• proteins
• proteomics
• viral infection

• Humans
• SARS-CoV-2
• COVID-19
• Proteomics
• Pandemics

• 2274/20 Fundo de Apoio ao Ensino, à Pesquisa e Extensão, Universidade Estadual de Campinas
• 2019/05155-9 Fundação de Amparo à Pesquisa do Estado de São Paulo
• 2020/04746-0 Fundação de Amparo à Pesquisa do Estado de São Paulo
• 2020/04558-0 Fundação de Amparo à Pesquisa do Estado de São Paulo
• 2020/04579-7 Fundação de Amparo à Pesquisa do Estado de São Paulo
• 2020/05040-4 Fundação de Amparo à Pesquisa do Estado de São Paulo
• 2020/04919-2 Fundação de Amparo à Pesquisa do Estado de São Paulo
• 2020/04583-4 Fundação de Amparo à Pesquisa do Estado de São Paulo
• 88887·495565/2020-00 Conselho Nacional de Desenvolvimento Científico e Tecnológico

• Brain organoids
• Human pluripotent stem cells
• Neural cells
• Neuroproteomics
• Schizophrenia

• COVID-19
• NRP1
• SARS-CoV-2
• astrocytes
• neurological symptoms

Dopamine signaling has numerous roles during brain development. In addition, alterations in dopamine signaling may be also involved in the pathophysiology of psychiatric disorders. Neurodevelopment is modulated in multiple steps by reactive oxygen species (ROS), byproducts of oxidative metabolism that are signaling factors involved in proliferation, differentiation, and migration. Hexokinase (HK), when associated with the mitochondria (mt-HK), is a potent modulator of the generation of mitochondrial ROS in the brain. In the present study, we investigated whether dopamine could affect both the activity and redox function of mt-HK in human neural progenitor cells (NPCs). We found that dopamine signaling via D₁R decreases mt-HK activity and impairs ROS modulation, which is followed by an expressive release of H₂O₂ and impairment in calcium handling by the mitochondria. Nevertheless, mitochondrial respiration is not affected, suggesting specificity for dopamine on mt-HK function. In neural stem cells (NSCs) derived from induced-pluripotent stem cells (iPSCs) of schizophrenia patients, mt-HK is unable to decrease mitochondrial ROS, in contrast with NSCs derived from healthy individuals. Our data point to mitochondrial hexokinase as a novel target of dopaminergic signaling, as well as a redox modulator in human neural progenitor cells, which may be relevant to the pathophysiology of neurodevelopmental disorders such as schizophrenia.

• ROS
• dopamine
• mitochondria
• mt-HK
• neurodevelopment

Retinitis pigmentosa is the most common hereditary retinal disease. Dietary supplements, neuroprotective agents, cytokines, and lately, prosthetic devices, gene therapy, and optogenetics have been employed to slow down the retinal degeneration or improve light perception. Completing retinal circuitry by transplanting photoreceptors has always been an appealing idea in retinitis pigmentosa. Recent developments in stem cell technology, retinal imaging techniques, tissue engineering, and transplantation techniques have brought us closer to accomplish this goal. The eye is an ideal organ for cell transplantation due to a low number of cells required to restore vision, availability of safe surgical and imaging techniques to transplant and track the cells in vivo, and partial immune privilege provided by the subretinal space. Human embryonic stem cells, induced pluripotential stem cells, and especially retinal organoids provide an adequate number of cells at a desired developmental stage which may maximize integration of the graft to host retina. However, stem cells must be manufactured under strict good manufacturing practice protocols due to known tumorigenicity as well as possible genetic and epigenetic stabilities that may pose a danger to the recipient. Immune compatibility of stem cells still stands as a problem for their widespread use for retinitis pigmentosa. Transplantation of stem cells from different sources revealed that some of the transplanted cells may not integrate the host retina but slow down the retinal degeneration through paracrine mechanisms. Discovery of a similar paracrine mechanism has recently opened a new therapeutic path for reversing the cone dormancy and restoring the sight in retinitis pigmentosa.

