Efficacy Of Reprogramming Research Articles

SOXC Enhances NGN2-Mediated Reprogramming of Glioblastoma Cells Into Neuron-Like Cells by Modulating RhoA and RAC1/CDC42 Pathway Activity.

Glioblastoma represents the most frequently diagnosed malignant neoplasm within the central nervous system. Human glioblastoma cells can be phenotypically reprogrammed into neuron-like cells through the forced expression of NEUROG2 and SOXC factors. NEUROG2 serves as a pioneer factor, establishing an initial framework for this transformation. However, the specific role of SOXC factors has not been fully elucidated. In this study, we used ChIP-seq to determine the potential target gene of NGN2. RNA-seq has been used to evaluate the transcriptional change during NGN2-SOX11-mediated neuron reprogramming. Immunofluorescence was used to determine the neuron reprogramming efficacy and cell proliferation ability. ChIP-qPCR, Co-IP, and Western Blot were performed to investigate the mechanism. Our findings reveal that SOXC factors, in contrast to their previously identified function as transcriptional activators, act as transcriptional repressors. They achieve this by recruiting TRIM28 to suppress the expression of ECT2, a RhoGEF. This suppression results in the differential regulation of RhoA, RAC1, and CDC42 activities throughout the reprogramming process. We further establish that small molecules targeting RhoA and its effectors can substitute for SOXC factors in facilitating the neuronal reprogramming of glioblastoma cells. These results underscore the pivotal role of SOXC factors' transcriptional repression and illuminate one of their specific downstream targets.

Endogenous retroviral ERVH48-1 promotes human urine cell reprogramming

Endogenous retroviruses (ERVs), once thought to be mere remnants of ancient viral integrations in the mammalian genome, are now recognized for their critical roles in various physiological processes, including embryonic development, innate immunity, and tumorigenesis. Their impact on host organisms is significant driver of evolutionary changes, offering insight into evolutionary mechanisms. In our study, we explored the functionality of ERVs by examining single-cell transcriptomic profiles from human embryonic stem cells and urine cells. This led to the discovery of a unique ERVH48-1 expression pattern between these cell types. Additionally, somatic cell reprogramming efficacy was enhanced when ERVH48-1 was overexpressed in a urine cell-reprogramming system. Induced pluripotent stem cells (iPSCs) generated with ERVH48-1 overexpression recapitulated the traits of those produced by traditional reprogramming approaches, and the resulting iPSCs demonstrated the capability to differentiate into all three germ layers in vitro. Our research elucidated the role of ERVs in somatic cell reprogramming.

Modifying miRs for effective reprogramming of fibroblasts to cardiomyocytes

Reprogramming scar fibroblasts into cardiomyocytes has been proposed to reverse the damage associated with myocardial infarction (MI). However, the limited improvement in cardiac function calls for enhanced strategies. We reported enhanced efficacy of our miR reprogramming cocktail miR combo (miR-1, miR-133a, miR-208a and miR-499) via RNA-sensing receptor stimulation. We hypothesized that we could combine RNA-sensing receptor activation with fibroblast reprogramming by chemically modifying miR combo. To test the hypothesis, miR combo was modified to enhance interaction with the RNA-sensing receptor Rig1 via the addition of a 5'-triphosphate (5'ppp) group. Importantly, when compared to unmodified miR combo, 5’ppp modified miR combo markedly improved reprogramming efficacy in vitro. Enhanced reprogramming efficacy correlated with a type-I interferon immune response with strong and selective secretion of Interferon β (IFNβ). Antibody blocking studies and media replacement experiments indicated that 5’ppp-miR combo utilized IFNβ to enhance fibroblast reprogramming efficacy. In conclusion, miRs can acquire powerful additional roles through chemical modification which potentially increases their clinical applications.

