Reprogramming Procedures Research Articles

Establishment of Transgene-Free Porcine Induced Pluripotent Stem Cells.

Although protocols to generate authentic transgene-free mouse and human induced pluripotent stem cells (iPSCs) are now well established, standard methods for reprogramming porcine somatic cells still suffer from low efficiency and transgene retention. The Basic Protocol describes reprogramming procedures to establish transgene-free porcine iPSCs (PiPSCs) from porcine fibroblasts. This method uses episomal plasmids encoding POU5F1, SOX2, NANOG, KLF4, SV40LT, c-MYC, LIN28A, and microRNA-302/367, combined with an optimized medium, to establish PiPSC lines. Support protocols describe the establishment and characterization of clonal PiPSC lines, as well as the preparation of feeder cells and EBNA1 mRNA. This optimized, step-by-step approach tailored to this species enables the efficient derivation of PiPSCs in ∼4 weeks. The establishment of transgene-free PiPSCs provides a new and valuable model for studies of larger mammalian species' development, disease, and regenerative biology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Reprogramming of porcine fibroblasts with episomal plasmids Support Protocol 1: Preparation of mouse embryonic fibroblasts for feeder layer Support Protocol 2: Preparation of in vitro-transcribed EBNA1 mRNA Support Protocol 3: Establishment of clonal porcine induced pluripotent stem cell (PiPSC) lines Support Protocol 4: PiPSC characterization: Genomic DNA PCR and RT-PCR Support Protocol 5: PiPSC characterization: Immunostaining.

Generation of chimpanzee induced pluripotent stem cell lines for cross-species comparisons.

As humans' closest living relatives, chimpanzees offer valuable insights into human evolution. However, technical and ethical limitations hinder investigations into the molecular and cellular foundations that distinguish chimpanzee and human traits. Recently, induced pluripotent stem cells (iPSCs) have emerged as a novel model for functional comparative studies and provided a non-invasive alternative for studying embryonic phenomena. In this study, we generated five new chimpanzee iPSC lines from peripheral blood cells and skin fibroblasts with SeV vectors carrying four reprogramming factors (human OCT3/4, SOX2, KLF4, and L-MYC) and characterized their pluripotency and differentiation potential. We also examined the expression of a human-specific non-coding RNA, HSTR1, which is predicted to be involved in human brain development. Our results show that the chimpanzee iPSCs possess pluripotent characteristics and can differentiate into various cell lineages. Moreover, we found that HSTR1 is expressed in human iPSCs and their neural derivatives but not in chimpanzee counterparts, supporting its possible role in human-specific brain development. As iPSCs are inherently variable due to genetic and epigenetic differences in donor cells or reprogramming procedures, it is essential to expand the number of chimpanzee iPSC lines to comprehensively capture the molecular and cellular properties representative of chimpanzees. Hence, our cells provide a valuable resource for investigating the function and regulation of human-specific transcripts such as HSTR1 and for understanding human evolution more generally.

Electrical storm management in the cardiac care unit

Electrical storm (ES) is characterized by at least three separate episodes of ventricular arrhythmia (VA) over 24 h that require treatment or an incessant VA lasting >12 h. The incidence is higher in patients with implantable cardioverter-defibrillators (ICDs) in secondary prevention and the main manifestation is monomorphic VA. ES onset represents a major event in the history of patients with cardiomyopathies that significantly worsens prognosis. The management of ES is complex and requires a multidisciplinary approach including a comprehensive clinical assessment, resuscitation and sedation management skills, ICD reprogramming, ablation, and neuromodulation procedures. ES early recognition and prompt treatment initiation increase the chances of therapeutic success. Each one of these aspects will be properly discussed in the present decalogue. Notably, ES management remains a challenge, with only limited available evidence from small retrospective series and a substantial lack/limited number of randomized or prospective trials. The spectrum of available antiarrhythmic drugs is limited, as well as their efficacy. The future hope is that larger prospective studies will be able to answer important questions, concerning the most effective pharmacologic strategies, the timing for the invasive treatment, the indications for acute neuromodulation strategies and for the circulatory support tools.

