Highly Defined Induced Pluripotent Stem Cell Lines Mimic Donor Red Blood Cell Antigen Profiles for Therapeutic and Diagnostic Use.

Our group generated two induced pluripotent stem cell (iPSC) lines for in vitro red blood cell (RBC) production from blood donors with extensively known erythrocyte antigen profiles. One line was intended to give rise to RBCs for transfusions in patients with sickle cell disease (SCD), while the other was developed to create RBC panel reagents. Two blood donors were selected based on their RBC phenotypes, further complemented by high-throughput DNA array analysis to obtain a more comprehensive erythrocyte antigen profile. Enriched erythroblast populations from the donors' peripheral blood mononuclear cells were reprogrammed into iPSCs using nonintegrative plasmid vectors. The iPSC lines were characterized and subsequently subjected to hematopoietic differentiation. iPSC PB02 and iPSC PB12 demonstrated in vitro and in vivo iPSC features and retained the genotype of each blood donor's RBC antigen profile. Colony-forming cell assays confirmed that iPSC PB02 and iPSC PB12 generated hematopoietic progenitors. These two iPSC lines were generated with defined erythrocyte antigen profiles, self-renewal capacity, and hematopoietic differentiation potential. With improvements in hematopoietic differentiation, these cells could potentially be more efficiently differentiated into RBCs in the future. They could serve as a complementary approach for obtaining donor-independent RBCs and addressing specific demands for blood transfusions.

CDK5 Deficiency Does not Impair Neuronal Differentiation of Human Induced Pluripotent Stem Cells but Affects Neurite Outgrowth.

Cyclin-dependent kinase 5 (CDK5) is a protein kinase involved in neuronal homeostasis and development critical for neuronal survival. Besides, its deregulation is linked to neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases. For that reason, we aimed to generate a deficient CDK5 genetic model in neurons derived from human-induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 technology. We obtained a heterozygous CDK5+/- clone for the FN2.1 hiPSC line that retained hiPSC stemness and pluripotent potential. Then, neural stem cells (NSCs) and further neurons were derived from the CDK5+/- KO FN2.1 hiPSCs, and their phenotype was validated by immunofluorescence staining using antibodies that recognize lineage-specific markers (SOX-1, SOX-2, and NESTIN for NSCs and TUJ-1, MAP-5, and MAP-2 for neurons). We found that the proliferation rate increased in CDK5+/- KO hiPSC-derived neurons concomitantly with a reduction in NEUN and P35 expression levels. However, the morphometric analysis revealed that CDK5 deficiency caused an increase in the length of the main, primary, and secondary neurites and the neuronal soma area. As a whole, we found that a deficit in CDK5 does not impair hiPSC neuronal differentiation but deregulates proliferation and neurite outgrowth, favoring elongation. The misregulated activity of specific kinases leads to abnormalities such as impaired axonal connectivity in neurodegenerative diseases. Thus, therapeutic approaches aimed at normalizing the activity of kinases, such as CDK5, may help prevent the degeneration of vulnerable neurons.

Alpha-Synuclein Gene Alterations Modulate Tyrosine Hydroxylase in Human iPSC-Derived Neurons in a Parkinson's Disease Animal Model.

Parkinson's disease (PD) caused by SNCA gene triplication (3XSNCA) leads to early onset, rapid progression, and often dementia. Understanding the impact of 3XSNCA and its absence is crucial. This study investigates the differentiation of human induced pluripotent stem cell (hiPSC)-derived floor-plate progenitors into dopaminergic neurons. Three different genotypes were evaluated in this study: patient-derived hiPSCs with 3XSNCA, a gene-edited isogenic line with a frame-shift mutation on all SNCA alleles (SNCA 4KO), and a normal wild-type control. Our aim was to assess how the substantia nigra pars compacta (SNpc) microenvironment, damaged by 6-hydroxydopamine (6-OHDA), influences tyrosine hydroxylase-positive (Th+) neuron differentiation in these genetic variations. This study confirms successful in vitro differentiation into neuronal lineage in all cell lines. However, the SNCA 4KO line showed unusual LIM homeobox transcription factor 1 alpha (Lmx1a) extranuclear distribution. Crucially, both 3XSNCA and SNCA 4KO lines had reduced Th+ neuron expression, despite initial successful neuronal differentiation after two months post-transplantation. This indicates that while the SNpc environment supports early neuronal survival, SNCA gene alterations-either amplification or knock-out-negatively impact Th+ dopaminergic neuron maturation. These findings highlight SNCA's critical role in PD and underscore the value of hiPSC models in studying neurodegenerative diseases.

