iPSC Generation Research Articles

Alternative Method for Obtaining Human-Induced Pluripotent Stem Cell Lines and Three-Dimensional Growth: A Simplified, Passage-Free Approach that Minimizes Labor.

Induced pluripotent stem cells (iPSCs) hold significant promise for numerous applications in regenerative medicine, disease modeling, and drug discovery. However, the conventional workflow for iPSC generation, with cells grown under two-dimensional conditions, presents several challenges, including the need for specialized scientific skills such as morphologically assessing and picking colonies and removing differentiated cells during the establishment phase. Furthermore, maintaining established iPSCs in three-dimensional culture systems, while offering scalability, necessitates an enzymatic dissociation step for their further growth in a complex and time-consuming protocol. In this study, we introduce a novel approach to address these challenges by reprogramming somatic cells grown under three-dimensional conditions as spheres using a bioreactor, thereby eliminating the need for two-dimensional culture and colony picking. The iPSCs generated in this study were maintained under three-dimensional conditions simply by transferring spheres to the next bioreactor, without the need for an enzymatic dissociation step. This streamlined method simplifies the workflow, reduces technical variability and labor, and paves the way for future advancements in iPSC research and its wider applications. Key features • Establishment of induced pluripotent stem cells in a three-dimensional environment. • Maintenance and cryopreservation of iPSCs without the need for a dissociation step.

CHIR99021 and Brdu Are Critical in Chicken iPSC Reprogramming via Small-Molecule Screening.

Background/Objectives: Induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells into cells with most of the ESC (embryonic stem cell) characteristics show promise toward solving ethical problems currently facing stem cell research and eventually yield clinical grade pluripotent stem cells for therapies and regenerative medicine. In recent years, an increasing body of research suggests that the chemical induction of pluripotency (CIP) method can yield iPSCs in vitro, yet its application in avian species remains unreported. Methods: Herein, we successfully obtained stably growing chicken embryonic fibroblasts (CEFs) using the tissue block adherence method and employed 12 small-molecule compounds to induce chicken iPSC formation. Results: The final optimized iPSC induction system was bFGF (10 ng/mL), CHIR99021 (3 μM), RepSox (5 μM), DZNep (0.05 μM), BrdU (10 μM), BMP4 (10 ng/mL), vitamin C (50 μg/mL), EPZ-5676 (5 μM), and VPA (0.1 mM). Optimization of the induction system revealed that the highest number of clones was induced with 8 × 104 cells per well and at 1.5 times the original concentration. Upon characterization, these clones exhibited iPSC characteristics, leading to the development of a stable compound combination for iPSC generation in chickens. Concurrently, employing a deletion strategy to investigate the functionality of small-molecule compounds during induction, we identified CHIR99021 and BrdU as critical factors for inducing chicken iPSC formation. Conclusions: In conclusion, this study provides a reference method for utilizing small-molecule combinations in avian species to reprogram cells and establish a network of cell fate determination mechanisms.

Revolutionizing Cancer Treatments through Stem Cell-Derived CAR T Cells for Immunotherapy: Opening New Horizons for the Future of Oncology.

Recent advances in cellular therapies have paved the way for innovative treatments of various cancers and autoimmune disorders. Induced pluripotent stem cells (iPSCs) represent a remarkable breakthrough, offering the potential to generate patient-specific cell types for personalized as well as allogeneic therapies. This review explores the application of iPSC-derived chimeric antigen receptor (CAR) T cells, a cutting-edge approach in allogeneic cancer immunotherapies. CAR T cells are genetically engineered immune cells designed to target specific tumor antigens, and their integration with iPSC technology holds immense promise for enhancing the efficacy, safety, and scalability of cellular therapies. This review begins by elucidating the principles behind iPSC generation and differentiation into T cells, highlighting the advantage of iPSCs in providing a uniform, inexhaustible source of CAR T cells. Additionally, we discuss the genetic modification of iPSC-derived T cells to express various CARs, emphasizing the precision and flexibility this affords in designing customized therapies for a diverse range of malignancies. Notably, iPSC-derived CAR T cells demonstrate a superior proliferative capacity, persistence, and anti-tumor activity compared to their conventionally derived counterparts, offering a potential solution to challenges associated with conventional CAR T cell therapies. In conclusion, iPSC-derived CAR T cells represent a groundbreaking advancement in cellular therapies, demonstrating unparalleled potential in revolutionizing the landscape of immunotherapies. As this technology continues to evolve, it holds the promise of providing safer, more effective, and widely accessible treatment options for patients battling cancer and other immune-related disorders. This review aims to shed light on the transformative potential of iPSC-derived CAR T cells and inspire further research and development in this dynamic field.

