Stem Cell


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 1131 Views Sep 5, 2022

Skeletal muscle stem cells differentiated from human-induced pluripotent stem cells (hiPSCs) serve as a uniquely promising model system for investigating human myogenesis and disease pathogenesis, and for the development of gene editing and regenerative stem cell therapies. Here, we present an effective and reproducible transgene-free protocol for derivation of human skeletal muscle stem cells, iMyoblasts, from hiPSCs. Our two-step protocol consists of 1) small molecule-based differentiation of hiPSCs into myocytes, and 2) stimulation of differentiated myocytes with growth factor-rich medium to activate the proliferation of undifferentiated reserve cells, for expansion and cell line establishment. iMyoblasts are PAX3+/MyoD1+ myogenic stem cells with dual potential to undergo muscle differentiation and to self-renew as a regenerative cell population for muscle regeneration both ex vivo and in vivo. The simplicity and robustness of iMyoblast generation and expansion have enabled their application to model the molecular pathogenesis of Facioscapulohumeral Muscular Dystrophy and Limb-Girdle Muscular Dystrophies, to both ex vivo and in vivo muscle xenografts, and to respond efficiently to gene editing, enabling the co-development of gene correction and stem cell regenerative therapeutic technologies for the treatment of muscular dystrophies and muscle injury.

Graphical abstract:

0 Q&A 1344 Views Jul 20, 2022

To optimize differentiation protocols for stem cell-based in vitro modeling applications, it is essential to assess the change in gene expression during the differentiation process. This allows controlling its differentiation efficiency into the target cell types. While RNA transcriptomics provides detail at a larger scale, timing and cost are prohibitive to include such analyses in the optimization process. In contrast, expression analysis of individual genes is cumbersome and lengthy.

Here, we developed a versatile and cost-efficient SYBR Green array of 27 markers along with two housekeeping genes to quickly screen for differentiation efficiency of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons. We first identified relevant pluripotency, neuroprogenitor, and neuronal markers for the array by literature search, and designed primers with a product size of 80-120 bp length, an annealing temperature of 60°C, and minimal predicted secondary structures. We spotted combined forward and reverse primers on 96-well plates and dried them out overnight. These plates can be prepared in advance in batches and stored at room temperature until use. Next, we added the SYBR Green master mix and complementary DNA (cDNA) to the plate in triplicates, ran quantitative PCR (qPCR) on a Quantstudio 6 Flex, and analyzed results with QuantStudio software.

We compared the expression of genes for pluripotency, neuroprogenitor cells, cortical neurons, and synaptic markers in a 96-well format at four different time points during the cortical differentiation. We found a sharp reduction of pluripotency genes within the first three days of pre-differentiation and a steady increase of neuronal markers and synaptic markers over time. In summary, we built a gene expression array that is customizable, fast, medium-throughput, and cost-efficient, ideally suited for optimization of differentiation protocols for stem cell-based in vitro modeling.

0 Q&A 1840 Views Mar 20, 2022

As a model to interrogate human macrophage biology, macrophages differentiated from human induced pluripotent stem cells (hiPSCs) transcend other existing models by circumventing the variability seen in human monocyte-derived macrophages, whilst epitomizing macrophage phenotypic and functional characteristics over those offered by macrophage-like cell lines (Mukherjee et al., 2018). Furthermore, hiPSCs are amenable to genetic manipulation, unlike human monocyte-derived macrophages (MDMs) (van Wilgenburg et al., 2013; Lopez-Yrigoyen et al., 2020), proposing boundless opportunities for specific disease modelling.

We outline an effective and efficient protocol that delivers a continual production of hiPSC-derived-macrophages (iMACs), exhibiting human macrophage surface and intracellular markers, together with functional activity.

The protocol describes the resuscitation, culture, and differentiation of hiPSC into mature terminal macrophages, via the initial and intermediate steps of expansion of hiPSCs, formation into embryoid bodies (EBs), and generation of hematopoietic myeloid precursors.

We offer a simplified, scalable, and adaptable technique that advances upon other protocols, utilizing feeder-free conditions and reduced growth factors, to produce high yields of consistent iMACs over a period of several months, economically.