Comparison of Extracellular Vesicles from Induced Pluripotent Stem Cell-Derived Brain Cells.

The pathophysiology of many neuropsychiatric disorders is still poorly understood. Identification of biomarkers for these diseases could benefit patients due to better classification and stratification. Exosomes excreted into the circulatory system can cross the blood-brain barrier and carry a cell type-specific set of molecules. Thus, exosomes are a source of potential biomarkers for many diseases, including neuropsychiatric disorders. Here, we investigated exosomal proteins produced from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neural stem cells, neural progenitors, neurons, astrocytes, microglia-like cells, and brain capillary endothelial cells. Of the 31 exosome surface markers analyzed, a subset of biomarkers were significantly enriched in astrocytes (CD29, CD44, and CD49e), microglia-like cells (CD44), and neural stem cells (SSEA4). To identify molecular fingerprints associated with disease, circulating exosomes derived from healthy control (HC) individuals were compared against schizophrenia (SCZ) patients and late-onset Alzheimer's disease (LOAD) patients. A significant epitope pattern was identified for LOAD (CD1c and CD2) but not for SCZ compared to HC. Thus, analysis of cell type- and disease-specific exosome signatures of iPSC-derived cell cultures may provide a valuable model system to explore proteomic biomarkers for the identification of novel disease profiles.

Obtaining Tissues of Human Amniotic Membrane and Identification of Pluripotent Markers.

The immunofluorescence technique has been used to identify pluripotent markers in the human amniotic epithelial cells (hAEC). hAEC belonging to human fetal membranes, specificamently to amnion layer, and are arising by epiblast, this sugest that the hAEC have characteristics of epiblast cells, in other words, characteristcs of pluripotent stem cells. Here we describe obtaining human amnion tissue and identifying pluripotent markers by immunofluorescence.

Manufacturing Parameters for the Creation of Clinical-Grade Human-Induced Pluripotent Stem Cell Lines From Umbilical Cord Mesenchymal Stromal Cells.

Induced pluripotent stem cells (iPSCs) are reprogrammed cells with a remarkable capacity for unlimited expansion and differentiation into various cell types. Companies worldwide are actively engaged in developing clinical-grade iPSC lines to address the needs of regenerative medicine, immunotherapies, and precision medicine. However, ensuring the safety and quality of iPSCs is essential, with adherence to Good Manufacturing Practices (GMP) and ethical considerations being paramount. Perinatal cell and tissue banks, such as umbilical cord (UC) blood and tissue banks, are emerging as ideal sources for generating iPSCs due to their unique characteristics and GMP compliance. These banks provide access to immature cells with limited environmental exposure, known family and medical histories of donors, and readily available resources, thereby reducing the time and cost associated with personalized treatment strategies. This study describes the establishment of the first clinical-grade iPSC lines from umbilical cord mesenchymal stromal cells in Brazil. The process involved rigorous quality control measures, safety assessments, and adherence to regulatory standards, resulting in iPSCs with the necessary characteristics for clinical use, including sterility, genomic integrity, and stability. Importantly, the study contributes to the development of a Current Good Manufacturing Practice-compliant iPSC production pipeline in Brazil, using commercially available, chemically defined, and xeno-free products, along with validation by national outsourced laboratories, thereby facilitating the adoption of this technology within the country. The study emphasizes Brazil's contribution to the progress of translational medicine and the promotion of scientific advancements within the field of regenerative and precision medicine.

New Perspectives in Stem Cell Transplantation and Associated Therapies to Treat Retinal Diseases: From Gene Editing to 3D Bioprinting.