The Efficiency of In Vitro Differentiation of Primate iPSCs into Cardiomyocytes Depending on Their Cell Seeding Density and Cell Line Specificity.

A thorough characterization of induced pluripotent stem cells (iPSCs) used with in vitro models or therapeutics is essential. Even iPSCs derived from a single donor can exhibit variability within and between cell lines, which can lead to heterogeneity in results and hinder the promising future of cell replacement therapies. In this study, the cell seeding density of human and rhesus monkey iPSCs was tested to maximize the cell line-specific yield of the generated cardiomyocytes. We found that, despite using the same iPSC generation and differentiation protocols, the cell seeding density for the cell line-specific best differentiation efficiency could differ by a factor of four for the four cell lines used here. In addition, the cell lines showed differences in the range of cell seeding densities that they could tolerate without the severe loss of differentiation efficiency. Overall, our data show that the cell seeding density is a critical parameter for the differentiation inefficiency of primate iPSCs to cardiomyocytes and that iPSCs generated with the same episomal approach still exhibit considerable heterogeneity. Therefore, individual characterization of iPSC lines is required, and functional comparability with in vivo processes must be ensured to warrant the translatability of in vitro research with iPSCs.

A streamlined method to generate endothelial cells from human pluripotent stem cells via transient doxycycline-inducible ETV2 activation.

The development of reliable methods for producing functional endothelial cells (ECs) is crucial for progress in vascular biology and regenerative medicine. In this study, we present a streamlined and efficient methodology for the differentiation of human induced pluripotent stem cells (iPSCs) into induced ECs (iECs) that maintain the ability to undergo vasculogenesis in vitro and in vivo using a doxycycline-inducible system for the transient expression of the ETV2 transcription factor. This approach mitigates the limitations of direct transfection methods, such as mRNA-mediated differentiation, by simplifying the protocol and enhancing reproducibility across different stem cell lines. We detail the generation of iPSCs engineered for doxycycline-induced ETV2 expression and their subsequent differentiation into iECs, achieving over 90% efficiency within four days. Through both in vitro and in vivo assays, the functionality and phenotypic stability of the derived iECs were rigorously validated. Notably, these cells exhibit key endothelial markers and capabilities, including the formation of vascular networks in a microphysiological platform in vitro and in a subcutaneous mouse model. Furthermore, our results reveal a close transcriptional and proteomic alignment between the iECs generated via our method and primary ECs, confirming the biological relevance of the differentiated cells. The high efficiency and effectiveness of our induction methodology pave the way for broader application and accessibility of iPSC-derived ECs in scientific research, offering a valuable tool for investigating endothelial biology and for the development of EC-based therapies.

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

IPS cell generation-associated point mutations include many C > T substitutions via different cytosine modification mechanisms

Genomic aberrations are a critical impediment for the safe medical use of iPSCs and their origin and developmental mechanisms remain unknown. Here we find through WGS analysis of human and mouse iPSC lines that genomic mutations are de novo events and that, in addition to unmodified cytosine base prone to deamination, the DNA methylation sequence CpG represents a significant mutation-prone site. CGI and TSS regions show increased mutations in iPSCs and elevated mutations are observed in retrotransposons, especially in the AluY subfamily. Furthermore, increased cytosine to thymine mutations are observed in differentially methylated regions. These results indicate that in addition to deamination of cytosine, demethylation of methylated cytosine, which plays a central role in genome reprogramming, may act mutagenically during iPSC generation.

Generation of iPSCs from a Patient with the M694V Mutation in the MEFV Gene Associated with Familial Mediterranean Fever and Their Differentiation into Macrophages.