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction

BackgroundDirect cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses.MethodsCardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry.ResultsWe found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair.ConclusionsFibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.Graphical

Neonatal and adult cardiac fibroblasts exhibit inherent differences in cardiac regenerative capacity

Directly reprogramming fibroblasts into cardiomyocytes improves cardiac function in the infarcted heart. However, the low efficacy of this approach hinders clinical applications. Unlike the adult mammalian heart, the neonatal heart has an intrinsic regenerative capacity. Consequently, we hypothesized that birth imposes fundamental changes in cardiac fibroblasts which limit their regenerative capabilities. In support, we found that reprogramming efficacy invitro was markedly lower with fibroblasts derived from adult mice versus those derived from neonatal mice. Notably, fibroblasts derived from adult mice expressed significantly higher levels of pro-angiogenic genes. Moreover, under conditions that promote angiogenesis, only fibroblasts derived from adult mice differentiated into tube-like structures. Targeted knockdown screening studies suggested a possible role for the transcription factor Epas1. Epas1 expression was higher in fibroblasts derived from adult mice, and Epas1 knockdown improved reprogramming efficacy in cultured adult cardiac fibroblasts. Promoter activity assays indicated that Epas1 functions as both a transcription repressor and an activator, inhibiting cardiomyocyte genes while activating angiogenic genes. Finally, the addition of an Epas1 targeting siRNA to the reprogramming cocktail markedly improved reprogramming efficacy invivo with both the number of reprogramming events and cardiac function being markedly improved. Collectively, our results highlight differences between neonatal and adult cardiac fibroblasts and the dual transcriptional activities of Epas1 related to reprogramming efficacy.

Abstract 4550: Assessing efficacy and immune-stimulatory effects of tumor-derived dendritic cell reprogramming using immuno-competent 3D tumor spheroid model

Abstract Immunotherapy has brought hope for cancer treatment, but its clinical success remains limited. Recently, overexpression of the transcription factors PU.1, IRF8 and BATF3 (PIB) was shown to induce direct reprogramming of tumor cells into antigen-presenting type 1 conventional dendritic cells (cDC1s), a rare subset of immune cells with pivotal role in anti-cancer immunity. This strategy might open avenues to enhance cancer cell recognition and elimination by the immune system. However, currently existing in-vitro and in-vivo testing platforms do not qualify to reproduce all complex cell interactions essential for the approbation of this hypothesis. Here, we report the development of the InSphero 3D InSight™ Oncology Platform for in-vitro assessment of efficacy and immune-stimulatory effects of this novel cancer immunotherapy approach. The feasibility of 3D spheroid formation for several GFP-expressing tumor cell lines was evaluated by varying seeding conditions in AKURA 96 well plate. We have measured the growth (ATP content) and GFP signal overtime and analyzed the morphology of the spheroids by IHC. With this, we have established spheroid models of T98G (glioblastoma), PK59 (pancreatic cancer), and A375 (melanoma) cell lines that are growing and viable for at least 10 days. In parallel, using 2D cultures, we have identified the optimal multiplicity of infection of a lentiviral vector encoding for PIB and mCherry to enable high transduction (mCherry+ cells), reprogramming efficiency (mCherry+CD45+HLA-DR+ cells), and cell viability, quantified by flow cytometry and IHC. Then, we have demonstrated that cDC1 reprogramming progresses in the context of 3D cancer spheroids and tumor cells acquire expression of CD45+ and HLA-DR+ cells using IHC and confocal microscopy analysis. We developed an algorithm enabling automated analysis of confocal images and quantification of cDC1 reprogramming efficiency from individual image stacks calculated as a ratio of mCherry+, CD45+ and HLA-DR+ cells versus the number of DAPI+ nuclei. Using the new algorithm we have evaluated the reprogramming efficacy of the different virus dosages in all three types of 3D tumor spheroids. Lastly, we have cocultured tumor spheroids transduced with PIB with naïve or activated HLA-matched PBMCs and evaluated cytokine secretion as a readout of immune cell activation. We observed that reprogramming induces activation of T cells and correlated it to the number of reprogrammed cells in the tumor spheroid, evaluated by the HC imaging. In summary, we developed the InSphero 3D InSight™ Oncology Platform that allowed us to demonstrate the effects of direct reprogramming of tumor cells into immunogenic dendritic cells. Combined with high-content imaging analysis, this platform offers a powerful solution for preclinical translational research. Citation Format: Irina Agarkova, Nadezhda Rotankova, Michal Rudnik, Silvan Strebel, Olga Zimmermannova, Andre Rosa, Fabio F. Rosa, Wolfgang Moritz, Carlos-Filipe Pereira, Christiana F. Pires. Assessing efficacy and immune-stimulatory effects of tumor-derived dendritic cell reprogramming using immuno-competent 3D tumor spheroid model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4550.