Inherited and non-inherited retinopathies can affect distinct cell types, leading to progressive cell death and visual loss. In the last years, new approaches have indicated exciting opportunities to treat retinopathies. Cell therapy in retinitis pigmentosa, age-related macular disease, and glaucoma have yielded encouraging results in rodents and humans. The first two diseases mainly impact the photoreceptors and the retinal pigmented epithelium, while glaucoma primarily affects the ganglion cell layer. Induced pluripotent stem cells and multipotent stem cells can be differentiated in vitro to obtain specific cell types for use in transplant as well as to assess the impact of candidate molecules aimed at treating retinal degeneration. Moreover, stem cell therapy is presented in combination with newly developed methods, such as gene editing, Müller cells dedifferentiation, sheet & drug delivery, virus-like particles, optogenetics, and 3D bioprinting. This review describes the recent advances in this field, by presenting an updated panel based on cell transplants and related therapies to treat retinopathies.

Induced Pluripotent Stem Cells in Drug Discovery and Neurodegenerative Disease Modelling.

Induced pluripotent stem cells (iPSCs) are derived from reprogrammed adult somatic cells. These adult cells are manipulated in vitro to express genes and factors essential for acquiring and maintaining embryonic stem cell (ESC) properties. This technology is widely applied in many fields, and much attention has been given to developing iPSC-based disease models to validate drug discovery platforms and study the pathophysiological molecular processes underlying disease onset. Especially in neurological diseases, there is a great need for iPSC-based technological research, as these cells can be obtained from each patient and carry the individual's bulk of genetic mutations and unique properties. Moreover, iPSCs can differentiate into multiple cell types. These are essential characteristics, since the study of neurological diseases is affected by the limited access to injury sites, the need for in vitro models composed of various cell types, the complexity of reproducing the brain's anatomy, the challenges of postmortem cell culture, and ethical issues. Neurodegenerative diseases strongly impact global health due to their high incidence, symptom severity, and lack of effective therapies. Recently, analyses using disease specific, iPSC-based models confirmed the efficacy of these models for testing multiple drugs. This review summarizes the advances in iPSC technology used in disease modelling and drug testing, with a primary focus on neurodegenerative diseases, including Parkinson's and Alzheimer's diseases.

iPSC-derived cells for whole liver bioengineering.

Liver bioengineering stands as a prominent alternative to conventional hepatic transplantation. Through liver decellularization and/or bioprinting, researchers can generate acellular scaffolds to overcome immune rejection, genetic manipulation, and ethical concerns that often accompany traditional transplantation methods, in vivo regeneration, and xenotransplantation. Hepatic cell lines derived from induced pluripotent stem cells (iPSCs) can repopulate decellularized and bioprinted scaffolds, producing an increasingly functional organ potentially suitable for autologous use. In this mini-review, we overview recent advancements in vitro hepatocyte differentiation protocols, shedding light on their pivotal role in liver recellularization and bioprinting, thereby offering a novel source for hepatic transplantation. Finally, we identify future directions for liver bioengineering research that may allow the implementation of these systems for diverse applications, including drug screening and liver disease modeling.

Induced-pluripotent stem cells and neuroproteomics as tools for studying neurodegeneration.

The investigation of neurodegenerative diseases advanced significantly with the advent of cell-reprogramming technology, leading to the creation of new models of human illness. These models, derived from induced pluripotent stem cells (iPSCs), facilitate the study of sporadic as well as hereditary diseases and provide a comprehensive understanding of the molecular mechanisms involved with neurodegeneration. Through proteomics, a quantitative tool capable of identifying thousands of proteins from small sample volumes, researchers have attempted to identify disease mechanisms by detecting differentially expressed proteins and proteoforms in disease models, biofluids, and postmortem brain tissue. The integration of these two technologies allows for the identification of novel pathological targets within the realm of neurodegenerative diseases. Here, we highlight studies from the past 5 years on the contributions of iPSCs within neuroproteomic investigations, which uncover the molecular mechanisms behind these illnesses.

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.

AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.

AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT.