Case Report: Optical Coherence Tomography, as a Non-Invasive Approach to Evaluate Intracranial Pressure in Normal Pressure Hydrocephalus Patients, as with their Treatment Follow-ups and VP Shunts Reprogramming Procedures

Case Report: Optical Coherence Tomography, as a Non-Invasive Approach to Evaluate Intracranial Pressure in Normal Pressure Hydrocephalus Patients, as with their Treatment Follow-ups and VP Shunts Reprogramming Procedures

The relevance of mitochondrial DNA variants fluctuation during reprogramming and neuronal differentiation of human iPSCs.

SummaryThe generation of inducible pluripotent stem cells (iPSCs) is a revolutionary technique allowing production of pluripotent patient-specific cell lines used for disease modeling, drug screening, and cell therapy. Integrity of nuclear DNA (nDNA) is mandatory to allow iPSCs utilization, while quality control of mitochondrial DNA (mtDNA) is rarely included in the iPSCs validation process. In this study, we performed mtDNA deep sequencing during the transition from parental fibroblasts to reprogrammed iPSC and to differentiated neuronal precursor cells (NPCs) obtained from controls and patients affected by mitochondrial disorders. At each step, mtDNA variants, including those potentially pathogenic, fluctuate between emerging and disappearing, and some having functional implications. We strongly recommend including mtDNA analysis as an unavoidable assay to obtain fully certified usable iPSCs and NPCs.

Myogenic progenitor cells derived from human induced pluripotent stem cell are immune-tolerated in humanized mice.

It is still unclear if immune responses will compromise the large‐scale utilization of human induced pluripotent stem cells (hiPSCs)‐derived cell therapies. To answer this question, we used humanized mouse models generated by the adoptive transfer of peripheral blood mononuclear cells or the cotransplantation of hematopoietic stem cells and human thymic tissue. Using these mice, we evaluated the engraftment in skeletal muscle of myoblasts derived either directly from a muscle biopsy or differentiated from hiPSCs or fibroblasts. Our results showed that while allogeneic grafts were mostly rejected and highly infiltrated with human T cells, engraftment of autologous cells was tolerated. We also observed that hiPSC‐derived myogenic progenitor cells (MPCs) are not targeted by autologous T cells and natural killer cells in vitro. These findings suggest that the reprogramming and differentiation procedures we used are not immunogenic and that hiPSC‐derived MPCs will be tolerated in the presence of a competent human immune system.

Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts.

Induced pluripotent stem cell (iPSC)-technology is an important platform in medicine and disease modeling. Physiological degeneration and disease onset are common occurrences in the aging population. iPSCs could offer regenerative medical options for age-related degeneration and disease in the elderly. However, reprogramming somatic cells from the elderly is inefficient when successful at all. Perhaps due to their low rates of replication in culture, traditional transduction and reprogramming approaches with centenarian fibroblasts met with little success. A simple and reproducible reprogramming process is reported here which enhances interactions of the cells with the viral vectors that leads to improved iPSC generation. The improved methods efficiently generates fully reprogrammed iPSC lines from 105–107 years old subjects in feeder-free conditions using an episomal, Sendai-Virus (SeV) reprogramming vector expressing four reprogramming factors.In conclusion, dermal fibroblasts from human subjects older than 100 years can be efficiently and reproducibly reprogrammed to fully pluripotent cells with minor modifications to the standard reprogramming procedures. Efficient generation of iPSCs from the elderly may provide a source of cells for the regeneration of tissues and organs with autologous cells as well as cellular models for the study of aging, longevity and age-related diseases.

Loss of hierarchical imprinting regulation at the Prader-Willi/Angelman syndrome locus in human iPSCs.

The human chr15q11-q13 imprinted cluster is linked to several disorders, including Prader–Willi (PWS) and Angelman (AS) syndromes. Recently, disease modeling approaches based on induced pluripotent stem cells (iPSCs) have been used to study these syndromes. A concern regarding the use of these cells for imprinted disease modeling is the numerous imprinting defects found in many iPSCs. Here, by reprogramming skin fibroblasts from a control and AS individuals, we generated several iPSC lines and addressed the stability of imprinting status across the PWS/AS domain. We focused on three important regulatory DNA elements which are all differentially methylated regions (DMRs), methylated on the maternal allele: the PWS imprinting center (PWS-IC), which is a germline DMR and the somatic NDN and MKRN3 DMRs, hierarchically controlled by PWS-IC. Normal PWS-IC methylation pattern was maintained in most iPSC lines; however, loss of maternal methylation in one out of five control iPSC lines resulted in a monoallelic to biallelic switch for many imprinted genes in this domain. Surprisingly, MKRN3 DMR was found aberrantly hypermethylated in all control and AS iPSCs, regardless of the methylation status of the PWS-IC master regulator. This suggests a loss of hierarchical control of imprinting at PWS/AS region. We confirmed these results in established iPSC lines derived using different reprogramming procedures. Overall, we show that hierarchy of imprinting control in donor cells might not apply to iPSCs, accounting for their spectrum of imprinting alterations. Such differences in imprinting regulation should be taken into consideration for the use of iPSCs in disease modeling.