To establish a straightforward single-cell passaging cultivation method that enables high-quality maintenance of human induced pluripotent stem cells without the appearance of karyotypic abnormalities or loss of pluripotency.

Cells were kept in culture for over 50 passages, following a structured chronogram of passage and monitoring cell growth by population doubling time calculation and cell confluence. Standard procedures for human induced pluripotent stem cells monitoring as embryonic body formation, karyotyping and pluripotency markers expression were evaluated in order to assess the cellular state in long-term culture. Cells that underwent these tests were then subjected to differentiation into keratinocytes, cardiomyocytes and definitive endoderm to evaluate its differentiation capacity.

Human induced pluripotent stem cells clones maintained its pluripotent capability as well as chromosomal integrity and were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes, cardiomyocytes and definitive endoderm.

Our findings support the routine of human induced pluripotent stem cells single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy human induced pluripotent stem cells to be used in drug discovery, toxicity, and disease modeling as well as for therapeutic approaches.

• Human induced pluripotent stem cells
• human induced pluripotent stem cells differentiation
• human induced pluripotent stem cells maintenance

• genetic circuit engineering
• synthetic biology
• expression systems

• Animals
• Arabidopsis/enzymology
• Cattle
• Fibroblasts/enzymology
• Humans
• Integrases/genetics
• Integrases/metabolism
• Leukocytes, Mononuclear/enzymology
• Plasmids/genetics
• Promoter Regions, Genetic
• Protoplasts/enzymology
• Recombination, Genetic
• Serine/genetics
• Serine/metabolism

• INCT BioSyn (National Institute of Science and Technology in Synthetic Biology); CNPq (National Council for Scientific and Technological Development); CAPES (Coordination for the Improvement of Higher Education Personnel); Brazilian Ministry of Health; FAPDF (Research Support Foundation of the Federal District)

• brain organoids
• neural cells
• oligodendrocyte progenitors
• proteomics
• stem cells

Ten years after the initial generation of induced pluripotent stem cells (hiPSCs) from human tissues, their potential is no longer questioned, with over 15000 publications listed on PubMed, covering various fields of research; including disease modeling, cell therapy strategies, pharmacology/toxicology screening and 3D organoid systems. However, despite evidences that the presence of mutations in hiPSCs should be a concern, publications addressing genomic integrity of these cells represent less than 1% of the literature. After a first overview of the mutation types currently reported in hiPSCs, including karyotype abnormalities, copy number variations, single point mutation as well as uniparental disomy, this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity. Then, a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest. Finally, in a last section, the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed. We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.

• Induced pluripotent stem cells
• Genomic integrity
• Mutations
• Karyotype
• Differentiation
• Cell therapy
• Quality control
• Reprogramming

• Functional assessment
• Immune privilege
• Photoreceptor
• Pluripotent stem cell
• Retinal degeneration
• Retinal organoid
• Transplantation

• Animals
• Cell Communication/physiology
• Cytoplasm/transplantation
• Humans
• Photoreceptor Cells, Vertebrate/immunology
• Photoreceptor Cells, Vertebrate/transplantation
• Pluripotent Stem Cells/transplantation
• Retinal Degeneration/therapy
• Stem Cell Transplantation/methods
• Stem Cell Transplantation/trends

Astrocytes play a critical role in the development and homeostasis of the central nervous system (CNS). Astrocyte dysfunction results in several neurological and degenerative diseases. However, a major challenge to our understanding of astrocyte physiology and pathology is the restriction of studies to animal models, human post-mortem brain tissues, or samples obtained from invasive surgical procedures. Here, we report a protocol to generate human functional astrocytes from cerebral organoids derived from human pluripotent stem cells. The cellular isolation of cerebral organoids yielded cells that were morphologically and functionally like astrocytes. Immunolabelling and proteomic assays revealed that human organoid-derived astrocytes express the main astrocytic molecular markers, including glutamate transporters, specific enzymes and cytoskeletal proteins. We found that organoid-derived astrocytes strongly supported neuronal survival and neurite outgrowth and responded to ATP through transient calcium wave elevations, which are hallmarks of astrocyte physiology. Additionally, these astrocytes presented similar functional pathways to those isolated from adult human cortex by surgical procedures. This is the first study to provide proteomic and functional analyses of astrocytes isolated from human cerebral organoids. The isolation of these astrocytes holds great potential for the investigation of developmental and evolutionary features of the human brain and provides a useful approach to drug screening and neurodegenerative disease modelling.

