iPSCs

Dua tokoh utama adalah yang sangat populer menjembatani pengetahuan medis yang sangat reduksionis dan basic science, biolgi, yaitu Sir John Gurdon dan Shinya Yamanaka. Sir John Gurdon adalah professeor biologi pertama yang mendapatkan sel embrional dari transplantasi inti sel intestinal katak ke sel telur yang dienukleasi. Hal ini membuka pandangan manusia bahwa faktor-faktor tertentu di sel-sel yang belum terdiferensiasi dapat memunculkan sifat embrional layaknya sel-sel embri. Shinya Yamanaka adalah seorang dokter bedah ortopedi Osaka dan Ph.D. di bidang biologi molekuler. Yamanaka mendapat tantangan untuk menciptakan sesuatu yang unik dan sangat menantang ketika melamar pekerjaan di universitas, dan dengan idenya untuk mendapatkan kembali sel punsa dari sel-sel yang telah terdiferensiasi, Yamanaka akhirnya menemukan 4 faktor ranskripsi utama yang bertanggung jawab terhadap pluripotensi adalah Oct3/4, Sox1, cMyc, Kif4 yang tidak dimiliki oleh sel-sel terdiferensiasi. Dengan pengetahuan tersebut mereka pertama kali menemukan sel-sel pluripotesi dari sel-sel yang telah terdiferensiasi. Mereka mendapatkan hadiah nobel di tahun 2012.

Oculocutaneous albinism (OCA) is a group of hereditary recessive disorder recognized as a loss of pigmentation. OCA can derive from mutations in different genes that produce melanin. These mutations cause disturbances to get a standard melanin synthesis. There are 7 types of oculocutaneous albinism. These include OCA1, OCA2, OCA3, OCA4, OCA5, OCA6, and OCA7. To help OCA patients, it may include management of such as hats with brims and sunscreens. An effective therapy is unavailable for albinism at present. However, to fight OCA in the future, gene therapy can be used. Gene therapy can include use of such as retrovirus vectors, adenovirus vectors, and CRISPR/Cas9 system. Research results in animal models have shown remarkable advances. It means that the gene therapy will be helpful to treat people with albinism.

Human induced pluripotent stem cells (hiPSCs) provide a powerful platform for disease modeling and have unlocked new possibilities for understanding the mechanisms governing human biology, physiology, and genetics. However, hiPSC-derivatives have traditionally been utilized in two-dimensional monocultures, in contrast to the multi-systemic interactions that influence cells in the body. We will discuss recent advances in generating more complex hiPSC-based systems including three-dimensional organoids, tissue-engineered constructs, microfluidic organ-chip platforms, and humanized animal systems. While hiPSC differentiation still requires optimization, these next-generation multi-lineage technologies can augment the biomedical researcher’s toolkit and enable more realistic models of human tissue function.

Human-induced pluripotent stem cells (hiPSCs) and CRISPR/Cas9 gene editing system represent two instruments of basic and translational research, which both allow to acquire deep insight about the molecular bases of many diseases but also to develop pharmacological research.This review is focused to draw up the latest technique of gene editing applied on hiPSCs, exploiting some of the genetic manipulation directed to the discovery of innovative therapeutic strategies. There are many expediencies provided by the use of hiPSCs, which can represent a disease model clinically relevant and predictive, with a great potential if associated to CRISPR/Cas9 technology, a gene editing tool powered by ease and precision never seen before.Here, we describe the possible applications of CRISPR/Cas9 to hiPSCs: from drug development to drug screening and from gene therapy to the induction of the immunological response to specific virus infection, such as HIV and SARS-Cov-2.

