The authors would like to thank Dr. Marc Peschanski (I-Stem, &#201;vry, France), for providing the IMR90-hiPSCs; Dr. Giovanna Lazzari and Dr. Silvia Colleoni (Avantea srl, Cremona, Italy); Dr. Simone Haupt (University of Bonn, Germany); Dr. Tiziana Santini (Italian Institute of Technology, Rome), for providing advice on immunofluorescence staining evaluation; Dr. Benedetta Accordi, Dr. Elena Rampazzo, and Dr. Luca Persano (University of Padua, Italy), for their contributions to the RPPA analysis and antibody validation. Funding: this work was supported by the EU-funded project "SCR&amp;Tox" (Grant Agreement N&#176;. 266753).

<ol>
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The authors have nothing to disclose.

This work describes a robust and relatively rapid protocol for the differentiation of IMR90-hiPSCs into post-mitotic neurons and glial cells. Previously published neuronal differentiation protocols based on hESCs and hiPSCs usually yield high percentages of neural precursors25,26 and a significant number of neuronal target cells27,28,29,30,31,32,33. Analogously, the differentiation protocol described here is suitable to generate heterogeneous cultures of GABAergic, glutamatergic, and dopaminergic neuronal cells, together with glia and a discrete proportion of nestin+ cells. The presence of glutamatergic (~35-42%) and GABAergic (~15-20%) neural cells suggests that this culture possesses forebrain, cortical-like features, and the presence of a discrete number of dopaminergic neurons (~13-20%) may also indicate midbrain specificity. Additionally, the permanence of a modest proportion of nestin+ cells may prove suitable for the study of neurogenesis and the possible effects of chemicals on NSCs, which are primarily confined to both the hippocampus and the subventricular zone (SVZ) of the forebrain34. Further immunocytochemical and gene expression analyses would help to better define the regional specificity of the differentiated cell derivatives.
The two most critical steps in the differentiation protocol described in this document are: (i) the cutting of hiPSC colonies into homogenous fragments (which is critical for the generation of EBs with homogenous sizes) and (ii) the cutting of neuroectodermal structures (rosettes) for NSC differentiation, which requires significant manual skill and precision to avoid collecting mesodermal and endodermal cells that may reduce the proportions of neurons and glial cells obtained upon differentiation.
It is crucial to characterize the phenotypes of the cells during expansion (as undifferentiated colonies or NSCs) and during all differentiation steps. In particular, the gene and protein expression profiles of the neuronal/glial cell derivatives should show upregulation and activation of neuron-related signaling pathways, whereas the expression of pluripotency markers should be decreased.
The generation of EBs and neuroectodermal derivatives (rosettes) can be manually challenging and prone to variability. For this reason, we developed a protocol for the expansion of rosette-derived NSCs and their further differentiation into neuronal/glial cells.
Possible limitations of this differentiation protocol are mainly (i) the relatively low percentage of differentiated glial derivatives and (ii) the lack of mature neuronal network functions (as shown by the lack of bursts). Moreover, specific subpopulations of astrocytes can function as primary progenitors or NSCs35. While nestin/GFAP double-positive cells were not observed in this differentiated cell culture (data not shown), it is hypothesized that the GFAP+ cells in these mixed cultures are astrocytic progenitors and astrocytes. It is plausible that by extending the time of differentiation, the number of astrocytes may increase, and their morphology may become more mature, as already indicated by previous works from Zhang&#39;s group36,37.
In the new toxicity testing paradigm, knowledge on chemical-induced perturbations of biological pathways is of the utmost importance when assessing chemical adversity. Therefore, in vitro test systems should be able to relate adverse effects to disturbances of signaling pathways, according to the concept of the adverse outcome pathway (AOP). As a proof-of-concept, rotenone can be used to assess the activation of the Nrf2 pathway, which is involved in the cellular defense against oxidative or electrophilic stress38, and oxidative stress is an important and common key event in various AOPs relevant to developmental and adult neurotoxicity39.
Rotenone should elicit the activation of the Nrf2 pathway, which can be demonstrated by Nrf2 protein nuclear translocation and increased expression of Nrf2-target enzymes, including NQO1 and SRXN1. It has been found that rotenone induces a dose-dependent increase of GFAP protein levels, indicative of astrocyte activation40,41. Rotenone also decreases the number of dopaminergic (TH+) cells, which is in agreement with previous in vitro and in vivo studies showing rotenone-dependent dopaminergic cell death, since this type of neuron is particularly sensitive to oxidative stress21,22,23.
In conclusion, this hiPSC-derived neuronal and glial cell culture model is a valuable tool to assess the neurotoxic effects of chemicals that elicit oxidative stress resulting in Nrf2 pathway activation. As this differentiation protocol allows for the generation of mixed cultures of neuronal cells (GABAergic, dopaminergic, and glutamatergic neurons) and astrocytes, it may prove suitable for studying the crosstalk between neurons and glia in physiological and pathological conditions, such as in neurodegenerative diseases (e.g., Parkinson&#39;s disease). Moreover, the presence of a significant proportion of NSCs may help to assess the possible effects of chemicals on neural progenitors, which are known to be the main target of chemically-induced mutations or viral infections42.

The US National Research Council report1 envisioned a new toxicity testing paradigm in which regulatory toxicity testing would be shifted from an approach relying on phenotypic changes observed in animals to an approach focusing on mechanistic in vitro assays using human cells. Pluripotent stem cell (PSC) derivatives may represent alternatives to cancer cell models, as the obtained cells may more closely resemble the physiological conditions of human tissues and provide more relevant tools to study chemical-induced adverse effects. The two major types of PSC cultures that are most promising for toxicity testing are human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), which are currently widely used in the areas of basic research and regenerative medicine2,3. This expertise can now be harnessed for the development of a new class of toxicological in vitro tests aimed at identifying the perturbed physiological pathways involved with the development of adverse effects in vivo. However, test methods for regulatory safety assessments based on hESCs would be unlikely to be accepted by all EU Member States and by countries worldwide due to possible ethical concerns and diverse national legislative policies regulating the use of embryo-derived cells.
hiPSCs share characteristics similar to hESCs4,5 and hold great potential for in vitro methods, both for identifying therapeutic targets, as well as for safety assessments. In addition, hiPSC technology mitigates the constraints of a limited donor pool and the ethical concerns associated with embryo-derived cells. A major challenge for hiPSCs is the demonstration that these cells can reproducibly generate a significant range of toxicologically relevant cell derivatives, with characteristics and responses typical of human tissues. Predefined levels of the selected markers are generally used to characterize cell populations after the differentiation process and to provide insights into the stability of the differentiation process.
Previous works evaluated the suitability of hiPSCs to generate mixed cultures of neuronal and glial cells and to assess the effects of rotenone, an inhibitor of mitochondrial respiratory complex I, on the activation of the Nrf2 pathway, a key regulator of anti-oxidant defense mechanisms in many cell types6,7.
This work describes a protocol used for the differentiation of hiPSCs into mixed neuronal and glial cultures, providing details on the signaling pathways (gene and protein level) that are activated upon neuronal/glial differentiation. Additionally, the work shows representative results demonstrating how this hiPSC-derived neuronal and glial cell model can be used to assess Nrf2 signaling activation induced by acute (24-h) treatment with rotenone, allowing for the assessment of oxidative stress induction.
IMR90 fibroblasts were reprogrammed into hiPSCs at I-Stem (France) by the viral transduction of 2 transcription factors (Oct4 and Sox2) using pMIG vectors6. Analogous hiPSC models can also be applied. The protocols described below summarize all the stages of differentiation of hiPSCs into neural stem cells (NSCs) and further into mixed cultures of post-mitotic neurons and glial cells (steps 1 and 2, also see the EURL ECVAM DBALM website for a detailed description of the protocol)8.
An additional protocol for the isolation, expansion, cryopreservation, and further differentiation of NSCs into mixed neurons and glial cells is detailed in steps 3 and 4 (also refer to the EURL ECVAM DBALM website for a detailed description of this protocol)9. Step 5 describes the analyses that can be done to assess the phenotypic identity of the cells during the several stages of commitment and differentiation.

protocol for the differentiation of human induced pluripotent stem cells into mixed cultures of neurons and glia for neurotoxicity testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

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Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

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Embryonic and Induced Pluripotent Stem Cells

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22.9K Views.  Joint Research Centre. Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Video: Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...