Reprogrammed chondrocytes engineered to produce IL-12 provide novel ex vivo immune-gene therapy for cancer.

The somatic cell reprogramming technology was applied to a novel and promising ex vivo immune-gene therapy strategy for cancer. To establish a novel ex vivo cytokine gene therapy of cancer using the somatic cell reprogramming procedures. Mouse fibroblasts were converted into chondrocytes and subsequently transduced with IL-12 gene. The resultant IL-12 induced chondrogenic cells were irradiated with x-ray and inoculated into mice bearing CT26 colon cancer. The irradiation at 20 Gy or higher totally eliminated the proliferative potential of the cells, while less significantly influencing the IL-12 production from the cells. An inoculation of the irradiated IL-12 induced chondrogenic cells significantly suppressed tumor by inducing tumor-specific cytotoxic T lymphocytes, enhancing natural killer tumoricidal activity and inhibiting tumor neoangiogenesis in the mice. The somatic cell reprogramming procedures may provide a novel and effective means to treat malignancies.

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

Direct Conversion of Human Fibroblasts into Schwann Cells that Facilitate Regeneration of Injured Peripheral Nerve In Vivo.

Schwann cells (SCs) play pivotal roles in the maintenance and regeneration of the peripheral nervous system. Although transplantation of SCs enhances repair of experimentally damaged peripheral and central nerve tissues, it is difficult to prepare a sufficient number of functional SCs for transplantation therapy without causing adverse events for the donor. Here, we generated functional SCs by somatic cell reprogramming procedures and demonstrated their capability to promote peripheral nerve regeneration. Normal human fibroblasts were phenotypically converted into SCs by transducing SOX10 and Krox20 genes followed by culturing for 10 days resulting in approximately 43% directly converted Schwann cells (dSCs). The dSCs expressed SC‐specific proteins, secreted neurotrophic factors, and induced neuronal cells to extend neurites. The dSCs also displayed myelin‐forming capability both in vitro and in vivo. Moreover, transplantation of the dSCs into the transected sciatic nerve in mice resulted in significantly accelerated regeneration of the nerve and in improved motor function at a level comparable to that with transplantation of the SCs obtained from a peripheral nerve. The dSCs induced by our procedure may be applicable for novel regeneration therapy for not only peripheral nerve injury but also for central nerve damage and for neurodegenerative disorders related to SC dysfunction. Stem Cells Translational Medicine 2017;6:1207–1216

Use of Contextual Information to Optimize Wireless Sensor Node’s Energy Consumption Rate during Reprogramming Procedures

This research work explores the use of context information to improve upon wireless sensor networks performance by reducing the energy consumption rate during reprogramming processes. A software system that dynamically reconfigures wireless sensor network operational functionalities optimally based on evolving application context is developed. In order to demonstrate the benefits of the context based reconfiguration model, two contexts related input variables were used. The first variable is obtained using a metric tool (PDE) devised for extracting the delta of two files (application related context). The second variable entails the battery energy level state of the sensor node taken as an operationaldemand related context. A robust inference engine was developed based on the inferred expert knowledge on memory related energy consumption pattern during the reconfiguration process. The resulting output from the fuzzy logic controller decides when and which of the reconfiguration approach is to be implemented in order to prolong the battery life. The model's performance was evaluated on an OMNet++ simulation platform using pilot data obtained from a test bed composed of Microchips‟ PIC32MX320F128H microcontroller and MRF24J40MB transceiver. In a network of six nodes, two were equipped with the developed model capability and the others were not. The overall energy expended as read, erase and write were obtained from each node for the purpose of comparison. Results obtained show that 65% of energy expended during the erasure procedure is saved in nodes that adopt the context-based reconfiguration model. Similarly, 45% and 69% reduction in energy consumption were obtained for the read and write procedures respectively.