Familial Mediterranean fever (FMF) is a systemic autoinflammatory disorder caused by inherited mutations in the MEFV (Mediterranean FeVer) gene, located on chromosome 16 (16p13.3) and encoding the pyrin protein. Despite the existing data on MEFV mutations, the exact mechanism of their effect on the development of the pathological processes leading to the spontaneous and recurrent autoinflammatory attacks observed in FMF, remains unclear. Induced pluripotent stem cells (iPSCs) are considered an important tool to study the molecular genetic mechanisms of various diseases due to their ability to differentiate into any cell type, including macrophages, which contribute to the development of FMF. In this study, we developed iPSCs from an Armenian patient with FMF carrying the M694V, p.(Met694Val) (c.2080A>G, rs61752717) pathogenic mutation in exon 10 of the MEFV gene. As a result of direct differentiation, macrophages expressing CD14 and CD45 surface markers were obtained. We found that the morphology of macrophages derived from iPSCs of a patient with the MEFV mutation significantly differed from that of macrophages derived from iPSCs of a healthy donor carrying the wild-type MEFV gene.

Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi

Comparisons of molecular phenotypes across primates provide unique information to understand human biology and evolution, and single-cell RNA-seq CRISPR interference (CRISPRi) screens are a powerful approach to analyze them. Here, we generate and validate three human, three gorilla, and two cynomolgus iPS cell lines that carry a dox-inducible KRAB-dCas9 construct at the AAVS1 locus. We show that despite variable expression levels of KRAB-dCas9 among lines, comparable downregulation of target genes and comparable phenotypic effects are observed in a single-cell RNA-seq CRISPRi screen. Hence, we provide valuable resources for performing and further extending CRISPRi in human and non-human primates.

Male-pronuclei-specific granulin facilitates somatic cells reprogramming via mitigating excessive cell proliferation and enhancing lysosomal function.

Critical reprogramming factors resided predominantly in the oocyte or male pronucleus can enhance the efficiency or the quality of induced pluripotent stem cells (iPSCs) induction. However, few reprogramming factors exist in the male pronucleus had been verified. Here, we demonstrated that granulin (Grn), a factor enriched specifically in male pronucleus, can significantly improve the generation of iPSCs from mouse fibroblasts. Grn is highly expressed on Day 1, Day 3, Day 14 of reprogramming induced by four Yamanaka factors and functions at the initial stage of reprogramming. Transcriptome analysis indicates that Grn can promote the expression of lysosome-related genes, while inhibit the expression of genes involved in DNA replication and cell cycle at the early reprogramming stage. Further verification determined that Grn suppressed cell proliferation due to the arrest of cell cycle at G2/M phase. Moreover, ectopic Grn can enhance the lysosomes abundance and rescue the efficiency reduction of reprogramming resulted from lysosomal protease inhibition. Taken together, we conclude that Grn serves as an activator for somatic cell reprogramming through mitigating cell hyperproliferation and promoting the function of lysosomes.

Induced Pluripotent Stem Cells and Organoids in Advancing Neuropathology Research and Therapies.

This review delves into the groundbreaking impact of induced pluripotent stem cells (iPSCs) and three-dimensional organoid models in propelling forward neuropathology research. With a focus on neurodegenerative diseases, neuromotor disorders, and related conditions, iPSCs provide a platform for personalized disease modeling, holding significant potential for regenerative therapy and drug discovery. The adaptability of iPSCs, along with associated methodologies, enables the generation of various types of neural cell differentiations and their integration into three-dimensional organoid models, effectively replicating complex tissue structures in vitro. Key advancements in organoid and iPSC generation protocols, alongside the careful selection of donor cell types, are emphasized as critical steps in harnessing these technologies to mitigate tumorigenic risks and other hurdles. Encouragingly, iPSCs show promising outcomes in regenerative therapies, as evidenced by their successful application in animal models.

Generation of iPSCs from identical twin, one affected by LHON and one unaffected, both carrying a combination of two mitochondrial variants: m.14484 T>C and m.10680G>A

Leber hereditary optic neuropathy (LHON) is one of the most common mitochondrial illness, causing retinal ganglion cell degeneration and central vision loss. It stems from point mutations in mitochondrial DNA (mtDNA), with key mutations being m.3460G > A, m.11778G > A, and m.14484 T > C. Fibroblasts from identical twins, sharing m.14484 T > C and m.10680G > A variants each with 70 % heteroplasmy, were used to generate iPSC lines. Remarkably, one twin, a LHON patient, displayed symptoms, while the other, a carrier, remained asymptomatic. These iPSCs offer a valuable tool for studying factors influencing disease penetrance and unravelling the role of m.10680G > A, which is still debated.

In Vitro Modelling of Osteogenesis Imperfecta with Patient-Derived Induced Mesenchymal Stem Cells.

(1) Mesenchymal stem cells (MSCs) are a valuable cell model to study the bone pathology of Osteogenesis Imperfecta (OI), a rare genetic collagen-related disorder characterized by bone fragility and skeletal dysplasia. We aimed to generate a novel OI induced mesenchymal stem cell (iMSC) model from induced pluripotent stem cells (iPSCs) derived from human dermal fibroblasts. For the first time, OI iMSCs generation was based on an intermediate neural crest cell (iNCC) stage. (2) Skin fibroblasts from healthy individuals and OI patients were reprogrammed into iPSCs and subsequently differentiated into iMSCs via iNCCs. (3) Successful generation of iPSCs from acquired fibroblasts was confirmed with changes in cell morphology, expression of iPSC markers SOX2, NANOG, and OCT4 and three germ-layer tests. Following differentiation into iNCCs, cells presented increased iNCC markers including P75NTR, TFAP2A, and HNK-1 and decreased iPSC markers, shown to reach the iNCC stage. Induction into iMSCs was confirmed by the presence of CD73, CD105, and CD90 markers, low expression of the hematopoietic, and reduced expression of the iNCC markers. iMSCs were trilineage differentiation-competent, confirmed using molecular analyses and staining for cell-type-specific osteoblast, adipocyte, and chondrocyte markers. (4) In the current study, we have developed a multipotent in vitro iMSC model of OI patients and healthy controls able to differentiate into osteoblast-like cells.

A passage-free, simplified, and scalable novel method for iPSC generation in three-dimensional culture

Induced pluripotent stem cells (iPSCs) have immense potential for use in disease modeling, etiological studies, and drug discovery. However, the current workflow for iPSC generation and maintenance poses challenges particularly during the establishment phase when specialized skills are required. Although three-dimensional culture systems offer scalability for maintaining established iPSCs, the enzymatic dissociation step is complex and time-consuming. In this study, a novel approach was developed to address these challenges by enabling iPSC generation, maintenance, and differentiation without the need for two-dimensional culture or enzymatic dissociation. This streamlined method offers a more convenient workflow, reduces variability and labor for technicians, and opens up avenues for advancements in iPSC research and broader applications.

An iPSC model for POLR3A-associated spastic ataxia: Generation of three unrelated patient cell lines

Spastic Ataxias (SA) are a group of neurodegenerative disorders with combined pyramidal and cerebellar system affection, leading to an overlap phenotype between Hereditary Spastic Paraplegias (HSP) and Cerebellar Ataxias (CA). Here we describe the generation of iPSCs from three unrelated patients with an ultra-rare subtype of SA caused by compound heterozygous mutations in POLR3A, that encodes the largest subunit of RNA polymerase III. iPSCs were reprogrammed from normal human dermal fibroblasts (NHDFs) using episomal reprogramming with integration-free plasmid vectors: HIHRSi004-A, derived from a 44 year-old male carrying the mutations c.1909 + 22G > A/c.3944_3945delTG, HIHRSi005-A obtained from a 66 year-old male carrying the mutations c.1909 + 22G > A/c.1531C > T, and HIHRSi006-A from a 27 year-old male carrying the mutations c.1909 + 22G > A/c.2472_2472delC (ENST00000372371.8).

Epigenetic regulation and factors that influence the effect of iPSCs-derived neural stem/progenitor cells (NS/PCs) in the treatment of spinal cord injury.

Spinal cord injury (SCI) is a severe neurological disorder that causes neurological impairment and disability. Neural stem/progenitor cells (NS/PCs) derived from induced pluripotent stem cells (iPSCs) represent a promising cell therapy strategy for spinal cord regeneration and repair. However, iPSC-derived NS/PCs face many challenges and issues in SCI therapy; one of the most significant challenges is epigenetic regulation and that factors that influence this mechanism. Epigenetics refers to the regulation of gene expression and function by DNA methylation, histone modification, and chromatin structure without changing the DNA sequence. Previous research has shown that epigenetics plays a crucial role in the generation, differentiation, and transplantation of iPSCs, and can influence the quality, safety, and outcome of transplanted cells. In this study, we review the effects of epigenetic regulation and various influencing factors on the role of iPSC-derived NS/PCs in SCI therapy at multiple levels, including epigenetic reprogramming, regulation, and the adaptation of iPSCs during generation, differentiation, and transplantation, as well as the impact of other therapeutic tools (e.g., drugs, electrical stimulation, and scaffolds) on the epigenetic status of transplanted cells. We summarize our main findings and insights in this field and identify future challenges and directions that need to be addressed and explored.