This review summarizes the structural and electrophysiological changes in the epileptic hippocampus in various animal models of epilepsy. Tornado hypothesis of epileptic hippocampus, states that there are many progressive detrimental changes in the hippocampus of the epileptic rats. PTZ, PTZ kindling, pilocarpine, electrical kindling, kainic acid, hyperthermia and in vitro models are taken as primary animal epilepsy models in this review. Paired pulse inhibition and GABAergic transmission is decreased in dentate gyrus (DG), CA3 and CA1 regions of the epileptic hippocampus. There is interneuron loss in the hilus, DG and CA3, while excitatory input from perforant path (PP) is increased and this is fed into the CA3 area by mossy fibers (MF) which have excessive sprouting that forms novel recurrent synapses with the pyramidal cells of CA3. Gate function of DG is either impaired or lost. CA3 area of hippocampus becomes an epileptic focus and sends ictal discharges. These ictal discharges are carried into CA1 by Schaffer collaterals and then into entorhinal cortex (EC). The input from EC is amplified in the hippocampal circuitry which is fed into hippocampus from EC into DG again successively. Thus, a weak electrical input into the hippocampal formation results in an amplified signal back into EC. Since the basic ultrastructural and electrophysiological feedback control mechanisms are impaired, this electrical tornado cannot be compensated for and an epileptic amplified ictal discharge spreads to the limbic system and other adjacent structures of the brain. Eventually the hippocampal circuitry, that has developed a vicious circle, becomes a bio-electrical amplifier which triggers an electrical tornado, under certain biochemical conditions. mossy fiber sprouting, in vitro model of epilepsy, perforant path, tonic clonic seizures, intractable temporal lobe epilepsy.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two age-dependent multifactorial neurodegenerative disorders, which are typically characterized by the selective death of motor neurons and cerebral cortex neurons, respectively. These two diseases share many clinical, genetic and pathological aspects. During the past decade, cell reprogramming technologies enabled researchers to generate human induced pluripotent stem cells (iPSCs) from somatic cells. This resulted in the unique opportunity to obtain specific neuronal and non-neuronal cell types from patients which could be used for basic research. Moreover, these in vitro models can mimic not only the familial forms of ALS/FTD, but also sporadic cases without known genetic cause. At present, there have been extensive technical advances in the generation of iPSCs, as well as in the differentiation procedures to obtain iPSC-derived motor neurons, cortical neurons and non-neuronal cells. The major challenge at ...

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two age-dependent multifactorial neurodegenerative disorders, which are typically characterized by the selective death of motor neurons and cerebral cortex neurons, respectively. These two diseases share many clinical, genetic and pathological aspects. During the past decade, cell reprogramming technologies enabled researchers to generate human induced pluripotent stem cells (iPSCs) from somatic cells. This resulted in the unique opportunity to obtain specific neuronal and non-neuronal cell types from patients which could be used for basic research. Moreover, these in vitro models can mimic not only the familial forms of ALS/FTD, but also sporadic cases without known genetic cause. At present, there have been extensive technical advances in the generation of iPSCs, as well as in the differentiation procedures to obtain iPSC-derived motor neurons, cortical neurons and non-neuronal cells. The major challenge at this moment is to determine whether these iPSC-derived cells show relevant phenotypes that recapitulate complex diseases. In this review, we will summarize the work related to iPSC models of ALS and FTD. In addition, we will discuss potential drawbacks and solutions for establishing more trustworthy iPSC models for both ALS and FTD.

Age related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease, however these do not exist for the dry form. Complement factor H (CFH) polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, retinal pigment epithelium (RPE) damage and visual decline. We have derived and characterised induced pluripotent stem cell (iPSCs) lines from two patients without AMD and low risk genotype and two pa‐ tients with advanced AMD and high risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H (FH), factor I (FI) and factor H like 1 (FHL‐1). The iPSC RPE cells derived from high risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy and deposition of " drüsen " like depos‐ its. The low and high risk RPE cells respond differently to intermittent ex‐ posure to UV light which leads to an improvement in cellular and function‐ al phenotype only in the high risk AMD‐RPE cells. Taken together our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. STEM CELLS 2017; 00:000–000 SIGNIFICANCE STATEMENT: Age related macular degeneration (AMD) is the one of the most common forms of blindness. Drugs that treat wet AMD have been a major break‐ through; however there is currently no treatment for the dry form. One of the problems with studying AMD is that the affected retinal tissue is difficult to obtain, there are no animal models that faithfully mimic the disease and human trials are long and costly. Herein, we report creation of a disease

