In the pursuit of identifying the underlying pathways of ocular diseases, the use of cell lines such as (retinal ganglion cell-5) RGC-5 and 661W became a valuable tool, including pathologies like retinal degeneration and glaucoma. In 2001, the establishment of the RGC-5 cell line marked a significant breakthrough in glaucoma research. Over time, however, concerns arose about the true nature of RGC-5 cells, with conflicting findings in the literature regarding their identity as retinal ganglion cells or photoreceptor-like cells. This study aimed to address the controversy surrounding the RGC-5 cell line\'s origin and properties by comparing it with the 661W cell line, a known cone photoreceptor model. Both cell lines were differentiated according to two prior published redifferentiation protocols under the same conditions using 500 nM of trichostatin A (TSA) and investigated for their morphological and neuronal marker properties. The results demonstrated that both cell lines are murine, and they exhibited distinct morphological and neuronal marker properties. Notably, the RGC-5 cells showed higher expression of the neuronal marker β-III tubulin and increased Thy-1-mRNA compared with the 661W cells, providing evidence of their different properties. The findings emphasize the importance of verifying the authenticity of cell lines used in ocular research and highlight the risks of contamination and altered cell properties.

About 15% of patients with parkinsonism have a hereditary form of Parkinson\'s disease (PD). Studies on the early stages of PD pathogenesis are challenging due to the lack of relevant models. The most promising ones are models based on dopaminergic neurons (DAns) differentiated from induced pluripotent stem cells (iPSCs) of patients with hereditary forms of PD. This work describes a highly efficient 2D protocol for obtaining DAns from iPSCs. The protocol is rather simple, comparable in efficiency with previously published protocols, and does not require viral vectors. The resulting neurons have a similar transcriptome profile to previously published data for neurons, and have a high level of maturity marker expression. The proportion of sensitive (SOX6+) DAns in the population calculated from the level of gene expression is higher than resistant (CALB+) DAns. Electrophysiological studies of the DAns confirmed their voltage sensitivity and showed that a mutation in the PARK8 gene is associated with enhanced store-operated calcium entry. The study of high-purity DAns differentiated from the iPSCs of patients with hereditary PD using this differentiation protocol will allow for investigators to combine various research methods, from patch clamp to omics technologies, and maximize information about cell function in normal and pathological conditions.

In the peripheral nervous system, Schwann cells (SCs) play a crucial role in axonal growth, metabolic support of neurons, and the production of myelin sheaths. Expansion of SCs after extraction from human or animal nerves is a long and often low-yielding process. We established a rapid cell culture method using a defined serum-free medium to differentiate human induced pluripotent stem cells (iPSCs) into SCs in only 21 days. The SC identity was characterized by expression of SRY-Box Transcription factor 10 (SOX10), S100b, glial fibrillary acidic protein (GFAP), P75, growth-associated protein 43 (GAP43), and early growth response 2 (EGR2) markers. The SC purity reached 87% as assessed by flow cytometry using the specific SOX10 marker, and 69% based on S100b expression. When SCs were cocultured with iPSC-derived motor neurons two-dimensionally or three-dimensionally (3D), they also expressed the markers of myelin MBP, MPZ, and gliomedin. Likewise, when they were seeded on the opposite side of a porous collagen sponge from motor neurons in the 3D model, they were able to migrate through it and colocalize with motor axons after 8 weeks of maturation. Moreover, they were shown by transmission electron microscopy to form myelin sheaths around motor axons. These results suggest that the use of autologous iPSC-derived SCs for clinical applications such as the repair of peripheral nerve damage, the treatment of spinal cord injuries, or for demyelinating diseases could be a valuable option. Impact Statement Peripheral nerve injuries can cause the complete paralysis of the upper or lower limbs, which considerably reduces the quality of life of patients. To repair this injury, many approaches have been developed by tissue engineering. Combining biomaterials with Schwann cells (SCs) has been shown to be an effective solution for stimulating nerve regeneration. However, the challenge faced concerns the strategy for obtaining autologous SCs to treat patients. A promising approach is to differentiate them from the patient\'s own cells, previously induced into pluripotent stem cells. We propose a fast culture method to generate functional SCs differentiated from induced pluripotent stem cells.

The multipotent pancreatic progenitor cells (MPCs) co-expressing the transcription factors, PDX1 and NKX6.1, are the source of functional pancreatic β-cells. The aim of this study was to examine the effect of p53 inhibition in MPCs on the generation of PDX1+/NKX6.1+ MPCs and pancreatic β-cell generation. Human embryonic stem cells (hESCs) were differentiated into MPCs and β-cells. hESC-MPCs (stage 4) were treated with different concentrations of p53 inhibitors, and their effect was evaluated using different approaches. NKX6.1 was overexpressed during MPCs specification. Inhibition of p53 using pifithrin-μ (PFT-μ) at the MPC stage resulted in a significant increase in the number of PDX1+/NKX6.1+ cells and a reduction in the number of CHGA+/NKX6.1- cells. Further differentiation of MPCs treated with PFT-μ into pancreatic β-cells showed that PFT-μ treatment did not significantly change the number of C-Peptide+ cells; however, the number of C-PEP+ cells co-expressing glucagon (polyhormonal) was significantly reduced in the PFT-μ treated cells. Interestingly, overexpression of NKX6.1 in hESC-MPCs enhanced the expression of key MPC genes and dramatically suppressed p53 expression. Our findings demonstrated that the p53 inhibition during stage 4 of differentiation enhanced MPC generation, prevented premature endocrine induction and favored the differentiation into monohormonal β-cells. These findings suggest that adding a p53 inhibitor to the differentiation media can significantly enhance the generation of monohormonal β-cells.

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modeling of hard-to-access tissues (such as the brain). Current protocols either direct neuronal differentiation with small molecules or use transcription-factor-mediated programming. In this study, we couple overexpression of transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced motor neurons (liMoNes/liMNs). This approach induces canonical MN markers including MN-specific Hb9/MNX1 in more than 95% of cells. liMNs resemble bona fide hPSC-derived MN, exhibit spontaneous electrical activity, express synaptic markers, and can contact muscle cells in vitro. Pooled, multiplexed single-cell RNA sequencing on 50 hPSC lines reveals reproducible populations of distinct subtypes of cervical and brachial MNs that resemble their in vivo, embryonic counterparts. Combining small molecule patterning with Ngn2 overexpression facilitates high-yield, reproducible production of disease-relevant MN subtypes, which is fundamental in propelling our knowledge of MN biology and its disruption in disease.

In order to use induced Pluripotent Stem Cells (iPSCs) to model neurodegenerative diseases, efficient and homogeneous generation of neurons in vitro represents a key step. Here we describe a method to obtain and characterize functional human spinal and cranial motoneurons using a combined approach of microfluidic chips and programs designed for scientific multidimensional imaging. We have used this approach to analyze axonal phenotypes. These tools are useful to investigate the cellular and molecular bases of neuromuscular diseases, including amyotrophic lateral sclerosis and spinal muscular atrophy.

Melanin is a black/brown pigment present in abundance in human skin. Its main function is photo-protection of underlying tissues from harmful UV light. Natural sources of isolated human melanin are limited; thus, in vitro cultures of human cells may be a promising source of human melanin. Here, we present an innovative in vitro differentiation protocol of induced pluripotent stem cells (iPS) into melanin-producing cells, delivering highly pigmented cells in quantity and quality incomparably higher than any other methods previously described. Pigmented cells constitute over 90% of a terminally differentiated population and exhibit features characteristic for melanocytes, i.e., expression of specific markers such as MITF-M (microphthalmia-associated transcription factor isoform M), TRP-1 (tyrosinase-related protein 1), and TYR (tyrosinase) and accumulation of black pigment in organelles closely resembling melanosomes. Black pigment is unambiguously identified as melanin with features corresponding to those of melanin produced by typical melanocytes. The advantage of our method is that it does not require any sophisticated procedures and can be conducted in standard laboratory conditions. Moreover, our protocol is highly reproducible and optimized to generate high-purity melanin-producing cells from iPS cells; thus, it can serve as an unlimited source of human melanin for modeling human skin diseases. We speculate that FGF-8 might play an important role during differentiation processes toward pigmented cells.

Oligodendrocytes are the main glial cell type in the central nervous system supporting the axonal part of neurons via myelin and lactate delivery. Both the conductive myelin formation and the energy support via lactate can be affected in diseases, such as multiple sclerosis and amyotrophic lateral sclerosis, respectively. Therefore, human disease modeling is needed to gain more mechanistic insights to drive drug discovery research. Here, patient-derived induced pluripotent stem cells (iPSCs) serve as a necessary tool providing an infinite cell source for patient-specific disease modeling, which allows investigation of oligodendrocyte involvement in human disease.Small molecule-based differentiation protocols to generate oligodendrocytes from pluripotent stem cells can last more than 90 days. Here, we provide a transcription factor-based, fast and efficient protocol for generating O4+ oligodendrocytes in just 20-24 days. After a neural induction phase of 8-12 days, SOX10 is overexpressed either with the use of lentiviral vectors or via engineered iPSCs, which inducibly overexpress SOX10 after doxycycline addition. Using this last method, a pure O4+ cell population is achieved after keeping the SOX10-overexpressing neural stem cells in culture for an additional 10 days. Furthermore, these O4+ cells can be co-cultured with iPSC-derived cortical neurons in 384-well format, allowing pro-myelinating drug screens. In conclusion, we provide a fast and efficient oligodendrocyte differentiation protocol allowing both in vitro human disease modeling and a high-throughput co-culture system for drug discovery.

Astrocytes, the main supportive cell type of the brain, show functional impairments upon ageing and in a broad spectrum of neurological disorders. Limited access to human astroglia for pre-clinical studies has been a major bottleneck delaying our understanding of their role in brain health and disease. We demonstrate here that functionally mature human astrocytes can be generated by SOX9 overexpression for 6 days in pluripotent stem cell (PSC)-derived neural progenitor cells. Inducible (i)SOX9-astrocytes display functional properties comparable to primary human astrocytes comprising glutamate uptake, induced calcium responses and cytokine/growth factor secretion. Importantly, electrophysiological properties of iNGN2-neurons co-cultured with iSOX9-astrocytes are indistinguishable from gold-standard murine primary cultures. The high yield, fast timing and the possibility to cryopreserve iSOX9-astrocytes without losing functional properties makes them suitable for scaled-up production for high-throughput analyses. Our findings represent a step forward to an all-human iPSC-derived neural model for drug development in neuroscience and towards the reduction of animal use in biomedical research.

Generation of kidney organoids from pluripotent stem cells (PSCs) is regarded as a potentially powerful way to study kidney development, disease, and regeneration. Direct differentiation of PSCs towards renal lineages is well studied; however, most of the studies relate to generation of nephron progenitor population from PSCs. Until now, differentiation of PSCs into ureteric bud (UB) progenitor cells has had limited success. Here, we describe a simple, efficient, and reproducible protocol to direct differentiation of mouse embryonic stem cells (mESCs) into UB progenitor cells. The mESC-derived UB cells were able to induce nephrogenesis when co-cultured with primary metanephric mesenchyme (pMM). In generated kidney organoids, the embryonic pMM developed nephron structures, and the mESC-derived UB cells formed numerous collecting ducts connected with the nephron tubules. Altogether, our study established an uncomplicated and reproducible platform to generate ureteric bud progenitors from mouse embryonic stem cells.