Rig1 receptor plays a critical role in cardiac reprogramming via YY1 signaling.

We discovered that innate immunity plays an important role in the reprogramming of fibroblasts into cardiomyocytes. In this report, we define the role of a novel retinoic acid-inducible gene 1 Yin Yang 1 (Rig1:YY1) pathway. We found that fibroblast to cardiomyocyte reprogramming efficacy was enhanced by specific Rig1 activators. To understand the mechanism of action, we performed various transcriptomic, nucleosome occupancy, and epigenomic approaches. Analysis of the datasets indicated that Rig1 agonists had no effect on reprogramming-induced changes in nucleosome occupancy or loss of inhibitory epigenetic motifs. Instead, Rig1 agonists were found to modulate cardiac reprogramming by promoting the binding of YY1 specifically to cardiac genes. To conclude, these results show that the Rig1:YY1 pathway plays a critical role in fibroblast to cardiomyocyte reprogramming.

Abstract 12047: miR-148-3p:mdfic Pathway Improves the Efficacy of Therapeutic Cardiac Reprogramming

We, and others, have demonstrated that directly reprogramming scar fibroblasts into new cardiomyocytes improves cardiac function in the infarcted heart. A major challenge is the relatively low conversion rate of fibroblasts into cardiomyocytes. We pioneered reprogramming method based on miRNAs, whereby fibroblasts were converted into cardiomyocytes via combination of four miRNAs which we called miR combo. We hypothesized that the efficacy of miR combo could be improved via additional miRNAs. To that end, we screened a number of miRNAs and found that miR-148-3p significantly enhanced the efficacy of miR combo with respect to the expression of cardiomyocyte genes (Actn2, Myh6 and Tnni3; 2 to 5 fold; N=5; P<0.05). Validating a role for miR-148a-3p, addition of the anti-miR-148a-3p inhibited the ability of miR-148a-3p to enhance reprogramming efficacy (N=5, P<0.05). In silico analysis, miR-148a-3p transfection studies, and luciferase reporter assays indicated that Pten, Pik3r3, Rock1 and Mdfic were miR-148a-3p targets. Of these targets, only the overexpression of Mdfic inhibited miR combo (N=4, P<0.05). In contrast, siRNA-mediated knockdown of Mdfic enhanced miR combo efficacy (Actn2, Myh6 and Tnni3; 2 to 10 fold; N=3; P<0.05). These studies suggested that Mdfic was a repressor of the cardiomyocyte phenotype. To provide further evidence, Mdfic mRNA levels were measured in freshly isolated cardiomyocytes and fibroblasts. Mdfic was found to be virtually absent in cardiomyocytes and strongly expressed in fibroblasts (N=3, P<0.05). Moreover, ChIP-qPCR and co-immunoprecipitation analysis indicated that in fibroblasts, Mdfic resided solely on cardiomyocyte genes where it inhibited gene expression by preventing the binding of transcription activator proteins (N=3, P<0.05). In summary, miR-148a-3p improves the efficacy of fibroblast to cardiomyocyte reprogramming via the down-regulation of Mdfic.

Abstract 12033: The IFNb-Gbp7 Pathway Plays an Important Role in Activating Cardiomyocyte Genes