The discovery of induced pluripotent stem cells (iPSCs) in 2006 has inspired almost all areas of the life sciences. In the first report about iPSCs from Takahashi & Yamanaka, terminally differentiated cells (adult skin fibroblasts)  were reprogrammed into pluripotent cells - very similar to embryonic stem cells (ESCs) - through the ectopic expression of four transcription factors: OCT3/4, SOX2, c-MYC and KFL4. Like ESCs, iPSCs have unlimited capacity to renew themselves; they also can differentiate into any other cell type (e.g. muscle cells, neurons). Since their discovery, an unprecedented high number of research groups have taken up this revolutionising technology, and generated iPSCs from patients with genetic diseases. Patient-specific iPSCs offer a new way to study the diseases on cell types which were not accessible for in vitro studies before: neurons from patients with neurological diseases such as Parkinson’s or Alzheimer’s disease, muscle cells from patients suffering from muscular dystrophy, diseases affecting retina cells, and many more examples. These cells provide an unlimited source of human material for modelling the disease and may facilitate screening for novel drugs. Moreover, using iPSCs for cellular replacement therapy or individualized cell therapy after genetic correction may become a viable option when general obstacles of cellular therapies can be overcome. This review summarizes and comments on approaches that have been taken in the last six years for the study and treatment of human diseases using iPSCs.

Hemophilia A is a hemorrhage disorder inherited according to the X-linked inheritance pattern. It affects about 1 in 4,000 to 10,000 males. Permanent changes in the F8 gene result in hemophilia A. These changes result in an abnormal version of coagulation factor VIII. This abnormal condition determines the kinds of the disorder, namely, severe and mild or moderate hemophilia A. Currently, to treat hemophilia A, infusions of plasma-derived or recombinant factor VIII can be used. However, the cost is extremely high. It makes heavy burdens on the patients, their families or the health care system. Therefore, advanced therapy as iPSCs technique has attracted attention of researchers to research for curing this disorder. For example, to obtain edited DNA segments due to hemophilia A, this technique can be used. Research results in animal models have shown important advances.

Work over several decades has laid solid foundations for the advancement of liver cell therapy. To date liver cell therapy in people has taken the form of hepatocyte transplantation for metabolic disorders with a hepatic basis, and for acute or chronic liver failure. Although clinical trials using various types of autologous cells have been implemented to promote liver regeneration or reduce liver fibrosis, clear evidence of therapeutic benefits have so far been lacking. Cell types that have shown efficacy in preclinical models include hepatocytes, liver sinusoidal endothelial cells, mesenchymal stem cells, endothelial progenitor cells, and macrophages. However, positive results in animal models have not always translated through to successful clinical therapies and more realistic preclinical models need to be developed. Studies defining the optimal repopulation by transplanted cells, including routes of cell transplantation, superior engraftment and proliferation of transplanted ce...

С появлением революционной технологии перепрограммирования соматических клеток в омоложенные стволовые клетки, называемые индуцированными стволовыми клетками (ИПСК),  человечество, казалось бы, получило фонтан вечной молодости и здоровья. Однако врачи не спешат использовать чудо-клетки для исцеления больных, так как  лечение с их помощью оказалось сопряжено с риском заболеть раком. Как же надежно  обезопасить терапию клетками полученными из ИПСК? Этот миниобзор посвящен новейшим разработкам и достижениям в разрешении этой задачи.

Reprogramming somatic cells into induced pluripotent stem (iPS) cells is nowadays approaching effectiveness and
clinical grade. Potential uses of this technology include predictive toxicology, drug screening, pathogenetic studies and
transplantation. Here, we review the basis of current iPS cell technology and potential applications in hematology,
ranging from disease modeling of congenital and acquired hemopathies to hematopoietic stem and other blood cell
transplantation.

Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2, and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.

Translation of pharmacological results from in vitro cell testing to clinical trials is challenging. One of the causes that may underlie these discrepant results is the lack of the phenotypic or species-specific relevance of the tested cells; today, this lack of relevance may be reduced by relying on cells differentiated from human pluripotent stem cells. To analyse the benefits provided by this approach, we chose to focus on Friedreich ataxia, a neurodegenerative condition for which the recent clinical testing of two compounds was not successful. These compounds, namely, resveratrol and nicotinamide, were selected because they had been shown to stimulate the expression of frataxin in fibroblasts and lymphoblastoid cells. Our results indicated that these compounds failed to do so in iPSC-derived neurons generated from two patients with Friedreich ataxia. By comparing the effects of both molecules on different cell types that may be considered to be non-relevant for the disease, such as fibroblasts, or more relevant to the disease, such as neurons differentiated from iPSCs, a differential
response was observed; this response suggests the importance of developing more predictive in vitro systems for drug discovery. Our results demonstrate the value of utilizing human iPSCs early in drug discovery to improve translational predictability.

Liver transplantation remains the only solution for patients suffering from severe end-stage liver disease. Only 33% of these patients on the transplant waiting list will receive a donation; furthermore, the demand for livers is expected to increase over 20% in the next two decades. Available liver donations are limited now more than ever. Stem cell therapies using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are being developed to provide cell therapies in order to bypass orthopedic liver transplantation. Additionally, hESCs and hiPSCs are applied in drug development and screening to help pharmaceuticals manufacture more efficacious drugs and in disease modeling allowing scientists to understand disease progression. Clinical cell therapy trials using hepatocyte transplantation have produced promising initial results. Despite continued advances, the effectiveness of transplanted hepatocytes declines within several months and a liver transplant is still necessary. The current advantage of using cell therapy models is to prolong functional use of a diseased liver until a donor liver is received. The potential use of hiPSCs differentiated into hepatocyte cells (hiPSC-HEPs) can generate an infinite supply of hepatic cells. HiPSCs provide a crucial research platform in the development of more effective protocols for cell therapy, drug development and disease modeling.

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known whether the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated induced pluripotent stem cells (iPSCs) from murine rod photoreceptors (r-iPSCs) and scored their ability to make retinae by using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs more efficiently produced differentiated retinae than did embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had fewer amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod-specific CTCF insulator protein-binding sites may promote r-iPSC retinogenesis. Together, our data suggest that the source of stem cells is important for producing retinal neurons in t...

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, in...

Tonic conductance mediated by extrasynaptic GABAA receptors has been implicated in the modulation of network oscillatory activity. Using an in vitro brain slice to produce oscillatory activity and a kinetic model of GABAA receptor dynamics, we show that changes in tonic inhibitory input to fast spiking interneurons underlie benzodiazepine-site mediated modulation of neuronal network synchrony in rat primary motor cortex. We found that low concentrations (10 nM) of the benzodiazepine site agonist, zolpidem, reduced the power of pharmacologically-induced beta-frequency (15-30 Hz) oscillatory activity. By contrast, higher doses augmented beta power. Application of the antagonist, flumazenil, also increased beta power suggesting endogenous modulation of the benzodiazepine binding site. Voltage-clamp experiments revealed that pharmacologically-induced rhythmic inhibitory postsynaptic currents were reduced by 10 nM zolpidem, suggesting an action on inhibitory interneurons. Further voltage...

Mitochondrial diseases are heterogeneous disorders, caused by mitochondrial dysfunction. Mitochondria are not regulated solely by nuclear genomic DNA but by mitochondrial DNA. It is difficult to develop effective therapies for mitochondrial disease because of the lack of mitochondrial disease models. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major mitochondrial diseases. The aim of this study was to generate MELAS-specific induced pluripotent stem cells (iPSCs) and to demonstrate that MELAS-iPSCs can be models for mitochondrial disease. We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243A>G heteroplasmy. MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels. The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPS...