knowledge of the molecular mechanisms that underlie the regulation of major adaptive responses to an unbalanced oxygen tension is central to understanding tissue homeostasis and disease. Hypoxia-inducible transcription factors (HIFs) coordinate changes in the transcriptome that control these adaptive responses. Here, we focused on the functional role of the transcriptional repressor basic-helix-loop-helix family member e40 (Bhlhe40), which we previously identified in a meta-analysis as one of the most consistently upregulated genes in response to hypoxia across various cell types. We investigated the role of Bhlhe40 in controlling proliferation and angiogenesis using a gene editing strategy in mouse embryonic stem cells (mESCs) that we differentiated in embryoid bodies (EBs). We observed that hypoxia-induced Bhlhe40 expression was compatible with the rapid proliferation of pluripotent mESCs under low oxygen tension. However, in EBs, hypoxia triggered a Bhlhe40-dependent cell cycle arrest in most progenitor cells and endothelial cells within vascular structures. Furthermore, Bhlhe40 knockout increased the basal vascularization of the EBs in normoxia and exacerbated the hypoxia-induced vascularization, supporting a novel role for Bhlhe40 as a negative regulator of blood vessel formation. Our findings implicate Bhlhe40 in mediating key functional adaptive responses to hypoxia, such as proliferation arrest and angiogenesis.

BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells.

Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.

Trisomy 12 compromises the mesendodermal differentiation propensity of human pluripotent stem cells.

Trisomy 12 is one of the most frequent chromosomal abnormalities in cultured human pluripotent stem cells (hPSCs). Although potential oncogenic properties and augmented cell cycle caused by trisomy 12 have been reported, the consequences of trisomy 12 in terms of cell differentiation, which is the basis for regenerative medicine, drug development, and developmental biology studies, have not yet been investigated. Here, we report that trisomy 12 compromises the mesendodermal differentiation of hPSCs. We identified sublines of hPSCs carrying trisomy 12 after their prolonged culture. Transcriptome analysis revealed that these hPSC sublines carried abnormal gene expression patterns in specific signaling pathways in addition to cancer-related cell cycle pathways. These hPSC sublines showed a lower propensity for mesendodermal differentiation in embryoid bodies cultured in a serum-free medium. BMP4-induced exit from the self-renewal state was impaired in the trisomy 12 hPSC sublines, with less upregulation of key transcription factor gene expression. As a consequence, the differentiation efficiency of hematopoietic and hepatic lineages was also impaired in the trisomy 12 hPSC sublines. We reveal that trisomy 12 disrupts the genome-wide expression patterns that are required for proper mesendodermal differentiation.

Differentiation of monocytes and polarized M1/M2 macrophages from human induced pluripotent stem cells.

Here, we present a protocol to differentiate induced pluripotent stem cell (iPSC) into adherent hematopoietic progenitors that release floating CD14+ CD45+ monocytes into the culture medium. We describe steps for iPSC expansion, embryoid body (EB) formation, suspension culture, plating EBs, and recurring harvests of monocytes, a.k.a. "monocyte factory." We then describe detailed procedures for freezing/thawing of monocytes and differentiation into polarized M1 and M2 macrophages. This protocol provides foundation to study iPSC monocytes and their progenies such as macrophages, microglial, and dendritic cells. For complete details on the use and execution of this protocol, please refer to Karlson et al.1 and Panicker et al.2.

Modelling post-implantation human development to yolk sac blood emergence.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.

The role of c-Jun for beating cardiomycyte formation in prepared embryonic body.

BACKGROUND: Morbidity and mortality associated with cardiovascular diseases, such as myocardial infarction, stem from the inability of terminally differentiated cardiomyocytes to regenerate, and thus repair the damaged myocardial tissue structure. The molecular biological mechanisms behind the lack of regenerative capacity for those cardiomyocytes remains to be fully elucidated. Recent studies have shown that c-Jun serves as a cell cycle regulator for somatic cell fates, playing a key role in multiple molecular pathways, including the inhibition of cellular reprogramming, promoting angiogenesis, and aggravation of cardiac hypertrophy, but its role in cardiac development is largely unknown. This study aims to delineate the role of c-Jun in promoting early-stage cardiac differentiation. METHODS: The c-Jun gene in mouse embryonic stem cells (mESCs) was knocked out with CRISPR-Cas9, and the hanging drop method used to prepare the resulting embryoid bodies. Cardiac differentiation was evaluated up to 9 days after c-Jun knockout (ko) via immunofluorescence, flow cytometric, and qPCR analyses. RESULTS: Compared to the wild-type control group, obvious beating was observed among the c-Jun-ko mESCs after 6 days, which was also associated with significant increases in myocardial marker expression. Additionally, markers associated with mesoderm and endoderm cell layer development, essential for further differentiation of ESCs into cardiomyocytes, were also up-regulated in the c-Jun-ko cell group. CONCLUSIONS: Knocking out c-Jun directs ESCs toward a meso-endodermal cell lineage fate, in turn leading to generation of beating myocardial cells. Thus, c-Jun plays an important role in regulating early cardiac cell development.

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Stress-free cell aggregation by using the CEPT cocktail enhances embryoid body and organoid fitness.

Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.

Arachidonic acid modulates the cellular energetics of human pluripotent stem cells and protects the embryoid bodies from embryotoxicity effects in vitro.

Arachidonic acid (AA), an ω-6 polyunsaturated fatty acid involved in signalling pathways that drive cell fate decisions, has an enhancing role in the immunomodulatory effect on mesenchymal stem cells and the vasculogenesis of embryonic stem cells. 3D embryoid bodies (EBs) from pluripotent stem cells (PSCs) have been used as in vitro models for embryotoxicity for various compounds/drugs. Valproic acid (VA), a common anti-epileptic drug, is known to be embryotoxic and cause malformations in embryos. As early embryogenesis depends on AA, we investigated the embryo protective effects of AA against the embryotoxic drug VA in this study. The effects of AA on the proliferation and cell cycle parameters of PSCs were studied. In particular, the potential of AA to abrogate VA-induced embryotoxicity in vitro was evaluated using ROS detection and antioxidant assays. In response to AA, we observed modulation in cell proliferation of induced pluripotent stem cells (iPSCs) and pluripotent NTERA-2 embryonal carcinoma (EC) cells. The present study substantiates the cytoprotective effects of AA against VA. These results imply that AA plays a critical role in the proliferation and differentiation of iPSCs and EC cells and protects the EBs from cytotoxic damage, thereby ensuring normal embryogenesis. Thus, the bioactive lipid AA may be explored for supplementation to benefit pregnant women treated with long-term anti-epileptic drugs to prevent in-utero fetal growth malformations.

Generation of a transgenic mouse embryonic stem cell line overexpressing DNMT1.

DNMT1 overexpression is reported in disorders like schizophrenia, bipolar, epilepsy and multiple cancer types. Here, we used non-homologous recombination to generate R1Dnmt1WT-1, a mouse embryonic stem cell (ESC) line carrying a Dnmt1 cDNA transgene to achieve about two-fold overexpression. This ESC line showed increased transcript levels of Sox2 pluripotency marker. R1Dnmt1WT-1 embryoid bodies showed increased levels of Lefty1 (endoderm), Tbxt and Acta2 (mesoderm), and Pax6 (ectoderm) transcripts. This new line showed normal karyotype and microsatellite profiles making it useful in studying carcinogenesis and abnormal neurogenesis due to DNMT1 overexpression.

Ovarian Malignant Mixed Germ Cell Tumor With Prominent Embryoid Bodies (Polyembryoma Background): A Case Report and Literature Review.

Ovarian malignant mixed germ cell tumors are rare tumors occurring in young women. The presence of prominent embryoid bodies in these tumors is extremely uncommon. Herein, we report such a case, with a histomorphologic description and immunohistochemical and fluorescence in situ hybridization analyses.

HDAC8 is implicated in embryoid body formation via canonical Hedgehog signaling and regulates neuronal differentiation.

Histone acetylation and deacetylation are associated with diverse biological phenomena via gene transcription, and histone deacetylases (HDACs) regulate protein deacetylation. HDAC8 is associated with childhood neurological disorders that develop in the uterus and may contribute to neurodevelopment. In our previous studies, we found that HDAC8 regulates neuronal differentiation in P19 pluripotent embryonic carcinoma cells (P19EC cells) by regulating embryoid body (EB) formation. However, the mechanism through which HDAC8 is involved in EB formation and neuronal differentiation remains unclear. Here, we show that HDAC8 regulates EB formation and neuronal differentiation by regulating the canonical Hedgehog (Hh) signaling pathway in P19EC cells. We found that HDAC8 is possibly involved in regulating the expression of the Smoothened receptor (Smo), an important receptor in canonical Hh signaling, and treatment with a Smo agonist restored EB formation ability, which was reduced in HDAC8 knockout P19EC cells. Our results demonstrate that HDAC8 functions in EB formation, which is involved in the Hh signaling pathway that is important for embryonic development.

Resveratrol's neural protective effects for the injured embryoid body and cerebral organoid.

OBJECTIVE: Resveratrol (RSV) is a polyphenol compound found in grapes, veratrum and other plants. It has been reported that RSV has anti-inflammatory, anti-oxidant, anti-cancer and other pharmacological effects. However, the impacts of RSV on development of nervous system are not understood well. The study aims to investigate RSV's neuroprotective effect during development and to provide a health care for pregnant women and their fetuses with RSV supplementation. METHODS: In this study, we induced human induced pluripotent stem cells (hiPSCs) to form the embryoid bodies (EBs) and cerebral organoids (COs) with 3 dimensional (3D) culture. In the meantime, D-galactose (D-gal, 5 mg/ml) was used to make nervous injury model, and on the other hand, RSV with various doses, such as 2 µm/L, 10 µm/L, 50 µm/L, were applied to understand its neuroprotection. Therefore, the cultures were divided into control group, D-gal nervous injury group and RSV intervention groups. After that, the diameters of EBs and COs were measured regularly under a reverted microscope. In the meantime, the neural proliferation, cell apoptosis and the differentiation of germ layers were detected via immunofluorescence. RESULTS: (1) D-gal could delay the development of EBs and COs; (2) RSV could rescue the atrophy of EBs and COs caused by D-gal; (3) RSV showed its neuroprotection, through promoting the neural cell proliferation, inhibiting apoptosis and accelerating the differentiation of germ layers. CONCLUSION: RSV has a neuroprotective effect on the development of the nervous system, suggesting RSV supplementation may be necessary during the health care of pregnancy and childhood.

[Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro].

OBJECTIVE: To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms. METHODS: EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, ß-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment. RESULTS: The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of ß-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, ß-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05). CONCLUSION: Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/ß-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor.

BACKGROUND: hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. METHODS: Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other's (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. RESULTS: A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. CONCLUSION: The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering.

MicroRNAs in Differentiation of Embryoid Bodies and the Teratoma Subtype of Testicular Cancer.

BACKGROUND: Testicular germ cell tumours (TGCTs) are the most frequent tumour type among young, adult men. TGCTs can be efficiently treated, but metastases of the teratoma subtype, for which there are no circulating biomarkers, represent a challenge. MATERIALS AND METHODS: Global microRNA expression in teratoma tissue and embryoid bodies was assessed using next-generation sequencing. Levels of microRNAs identified as potential biomarkers were obtained from serum of patients with teratoma and matched healthy men. RESULTS: We identified miR-222-5p, miR-200a-5p, miR-196b-3p and miR-454-5p as biomarker candidates from the tumour tissue and embryoid body screening but the expression of these microRNAs was very low in serum and not statistically different between patients and controls. miR-375-3p was highly expressed, being highest in patients with teratoma (p=0.012) but the levels of expression in serum from these patients and healthy controls overlapped. miR-371a-3p was not expressed in serum from patients with pure teratoma, only in patients with mixed tumours. CONCLUSION: The microRNA profiles of the teratoma subtype of TGCT and embryoid bodies were obtained and assessed for candidate circulating biomarkers, but none with high sensitivity and specificity for teratoma were identified in our study. We conclude that neither the proposed teratoma marker miR-375-3p nor miR-371a-3p are suitable as circulating teratoma markers.

A microfluidic hanging drop-based spheroid co-culture platform for probing tumor angiogenesis.

Co-culturing of embryoid bodies (EBs) and tumor spheroids (TSs) allows mimicking tumor angiogenesis in vitro. Here, we report a microfluidic hanging drop-based spheroid co-culture device (µ-CCD) that permits the generation and co-culturing of EBs and TSs using a simple manual operation procedure and setup. In brief, uniform-sized EBs and TSs can be generated on the device in eight pairs of hanging droplets from adjacent microfluidic channels, followed by the confrontation of EB and TS pairs by merging the droplet pairs to culture the EB-TS spheroids to investigate tumor-induced angiogenic sprouting. The physical parameters of the device were optimized to maintain the long-term stability of hanging droplets for up to ten days. The mouse embryonic stem cell line ES-D3 and breast cancer cell lines MDA-MB-231 and MCF-7 were used to generate EBs, invasive TSs, and non-invasive TSs respectively. Confocal imaging results showed that the vessel percentage area and total vessel length which are linked to tumor angiogenesis increased after 6 days of co-culturing. An anti-angiogenesis drug testing on the co-cultured EB-TS spheroids was also demonstrated in the device. The µ-CCD provides a simple yet high-efficiency method to generate and co-culture cell spheroids and may also be useful for other applications involving spheroid co-culturing.

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders.

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates.

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 µm from the outer rim of iPSC colonies. Single-cell RNA-sequencing and spatial reconstruction of gene expression demonstrated that OCT4high subsets, residing at the edge of the colony, have pronounced up-regulation of the TGF-ß pathway, particularly of NODAL and its inhibitor LEFTY. Interestingly, after 5-7 days, iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size without enzymatic or mechanical treatment. Within the early 3D aggregates, radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Early self-detached aggregates revealed up-regulated germline-specific gene expression patterns as compared to conventional EBs. However, there were no marked differences after further directed differentiation toward hematopoietic, mesenchymal, and neuronal lineages. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

Embryoid Body Formation from Mouse and Human Pluripotent Stem Cells for Transplantation to Study Brain Microenvironment and Cellular Differentiation.

Human embryonic stem cell (hESC) and human-induced pluripotent stem cell (hiPSC) technologies have a critical role in regenerative strategies for personalized medicine. Both share the ability to differentiate into almost any cell type of the human body. The study of their properties and clinical applications requires the development of robust and reproducible cell culture paradigms that direct cell differentiation toward a specific phenotype in vitro and in vivo. Our group evaluated the potential of mouse ESCs (mESCs), hESCs, and hiPSCs (collectively named pluripotent stem cells, PSCs) to analyze brain microenvironments through the use of embryoid body (EB)-derived cells from these cell sources. EB are cell aggregates in 3D culture conditions that recapitulate embryonic development. Our approach focuses on studying the midbrain dopaminergic phenotype and transplanting EB into the substantia nigra pars compacta (SNpc) in a Parkinson's disease rodent model. Here, we describe cell culture protocols for EB generation from PSCs that show significant in vivo differentiation toward dopaminergic neurons.