We discovered that reprogramming scar fibroblasts into cardiomyocytes requires activation of innate immunity. Recently, we found that innate immunity exerts its influence on fibroblast to cardiomyocyte reprogramming via the RNA-sensing receptor Rig1. However, the mechanism was not identified. Rig1 activation, whether by classical agonists or via rationally designed RNA molecules, enhanced the efficacy of cardiac reprogramming with cardiomyocyte-specific gene expression increasing by 10 to 50 fold (Actn2, Myh6, Tnni3; N=6; P<0.01) and cardiomyocyte generation increasing 5 fold (N=3, P<0.01). RNA-seq indicated that Rig1 activation in fibroblasts induced IFNβ signaling pathways. To verify a role for IFNβ, knockdown studies were performed. Transfection of fibroblasts with an IFNβ targeting siRNA completely ablated the effects of Rig1 agonists with respect to cardiac reprogramming (N=5, P<0.01). Further studies with specific blocking antibodies and media replacement showed that IFNβ was acting in a paracrine manner. To determine the mechanism whereby IFNβ regulated cardiomyocyte gene expression, we screened a number of candidates identified in the RNA-seq: Ccl5, Ccl10, Gbp5, and Gbp7. Knockdown of Ccl5, Ccl10, or Gbp5 had no effect on the ability of IFNβ to enhance cardiac reprogramming efficacy. In contrast, the ability of IFNβ to enhance cardiac reprogramming was lost in fibroblasts lacking Gbp7 (N=5, P<0.01). To conclude, the IFNβ-Gbp7 pathway plays a critical role in cardiac reprogramming. Manipulation of the IFNβ-Gbp7 pathway may enhance the effectiveness of cardiac reprogramming in vivo.

Is sacral nerve modulation reprogramming effective after permanent implantation for faecal incontinence?

Sacral nerve modulation (SNM) is recommended as a first-line surgical therapy for patients with faecal incontinence (FI). During patient follow-up, it is recommended that stimulation settings be reprogrammed to optimize patient outcomes. The aim of the present study was to evaluate the efficacy of stimulator reprogramming in patients with an implanted SNM device to treat FI. The data from patients who received a permanent SNM implant in a single centre from January 2008 to December 2019 were retrospectively analysed. Symptoms that occurred after implantation, the stimulator settings of the SNM device and changes made at each follow-up visit were noted. The efficacy of reprogramming was determined by assessing patient satisfaction. Of the 117 patients (male/female 4/113; mean age 59.5±11.8years) with a SNM implant for FI, 84 (72%) had at least one symptom requiring reprogramming of the stimulator, most often during the first year after implantation (p=0.05). The most frequently reported symptoms were loss of efficacy (68.5%; p=1×10-3 ) and pain (20.5%; p=1×10-3 ). Reprogramming was effective 53% of the time when treating loss of efficacy and 76% of the time when treating pain. When the stimulation parameters were reprogrammed at least four consecutive times to correct a symptom, the reprogramming was less effective in treating the symptom (p=0.02). Regular follow-up of patients with SNM device implants associated with reprogramming of stimulation parameters to improve the treatment of reported symptoms would optimize the efficacy of SNM.

Hippo Pathway Effector Tead1 Induces Cardiac Fibroblast to Cardiomyocyte Reprogramming

BackgroundThe conversion of fibroblasts into induced cardiomyocytes may regenerate myocardial tissue from cardiac scar through in situ cell transdifferentiation. The efficiency transdifferentiation is low, especially for human cells. We explored the leveraging of Hippo pathway intermediates to enhance induced cardiomyocyte generation.Methods and ResultsWe screened Hippo effectors Yap (yes‐associated protein), Taz (transcriptional activator binding domain), and Tead1 (TEA domain transcription factor 1; Td) for their reprogramming efficacy with cardio‐differentiating factors Gata4, Mef2C, and Tbx5 (GMT). Td induced nearly 3‐fold increased expression of cardiomyocyte marker cTnT (cardiac troponin T) by mouse embryonic and adult rat fibroblasts versus GMT administration alone (P<0.0001), while Yap and Taz failed to enhance cTnT expression. Serial substitution demonstrated that Td replacement of TBX5 induced the greatest cTnT expression enhancement and sarcomere organization in rat fibroblasts treated with all GMT substitutions (GMTd versus GMT: 17±1.2% versus 5.4±0.3%, P<0.0001). Cell contractility (beating) was seen in 6% of GMTd‐treated cells by 4 weeks after treatment, whereas no beating GMT‐treated cells were observed. Human cardiac fibroblasts likewise demonstrated increased cTnT expression with GMTd versus GMT treatment (7.5±0.3% versus 3.0±0.3%, P<0.01). Mechanistically, GMTd administration increased expression of the trimethylated lysine 4 of histone 3 (H3K4me3) mark at the promoter regions of cardio‐differentiation genes and mitochondrial biogenesis regulator genes in rat and human fibroblast, compared with GMT.ConclusionsThese data suggest that the Hippo pathway intermediate Tead1 is an important regulator of cardiac reprogramming that increases the efficiency of maturate induced cardiomyocytes generation and may be a vital component of human cardiodifferentiation strategies.