Systematic studies of micronutrients during brain formation are hindered by restrictions to animal models and adult post-mortem tissues. Recently, advances in stem cell biology have enabled recapitulation of the early stages of human telencephalon development in vitro. In the present work, we analyzed cerebral organoids derived from human pluripotent stem cells by synchrotron radiation X-ray fluorescence in order to measure biologically valuable micronutrients incorporated and distributed into the exogenously developing brain. Our findings indicate that elemental inclusion in organoids is consistent with human brain tissue and involves P, S, K, Ca, Fe and Zn. Occurrence of different concentration gradients also suggests active regulation of elemental transmembrane transport. Finally, the analysis of pairs of elements shows interesting elemental interaction patterns that change from 30 to 45 days of development, suggesting short- or long-term associations, such as storage in similar compartments or relevance for time-dependent biological processes. These findings shed light on which trace elements are important during human brain development and will support studies aimed to unravel the consequences of disrupted metal homeostasis for neurodevelopmental diseases, including those manifested in adulthood.

• Cerebral organoids
• Development
• Human pluripotent stem cells
• Neurogenesis
• SR-XRF
• Trace elements

• Antidepressant
• Ayahuasca
• DYRK1A
• Proliferation
• hNPC

Human pluripotent stem cells (hPSCs) may significantly improve drug development pipeline, serving as an in vitro system for the identification of novel leads, and for testing drug toxicity. Furthermore, these cells may be used to address the issue of differential drug response, a phenomenon greatly influenced by genetic factors. This application depends on the availability of hPSC lines from populations with diverse ancestries. So far, it has been reported that most lines of hPSCs derived worldwide are of European or East Asian ancestries. We have established 23 lines of hPSCs from Brazilian individuals, and we report the analysis of their genomic ancestry. We show that embryo-derived PSCs are mostly of European descent, while induced PSCs derived from participants of a national-wide Brazilian cohort study present high levels of admixed European, African and Native American genomic ancestry. Additionally, we use high density SNP data and estimate local ancestries, particularly those of CYP genes loci. Such information will be of key importance when interpreting variation among cell lines with respect to cellular phenotypes of interest. The availability of genetically admixed lines of hPSCs will be of relevance when setting up future in vitro studies of drug response.

• Cell metabolism
• DNA damage
• Human neural progenitor cells
• Low oxygen
• Mitochondria
• Reactive oxygen species

Aneuploid embryos diagnosed by FISH-based preimplantation genetic screening (PGS) have been shown to yield euploid lines of human embryonic stem cells (hESCs) with a relatively high frequency. Given that the diagnostic procedure is usually based on the analysis of 1–2 blastomeres of 5 to 10-cell cleavage-stage embryos, mosaicism has been a likely explanation for the phenomena. However, FISH-based PGS can have a significant rate of misdiagnosis, and therefore some of those lines may have been derived from euploid embryos misdiagnosed as aneuploid. More recently, coupling of trophectoderm (TE) biopsy at the blastocyst stage and array-CGH lead to a more informative form of PGS. Here we describe the establishment of a new line of hESCs from an embryo with a 43,XX,dup(9q),+12,-14,-15,-18,-21 chromosomal content based on array-CGH of TE biopsy. We show that, despite the complex chromosomal abnormality, the corresponding hESC line BR-6 is euploid (46,XX). Single nucleotide polymorphism analysis showed that the embryo´s missing chromosomes were not duplicated in BR-6, suggesting the existence of extensive mosaicism in the TE lineage.