0 Q&A 1831 Views Nov 5, 2021

High-throughput 3D spheroid formation from human induced pluripotent stem cells (hiPSCs) can be easily performed using the unique microfabric vessels EZSPHERE, resulting in effective and large scale generation of differentiated cells such as cardiomyocytes or neurons. Such hiPSC-derived cardiomyocytes (hiPSC-CMs) or neurons are very useful in the fields of regenerative medicine or cell-based drug safety tests. Previous studies indicated that 3D spheroids arising from hiPSCs are effectively differentiated into high quality hiPSC-CMs by controlling Wnt signals through utilization of the microfabric vessels EZSPHERE. Here, we describe a simple and highly efficient protocol for generating a large number of uniformly sized hiPSC spheroids and inducing them for cardiac differentiation using the EZSPHERE. This method comprises the collection and dissociation of the spheroids from cardiac differentiation medium, in the middle stage of the whole cardiac differentiation process, and re-seeding the obtained single cells into the EZSPHERE to re-aggregate them into uniform hiPSC-CM spheroids with controlled size. This re-aggregation process promotes non-canonical Wnt signal-related cardiac development and improves the purity and maturity of the hiPSC-CMs generated.

Graphic abstract:

Overview of cardiac differentiation from iPSCs by spheroid formation and reaggregation using EZSPHERE.

2 Q&A 5223 Views Mar 5, 2021

The high attrition rate in drug development processes calls for additional human-based model systems. However, in the context of brain disorders, sampling live neuronal cells for compound testing is not applicable. The use of human induced pluripotent stem cells (iPSCs) has revolutionized the field of neuronal disease modeling and drug discovery. Thanks to the development of iPSC-based neuronal differentiation protocols, including tridimensional cerebral organoids, it is now possible to molecularly dissect human neuronal development and human brain disease pathogenesis in a dish. These approaches may allow dissecting patient-specific treatment efficacy in a disease-relevant cellular context. For drug discovery approaches, however, a highly reproducible and cost-effective cell model is desirable. Here, we describe a step-by-step process for generating robust and expandable neural progenitor cells (NPCs) from human iPSCs. NPCs generated with this protocol are homogeneous and highly proliferative. These features make NPCs suitable for the development of high-throughput compound screenings for drug discovery. Human iPSC-derived NPCs show a metabolism dependent on mitochondrial activity and can therefore be used also to investigate neurological disorders in which mitochondrial function is affected. The protocol covers all steps necessary for the preparation, culture, and characterization of human iPSC-derived NPCs.

Graphic abstract:

Schematic of the protocol for the generation of human iPSC-derived NPCs

0 Q&A 3303 Views Dec 20, 2020

Defects in bone resorption by osteoclasts result in numerous rare genetic bone disorders as well as in some common diseases such as osteoporosis or osteopetrosis. The use of hiPSC-differentiated osteoclasts opens new avenues in this research field by providing an unlimited cell source and overcoming obstacles such as unavailability of human specimens and suitable animal models. Generation of hiPSCs is well established but efficient differentiation of hiPSCs into osteoclasts has been challenging. Published hiPSC-osteoclast differentiation protocols use a hiPSC-OP9 co-culture system or hiPSC-derived embryoid bodies (EBs) with multiple cytokines. Our three-stage protocol consists of 1) EB mesoderm differentiation, 2) expansion of myelomonocytic cells and 3) maturation of hiPSC-osteoclasts. We generate uniformly-sized EBs by culturing Accutase-dissociated hiPSCs on Nunclon Sphera microplates and promote EB mesoderm differentiation in a cytokine cocktail for 4 days. For Stage 2, EBs are transferred to gelatin-coated plates and cultured with hM-CSF and hIL-3 to expand the myelomonocytic population. By supplementing with vitamin D, hTGFβ, hM-CSF and hRANKL, cells collected at the end of Stage 2 are differentiated into mature osteoclasts (Stage 3). Compared to other techniques, our protocol does not require a co-culture system; induces EBs into mesoderm differentiation in a homogenous manner; uses less cytokines for differentiation; requires only a short time for osteoclast maturation and produces sufficient numbers of osteoclasts for subsequent molecular analyses.