Efficient induction and sustenance of pluripotent stem cells from bovine somatic cells.

ABSTRACTAlthough derivation of naïve bovine embryonic stem cells is unachieved, the possibility for generation of bovine induced pluripotent stem cells (biPSCs) has been generally reported. However, attempts to sustain biPSCs by promoting self-renewal have not been successful. Methods established for maintaining murine and human induced pluripotent stem cells (iPSCs) do not support self-renewal of iPSCs for any bovid species. In this study, we examined methods to enhance complete reprogramming and concurrently investigated signaling relevant to pluripotency of the bovine blastocyst inner cell mass (ICM). First, we identified that forced expression of SV40 large T antigen together with the reprogramming genes (OCT4, SOX2, KLF4 and MYC) substantially enhanced the reprogramming efficacy of bovine fibroblasts to biPSCs. Second, we uncovered that TGFβ signaling is actively perturbed in the ICM. Inhibition of ALK4/5/7 to block TGFβ/activin/nodal signaling together with GSK3β and MEK1/2 supported robust in vitro self-renewal of naïve biPSCs with unvarying colony morphology, steady expansion, expected pluripotency gene expression and committed differentiation plasticity. Core similarities between biPSCs and stem cells of the 16-cell-stage bovine embryo indicated a stable ground state of pluripotency; this allowed us to reliably gain predictive understanding of signaling in bovine pluripotency using systems biology approaches. Beyond defining a high-fidelity platform for advancing biPSC-based biotechnologies that have not been previously practicable, these findings also represent a significant step towards understanding corollaries and divergent aspects of bovine pluripotency. This article has an associated First Person interview with the joint first authors of the paper.

Tissue-Restricted Stem Cells as Starting Cell Source for Efficient Generation of Pluripotent Stem Cells: An Overview.

Induced pluripotent stem cells (iPSCs) have vast biomedical potential concerning disease modeling, drug screening and discovery, cell therapy, tissue engineering, and understanding organismal development. In the year 2006, a groundbreaking study reported the generation of iPSCs from mouse embryonic fibroblasts by viral transduction of four transcription factors, namely, Oct4, Sox2, Klf4, and c-Myc. Subsequently, human iPSCs were generated by reprogramming fibroblasts as a starting cell source using two reprogramming factor cocktails [(i) OCT4, SOX2, KLF4, and c-MYC, and (ii) OCT4, SOX2, NANOG, and LIN28]. The wide range of applications of these human iPSCs in research, therapeutics, and personalized medicine has driven the scientific community to optimize and understand this reprogramming process to achieve quality iPSCs with higher efficiency and faster kinetics. One of the essential criteria to address this is by identifying an ideal cell source in which pluripotency can be induced efficiently to give rise to high-quality iPSCs. Therefore, various cell types have been studied for their ability to generate iPSCs efficiently. Cell sources that can be easily reverted to a pluripotent state are tissue-restricted stem cells present in the fetus and adult tissues. Tissue-restricted stem cells can be isolated from fetal, cord blood, bone marrow, and other adult tissues or can be obtained by differentiation of embryonic stem cells or trans-differentiation of other tissue-restricted stem cells. Since these cells are undifferentiated cells with self-renewal potential, they are much easier to reprogram due to the inherent characteristic of having an endogenous expression of few pluripotency-inducing factors. This review presents an overview of promising tissue-restricted stem cells that can be isolated from different sources, namely, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, limbal epithelial stem cells, and spermatogonial stem cells, and their reprogramming efficacy. This insight will pave the way for developing safe and efficient reprogramming strategies and generating patient-specific iPSCs from tissue-restricted stem cells derived from various fetal and adult tissues.