• Cell Line
• Cytogenetic Analysis
• Embryo, Mammalian/cytology
• Embryonic Stem Cells/physiology
• Flow Cytometry
• Humans
• In Situ Hybridization, Fluorescence
• Karyotyping
• Ploidies
• Polymorphism, Single Nucleotide/genetics

• amyotrophic lateral sclerosis
• induced pluripotent stem cells
• microarray
• motor neuron differentiation
• sporadic ALS

Great hopes have been placed on human pluripotent stem (hPS) cells for therapy. Tissues or organs derived from hPS cells could be the best solution to cure many different human diseases, especially those who do not respond to standard medication or drugs, such as neurodegenerative diseases, heart failure, or diabetes. The origin of hPS is critical and the idea of creating a bank of well-characterized hPS cells has emerged, like the one that already exists for cord blood. However, the main obstacle in transplantation is the rejection of tissues or organ by the receiver, due to the three main immunological barriers: the human leukocyte antigen (HLA), the ABO blood group, and minor antigens. The problem could be circumvented by using autologous stem cells, like induced pluripotent stem (iPS) cells, derived directly from the patient. But iPS cells have limitations, especially regarding the disease of the recipient and possible difficulties to handle or prepare autologous iPS cells. Finally, reaching standards of good clinical or manufacturing practices could be challenging. That is why well-characterized and universal hPS cells could be a better solution. In this review, we will discuss the interest and the feasibility to establish hPS cells bank, as well as some economics and ethical issues.

• Biomedical Research/legislation & jurisprudence
• Biomedical Research/trends
• Cell Line
• Cluster Analysis
• Computer Simulation
• Embryonic Stem Cells/cytology
• Humans
• Pluripotent Stem Cells/cytology
• Public Policy
• Regenerative Medicine/legislation & jurisprudence
• Regenerative Medicine/trends
• Stem Cell Research/legislation & jurisprudence
• United States

The genomic integrity of two human pluripotent stem cells and their derived neuroprogenitor cell lines was studied, applying a combination of high-resolution genetic methodologies. The usefulness of combining array-comparative genomic hybridization (aCGH) and multiplex fluorescence in situ hybridization (M-FISH) techniques should be delineated to exclude/detect a maximum of possible genomic structural aberrations. Interestingly, in parts different genomic imbalances at chromosomal and subchromosomal levels were detected in pluripotent stem cells and their derivatives. Some of the copy number variations were inherited from the original cell line, whereas other modifications were presumably acquired during the differentiation and manipulation procedures. These results underline the necessity to study both pluripotent stem cells and their differentiated progeny by as many approaches as possible in order to assess their genomic stability before using them in clinical therapies.

• Array-based comparative genomic hybridization (aCGH)
• Fluorescence in situ hybridization (FISH)
• Neuroprogenitor cell lines
• Pluripotent stem cells

• Animals
• Brazil
• Humans
• Private Sector
• Regenerative Medicine/statistics & numerical data
• Stem Cell Research

• Cell Culture Techniques/methods
• Cell Line
• Embryonic Stem Cells/cytology
• Humans

• Animals
• Cell Dedifferentiation
• Cell Differentiation
• Cell Lineage
• Cell Separation/methods
• Cell Survival
• Embryonic Stem Cells/cytology
• Embryonic Stem Cells/immunology
• Embryonic Stem Cells/transplantation
• Fibroblasts/cytology
• Graft Rejection/prevention & control
• Heart/physiology
• Heart Failure/surgery
• Humans
• Mesenchymal Stem Cell Transplantation
• Mice
• Myocardial Contraction
• Myocytes, Cardiac/cytology
• Pluripotent Stem Cells
• Regeneration
• Stem Cell Transplantation/adverse effects
• Transplantation, Homologous

• Animals
• Biological Specimen Banks
• Cell Culture Techniques/methods
• Cell Culture Techniques/standards
• Cell Line
• Culture Media/chemistry
• Embryo Culture Techniques
• Embryo, Mammalian/chemistry
• Embryo, Mammalian/cytology
• Embryonic Stem Cells/chemistry
• Embryonic Stem Cells/cytology
• Embryonic Stem Cells/pathology
• Humans
• Mice
• Pluripotent Stem Cells/chemistry
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/pathology
• Quality Control
• Statistics as Topic
• Teratoma/pathology