Generation of iPS cell lines from schizophrenia patients using a non-integrative method

Skin biopsies were collected from three male patients (age 45, 44 and 44) with clinically diagnosed schizophrenia. The patients were diagnosed according to DSM-5 criteria by a trained psychiatrist. Dermal fibroblast cell lines were established and expanded for subsequent reprogramming procedures. Induced pluripotent stem (iPS) cells were derived using the integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, containing Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

Reprogrammed Functional Brown Adipocytes Ameliorate Insulin Resistance and Dyslipidemia in Diet-Induced Obesity and Type 2 Diabetes

SummaryBrown adipocytes (BAs) play important roles in body temperature regulation, energy balance, and carbohydrate and lipid metabolism. Activities of BAs are remarkably diminished in obese and diabetic patients, providing possibilities of transplanting functional BAs resulting in therapeutic benefit. Here, we show generation of functional BAs by cellular reprogramming procedures. Transduction of the PRDM16 gene into iPSC-derived embryoid bodies induced BA phenotypes (iBAs). Moreover, normal human fibroblasts were directly converted into BAs (dBAs) by C/EBP-β and C-MYC gene transduction. Approximately 90% of the fibroblasts were successfully converted within 12 days. The dBAs were highly active in mitochondrial biogenesis and oxidative metabolism. Mouse dBAs were induced by Prdm16, C/ebp-β, and L-myc genes, and after transplantation, they significantly reduced diet-induced obesity and insulin resistance in an UCP1-dependent manner. Thus, highly functional BAs can be generated by cellular reprogramming, suggesting a promising tailor-made cell therapy against metabolic disorders including type 2 diabetes mellitus.

Livestock models for exploiting the promise of pluripotent stem cells.

Livestock species are widely used as biomedical models. Pigs, in particular, are beginning to have a significant role in regenerative medicine for testing the applicability, success, and safety of grafts derived from induced pluripotent stem cells. Animal testing must always be performed before any clinical trials are performed in humans, and pigs may sometimes be the species of choice because of their physiological and anatomical similarities to humans. Induced pluripotent stem cells (iPSC) have been generated with some success from livestock species by a variety of reprogramming procedures, but authenticated embryonic stem cells (ESC) have not. There are now several studies in which porcine iPSC have been tested for their ability to provide functional grafts in pigs. Pigs have also served as recipients for grafts derived from human iPSC. There have also been recent advances in creating pigs with severe combined immunodeficiency (SCID). Like SCID mice, these pigs are expected to be graft tolerant. Additionally, chimeric, partially humanized pigs could be sources of human organs. Another potential application of pluripotent stem cells from livestock is for the purpose of differentiating the cells into skeletal muscle, which, in turn, could be used either to produce cultured meat or to engraft into damaged muscle. None of these technologies has advanced to a stage that they have become mainstream, however. Despite the value of livestock models in regenerative medicine, only a limited number of institutions are able to use these animals.

SAT0562 Directly Reprogrammed Osteoblasts Genetically Engineered to Produce Interleukin-10 Significantly Suppress Osteoclastgenesis

BackgroundRecent reports demonstrated that somatic cells such as fibroblasts can be directly reprogrammed into other cell types including neurons and cardiomyocytes by introducing critical transcription factors involved in the differentiation...

Smart sensor/actuator node reprogramming in changing environments using a neural network model

The techniques currently developed for updating software in sensor nodes located in changing environments require usually the use of reprogramming procedures, which clearly increments the costs in terms of time and energy consumption. This work presents an alternative to the traditional reprogramming approach based on an on-chip learning scheme in order to adapt the node behaviour to the environment conditions. The proposed learning scheme is based on C-Mantec, a novel constructive neural network algorithm especially suitable for microcontroller implementations as it generates very compact size architectures. The Arduino UNO board was selected to implement this learning algorithm as it is a popular, economic and efficient open source single-board microcontroller. C-Mantec has been successfully implemented in a microcontroller board by adapting it in order to overcome the limitations imposed by the limited resources of memory and computing speed of the hardware device. Also, this work brings an in-depth analysis of the solutions adopted to overcome hardware resource limitations in the learning algorithm implementation (e.g., data type), together with an efficiency assessment of this approach when the algorithm is tested on a set of circuit design benchmark functions. Finally, the utility, efficiency and versatility of the system is tested in three different-nature case studies in which the environmental conditions change its behaviour over time.

Defining the Diversity of Phenotypic Respecification Using Multiple Cell Lines and Reprogramming Regimens

To better understand the basis of variation in cellular reprogramming, we performed experiments with two primary objectives: first, to determine the degree of difference, if any, in reprogramming efficiency among cells lines of a similar type after accounting for technical variables, and second, to compare the efficiency of conversion of multiple similar cell lines to two separate reprogramming regimens-induced neurons and induced skeletal muscle. Using two reprogramming regimens, it could be determined whether converted cells are likely derived from a distinct subpopulation that is generally susceptible to reprogramming or are derived from cells with an independent capacity for respecification to a given phenotype. Our results indicated that when technical components of the reprogramming regimen were accounted for, reprogramming efficiency was reproducible within a given primary fibroblast line but varied dramatically between lines. The disparity in reprogramming efficiency between lines was of sufficient magnitude to account for some discrepancies in published results. We also found that the efficiency of conversion to one phenotype was not predictive of reprogramming to the alternate phenotype, suggesting that the capacity for reprogramming does not arise from a specific subpopulation with a generally "weak grip" on cellular identity. Our findings suggest that parallel testing of multiple cell lines from several sources may be needed to accurately assess the efficiency of direct reprogramming procedures, and that testing a larger number of fibroblast lines--even lines with similar origins--is likely the most direct means of improving reprogramming efficiency.

Nuclear reprogramming

There is currently particular interest in the field of nuclear reprogramming, a process by which the identity of specialised cells may be changed, typically to an embryonic-like state. Reprogramming procedures provide insight into many mechanisms of fundamental cell biology and have several promising applications, most notably in healthcare through the development of human disease models and patient-specific tissue-replacement therapies. Here, we introduce the field of nuclear reprogramming and briefly discuss six of the procedures by which reprogramming may be experimentally performed: nuclear transfer to eggs or oocytes, cell fusion, extract treatment, direct reprogramming to pluripotency and transdifferentiation.

Potential Application of Induced Pluripotent Stem Cells in Cell Replacement Therapy for Parkinsons Disease

Parkinson's disease (PD), a common degenerative disease in humans, is known to result from loss of dopamine neurons in the substantia nigra and is characterized by severe motor symptoms of tremor, rigidity, bradykinsia and postural instability. Although levodopa administration, surgical neural lesion, and deep brain stimulation have been shown to be effective in improving parkinsonian symptoms, cell replacement therapy such as transplantation of dopamine neurons or neural stem cells has shed new light on an alternative treatment strategy for PD. While the difficulty in securing donor dopamine neurons and the immuno-rejection of neural transplants largely hinder application of neural transplants in clinical treatment, induced pluripotent stem cells (iPS cells) derived from somatic cells may represent a powerful tool for studying the pathogenesis of PD and provide a source for replacement therapies in this neurodegenerative disease. Yamanaka et al. [2006, 2007] first succeeded in generating iPS cells by reprogramming fibroblasts with four transcription factors, Oct4, Sox2, Klf4, and c-Myc in both mouse and human. Animal studies have further shown that iPS cells from fibroblasts could be induced into dopamine neurons and transplantation of these cells within the central nervous system improved motor symptoms in the 6-OHDA model of PD. More interestingly, neural stem cells or fibroblasts from patients can be efficiently reprogrammed and subsequently differentiated into dopamine neurons. Derivation of patient-specific iPS cells and subsequent differentiation into dopamine neurons would provide a disease-specific in vitro model for disease pathology, drug screening and personalized stem cell therapy for PD. This review summarizes current methods and modifications in producing iPS cells from somatic cells as well as safety concerns of reprogramming procedures. Novel reprogramming strategies that deter abnormal permanent genetic and epigenetic alterations are essential for propagating clinically-qualified iPS cells. Future investigations into cell transforming and reprogramming processes are needed to generate the disease-specific iPS cells for personalized regeneration medicine of PD patients by disclosing detailed reprogramming mechanisms.