A chemical approach facilitates CRISPRa-only human iPSC generation and minimizes the number of targeted loci required.

Aim: We explored the generation of human induced pluripotent stem cells (iPSCs) solely through the transcriptional activation of endogenous genes by CRISPR activation (CRISPRa). Methods: Minimal number of human-specific guide RNAs targeting a limited set of loci were used with a unique cocktail of small molecules (CRISPRa-SM). Results: iPSC clones were efficiently generated by CRISPRa-SM, expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. iPSCs showed genomic stability and robust pluripotent potential as assessed by in vitro and invivo. Conclusion: CRISPRa-SM-generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine.

Inducing Pluripotency in Somatic Cells: Historical Perspective and Recent Advances.

Inducing pluripotency in somatic cells is mediated by the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc. The resulting induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine by virtue of their ability to differentiate into different types of functional cells. Specifically, iPSCs derived directly from patients offer a powerful platform for creating in vitro disease models. This facilitates elucidation of pathological mechanisms underlying human diseases and development of new therapeutic agents mitigating disease phenotypes. Furthermore, genetically and phenotypically corrected patient-derived iPSCs by gene-editing technology or the supply of specific pharmaceutical agents can be used for preclinical and clinical trials to investigate their therapeutic potential. Despite great advances in developing reprogramming methods, the efficiency of iPSC generation remains still low and varies between donor cell types, hampering the potential application of iPSC technology. This paper reviews histological timeline showing important discoveries that have led to iPSC generation and discusses recent advances in iPSC technology by highlighting donor cell types employed for iPSC generation.

The impact of induced pluripotent stem cells in animal conservation.

It is widely acknowledged that we are currently facing a critical tipping point with regards to global extinction, with human activities driving us perilously close to the brink of a devastating sixth mass extinction. As a promising option for safeguarding endangered species, induced pluripotent stem cells (iPSCs) hold great potential to aid in the preservation of threatened animal populations. For endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni), supply of embryos is often limited. After the death of the last male in 2019, only two females remained in the world. IPSC technology offers novel approaches and techniques for obtaining pluripotent stem cells (PSCs) from rare and endangered animal species. Successful generation of iPSCs circumvents several bottlenecks that impede the development of PSCs, including the challenges associated with establishing embryonic stem cells, limited embryo sources and immune rejection following embryo transfer. To provide more opportunities and room for growth in our work on animal welfare, in this paper we will focus on the progress made with iPSC lines derived from endangered and extinct species, exploring their potential applications and limitations in animal welfare research.

Avian iPSC Derivation to Recover Threatened Wild Species: A Comprehensive Review in Light of Well-Established Protocols.

Induced pluripotent stem cells (iPSCs) were first generated by Yamanaka in 2006, revolutionizing research by overcoming limitations imposed by the use of embryonic stem cells. In terms of the conservation of endangered species, iPSC technology presents itself as a viable alternative for the manipulation of target genetics without compromising specimens. Although iPSCs have been successfully generated for various species, their application in nonmammalian species, particularly avian species, requires further in-depth investigation to cover the diversity of wild species at risk and their different protocol requirements. This study aims to provide an overview of the workflow for iPSC induction, comparing well-established protocols in humans and mice with the limited information available for avian species. Here, we discuss the somatic cell sources to be reprogrammed, genetic factors, delivery methods, enhancers, a brief history of achievements in avian iPSC derivation, the main approaches for iPSC characterization, and the future perspectives and challenges for the field. By examining the current protocols and state-of-the-art techniques employed in iPSC generation, we seek to contribute to the development of efficient and species-specific iPSC methodologies for at-risk avian species. The advancement of iPSC technology holds great promise for achieving in vitro germline competency and, consequently, addressing reproductive challenges in endangered species, providing valuable tools for basic research, bird genetic preservation and rescue, and the establishment of cryobanks for future conservation efforts.