Graphic abstract

1 Q&A 5642 Views Sep 20, 2020
Induced pluripotent stem cell derived cardiovascular progenitor cells (iPSC-CVPCs) provide an unprecedented platform for examining the molecular underpinnings of cardiac development and disease etiology, but also have great potential to play pivotal roles in the future of regenerative medicine and pharmacogenomic studies. Biobanks like iPSCORE ( Stacey et al., 2013; Panopoulos et al., 2017), which contain iPSCs generated from hundreds of genetically and ethnically diverse individuals, are an invaluable resource for conducting these studies. Here, we present an optimized, cost-effective and highly standardized protocol for large-scale derivation of human iPSC-CVPCs using small molecules and purification using metabolic selection. We have successfully applied this protocol to derive iPSC-CVPCs from 154 different iPSCORE iPSC lines obtaining large quantities of highly pure cardiac cells. An important component of our protocol is Cell confluency estimates (ccEstimate), an automated methodology for estimating the time when an iPSC monolayer will reach 80% confluency, which is optimal for initiating iPSC-CVPC derivation, and enables the protocol to be readily used across iPSC lines with different growth rates. Moreover, we showed that cellular heterogeneity across iPSC-CVPCs is due to varying proportions of two distinct cardiac cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs), both of which have been shown to have a critical function in heart regeneration. This protocol eliminates the need of iPSC line-to-line optimization and can be easily adapted and scaled to high-throughput studies or to generate large quantities of cells suitable for regenerative medicine applications.
0 Q&A 4458 Views Sep 5, 2020
Human neuron transplantation offers novel opportunities for modeling human neurologic diseases and potentially replacement therapies. However, the complex structure of the human cerebral cortex, which is organized in six layers with tightly interconnected excitatory and inhibitory neuronal networks, presents significant challenges for in vivo transplantation techniques to obtain a balanced, functional and homeostatically stable neuronal network. Here, we present a protocol to introduce human induced pluripotent stem cell (hiPSC)-derived neural progenitors to rat brains. Using this approach, hiPSC-derived neurons structurally integrate into the rat forebrain, exhibit electrophysiological characteristics, including firing, excitatory and inhibitory synaptic activity, and establish neuronal connectivity with the host circuitry.
0 Q&A 8027 Views Feb 5, 2020
Developing protocols to obtain intestinal epithelial monolayers that recapitulate in vivo physiology to overcome the limitations of the organoids’ closed geometry has become of great interest during the last few years. Most of the developed culture models showed physiological-relevant cell composition but did not prove self-renewing capacities. Here, we show a simple method to obtain mouse small intestine-derived epithelial monolayers organized into proliferative crypt-like domains, containing stem cells, and differentiated villus-like regions, closely resembling the in vivo cell composition and distribution. In addition, we adapted our model to a tissue culture format compatible with functional studies and prove close to physiological barrier properties of our in vitro epithelial monolayers. Thus, we have set-up a protocol to generate physiologically relevant intestinal epithelial monolayers to be employed in assays where independent access to both luminal and basolateral compartments is needed, such as drug absorption, intracellular trafficking and microbiome-epithelium interaction assays.
0 Q&A 5931 Views Dec 20, 2019
Induced Pluripotent Stem Cells (iPSCs) serve as an excellent model system for studying the molecular underpinnings of tissue development. Human iPSC-derived retinal pigment epithelium (iPSC-RPE) cells have fetal-like molecular profiles. Hence, biobanks like iPSCORE, which contain iPSCs generated from hundreds of individuals, are an invaluable resource for examining how common genetic variants exert their effects during RPE development resulting in individuals having different propensities to develop Age-related Macular Degeneration (AMD) as adults. Here, we present an optimized, cost-effective and highly reproducible protocol for derivation of human iPSC-RPE cells using small molecules under serum-free condition and for their quality control using flow cytometry and immunofluorescence. While most previous protocols have required laborious manual selection to enrich for iPSC-RPE cells, our protocol uses whole culture passaging and yields a large number of iPSC-RPE cells with high purity (88-98.1% ZO-1 and MiTF double positive cells). The simplicity and robustness of this protocol would enable its adaption for high-throughput applications involving the generation of iPSC-RPE samples from hundreds of individuals.

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