Parkinson's disease: an update on preclinical studies of induced pluripotent stem cells.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among adults worldwide. It is characterised by the death of dopaminergic neurons in the substantia nigra pars compacta and, in some cases, presence of intracytoplasmic inclusions of α-synuclein, called Lewy bodies, a pathognomonic sign of the disease. Clinical diagnosis of PD is based on the presence of motor alterations. The treatments currently available have no neuroprotective effect. The exact causes of PD are poorly understood. Therefore, more precise preclinical models have been developed in recent years that use induced pluripotent stem cells (iPSC). In vitro studies can provide new information on PD pathogenesis and may help to identify new therapeutic targets or to develop new drugs.

Telomeric DNA breaks in human induced pluripotent stem cells trigger ATR-mediated arrest and telomerase-independent telomere damage repair.

Although mechanisms of telomere protection are well-defined in differentiated cells, it is poorly understood how stem cells sense and respond to telomere dysfunction. In particular, the broader impact of telomeric double-strand breaks (DSBs) in these cells is poorly characterized. Here, we report on DNA damage signaling, cell cycle, and transcriptome-level changes in human induced pluripotent stem cells (iPSCs) in response to telomere-internal DSBs. We engineered human iPSCs with an inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres. Using this model, we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DDR, which leads to p53-independent cell cycle arrest in G2. Using CRISPR-Cas9 to cripple the catalytic domain of telomerase, we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres, which instead are effectively repaired by robust homologous recombination (HR). In contrast to HR-based telomere maintenance in mouse embryonic stem cells, we find neither evidence that HR causes extension of telomeres beyond their initial lengths, nor an apparent role for ZSCAN4 in this process. Rather, HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length which is compatible with sustained survival of the cells over several days of TRF1-FokI induction. Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric damage.

Potential and Limitations of Induced Pluripotent Stem Cells-Derived Mesenchymal Stem Cells in Musculoskeletal Disorders Treatment.

The burden of musculoskeletal disorders (MSK) is increasing worldwide. It affects millions of people worldwide, decreases their quality of life, and can cause mortality. The treatment of such conditions is challenging and often requires surgery. Thus, it is necessary to discuss new strategies. The therapeutic potential of mesenchymal stem cells (MSC) in several diseases has been investigated with relative success. However, this potential is hindered by their limited stemness and expansion ability in vitro and their high donor variability. MSC derived from induced pluripotent stem cells (iPSC) have emerged as an alternative treatment for MSK diseases. These cells present distinct features, such as a juvenile phenotype, in addition to higher stemness, proliferation, and differentiation potential than those of MSC. Here, we review the opportunities, challenges, and applications of iPSC as relevant clinical therapeutic cell sources for MSK disorders. We discuss iPSC sources from which to derive iMSC and the advantages and disadvantages of iMSC over MSC as a therapeutic approach. We further summarize the main preclinical and clinical studies exploring the therapeutic potential of iMSC in MSK disorders.

Application of the Yamanaka Transcription Factors Oct4, Sox2, Klf4, and c-Myc from the Laboratory to the Clinic.

The transcription factors Oct4, Sox2, Klf4, and c-Myc enable the reprogramming of somatic cells into induced pluripotent cells. Reprogramming generates newly differentiated cells for potential therapies in cancer, neurodegenerative diseases, and rejuvenation processes. In cancer therapies, these transcription factors lead to a reduction in the size and aggressiveness of certain tumors, such as sarcomas, and in neurodegenerative diseases, they enable the production of dopaminergic cells in Parkinson's disease, the replacement of affected neuronal cells in olivopontocerebellar atrophy, and the regeneration of the optic nerve. However, there are limitations, such as an increased risk of cancer development when using Klf4 and c-Myc and the occurrence of abnormal dyskinesias in the medium term, possibly generated by the uncontrolled growth of differentiated dopaminergic cells and the impairment of the survival of the new cells. Therefore, the Yamanaka transcription factors have shown therapeutic potential through cell reprogramming for some carcinomas, neurodegenerative diseases, and rejuvenation. However, the limitations found in the studies require further investigation before the use of these transcription factors in humans.

The Impact of Biomaterial Surface Properties on Engineering Neural Tissue for Spinal Cord Regeneration.

Tissue engineering for spinal cord injury (SCI) remains a complex and challenging task. Biomaterial scaffolds have been suggested as a potential solution for supporting cell survival and differentiation at the injury site. However, different biomaterials display multiple properties that significantly impact neural tissue at a cellular level. Here, we evaluated the behavior of different cell lines seeded on chitosan (CHI), poly (ε-caprolactone) (PCL), and poly (L-lactic acid) (PLLA) scaffolds. We demonstrated that the surface properties of a material play a crucial role in cell morphology and differentiation. While the direct contact of a polymer with the cells did not cause cytotoxicity or inhibit the spread of neural progenitor cells derived from neurospheres (NPCdn), neonatal rat spinal cord cells (SCC) and NPCdn only attached and matured on PCL and PLLA surfaces. Scanning electron microscopy and computational analysis suggested that cells attached to the material's surface emerged into distinct morphological populations. Flow cytometry revealed a higher differentiation of neural progenitor cells derived from human induced pluripotent stem cells (hiPSC-NPC) into glial cells on all biomaterials. Immunofluorescence assays demonstrated that PCL and PLLA guided neuronal differentiation and network development in SCC. Our data emphasize the importance of selecting appropriate biomaterials for tissue engineering in SCI treatment.

Current overview of induced pluripotent stem cell-based blood-brain barrier-on-a-chip.

BACKGROUND: Induced pluripotent stem cells (iPSCs) show great ability to differentiate into any tissue, making them attractive candidates for pathophysiological investigations. The rise of organ-on-a-chip technology in the past century has introduced a novel way to make in vitro cell cultures that more closely resemble their in vivo environments, both structural and functionally. The literature still lacks consensus on the best conditions to mimic the blood-brain barrier (BBB) for drug screening and other personalized therapies. The development of models based on BBB-on-a-chip using iPSCs is promising and is a potential alternative to the use of animals in research. AIM: To analyze the literature for BBB models on-a-chip involving iPSCs, describe the microdevices, the BBB in vitro construction, and applications. METHODS: We searched for original articles indexed in PubMed and Scopus that used iPSCs to mimic the BBB and its microenvironment in microfluidic devices. Thirty articles were identified, wherein only 14 articles were finally selected according to the inclusion and exclusion criteria. Data compiled from the selected articles were organized into four topics: (1) Microfluidic devices design and fabrication; (2) characteristics of the iPSCs used in the BBB model and their differentiation conditions; (3) BBB-on-a-chip reconstruction process; and (4) applications of BBB microfluidic three-dimensional models using iPSCs. RESULTS: This study showed that BBB models with iPSCs in microdevices are quite novel in scientific research. Important technological advances in this area regarding the use of commercial BBB-on-a-chip were identified in the most recent articles by different research groups. Conventional polydimethylsiloxane was the most used material to fabricate in-house chips (57%), whereas few studies (14.3%) adopted polymethylmethacrylate. Half the models were constructed using a porous membrane made of diverse materials to separate the channels. iPSC sources were divergent among the studies, but the main line used was IMR90-C4 from human fetal lung fibroblast (41.2%). The cells were differentiated through diverse and complex processes either to endothelial or neural cells, wherein only one study promoted differentiation inside the chip. The construction process of the BBB-on-a-chip involved previous coating mostly with fibronectin/collagen IV (39.3%), followed by cell seeding in single cultures (36%) or co-cultures (64%) under controlled conditions, aimed at developing an in vitro BBB that mimics the human BBB for future applications. CONCLUSION: This review evidenced technological advances in the construction of BBB models using iPSCs. Nonetheless, a definitive BBB-on-a-chip has not yet been achieved, hindering the applicability of the models.

Human pluripotent stem cell-derived extracellular vesicles: From now to the future.

Extracellular vesicles (EVs) are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools. Accordingly, in various biological contexts, EVs are reported to engage in immune modulation, senescence, and cell proliferation and differentiation. Therefore, EVs could be key elements for potential off-the-shelf cell-free therapy. Little has been studied regarding EVs derived from human pluripotent stem cells (hPSC-EVs), even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability. In this review article, we provide an overview of studies using hPSC-EVs, focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs, how they are characterized, and applications already demonstrated. The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs' prospective applications as PSC-derived cell-free therapy products.