Ameliorating the Fibrotic Remodeling of the Heart through Direct Cardiac Reprogramming.

Coronary artery disease is the most common form of cardiovascular diseases, resulting in the loss of cardiomyocytes (CM) at the site of ischemic injury. To compensate for the loss of CMs, cardiac fibroblasts quickly respond to injury and initiate cardiac remodeling in an injured heart. In the remodeling process, cardiac fibroblasts proliferate and differentiate into myofibroblasts, which secrete extracellular matrix to support the intact structure of the heart, and eventually differentiate into matrifibrocytes to form chronic scar tissue. Discovery of direct cardiac reprogramming offers a promising therapeutic strategy to prevent/attenuate this pathologic remodeling and replace the cardiac fibrotic scar with myocardium in situ. Since the first discovery in 2010, many progresses have been made to improve the efficiency and efficacy of reprogramming by understanding the mechanisms and signaling pathways that are activated during direct cardiac reprogramming. Here, we overview the development and recent progresses of direct cardiac reprogramming and discuss future directions in order to translate this promising technology into an effective therapeutic paradigm to reverse cardiac pathological remodeling in an injured heart.

Inhibition of miRNA-212/132 improves the reprogramming of fibroblasts into induced pluripotent stem cells by de-repressing important epigenetic remodelling factors

MicroRNAs (miRNAs) repeatedly have been demonstrated to play important roles in the generation of induced pluripotent stem cells (iPSCs). To further elucidate the molecular mechanisms underlying transcription factor-mediated reprogramming we have established a model, which allows for the efficient screening of whole libraries of miRNAs modulating the generation of iPSCs from murine embryonic fibroblasts. Applying this model, we identified 14 miRNAs effectively inhibiting iPSC generation, including miR-132 and miR-212. Intriguingly, repression of these miRNAs during iPSC generation also resulted in significantly increased reprogramming efficacy. MiRNA target evaluation by qRT-PCR, Western blot, and luciferase assays revealed two crucial epigenetic regulators, the histone acetyl transferase p300 as well as the H3K4 demethylase Jarid1a (KDM5a) to be directly targeted by both miRNAs. Moreover, we demonstrated that siRNA-mediated knockdown of either p300 or Jarid1a recapitulated the miRNA effects and led to a significant decrease in reprogramming efficiency.Thus, conducting a full library miRNA screen we here describe a miRNA family, which markedly reduces generation of iPSC and upon inhibition in turn enhances reprogramming. These miRNAs, at least in part, exert their functions through repression of the epigenetic modulators p300 and Jarid1a, highlighting these two molecules as an endogenous epigenetic roadblock during iPSC generation.

Significant improvement of direct reprogramming efficacy of fibroblasts into progenitor endothelial cells by ETV2 and hypoxia.

BackgroundEndothelial progenitor cell (EPC) transplantation is a promising therapy for ischemic diseases such as ischemic myocardial infarction and hindlimb ischemia. However, limitation of EPC sources remains a major obstacle. Direct reprogramming has become a powerful tool to produce EPCs from fibroblasts. Some recent efforts successfully directly reprogrammed human fibroblasts into functional EPCs; however, the procedure efficacy was low. This study therefore aimed to improve the efficacy of direct reprogramming of human fibroblasts to functional EPCs.MethodsHuman fibroblasts isolated from foreskin were directly reprogrammed into EPCs by viral ETV2 transduction. Reprogramming efficacy was improved by culturing transduced fibroblasts in hypoxia conditions (5 % oxygen). Phenotype analyses confirmed that single-factor ETV2 transduction successfully reprogrammed dermal fibroblasts into functional EPCs.ResultsHypoxia treatment during the reprogramming procedure increased the efficacy of reprogramming from 1.21 ± 0.61 % in normoxia conditions to 7.52 ± 2.31 % in hypoxia conditions. Induced EPCs in hypoxia conditions exhibited functional EPC phenotypes similar to those in normoxia conditions, such as expression of CD31 and VEGFR2, and expressed endothelial gene profiles similar to human umbilical vascular endothelial cells. These cells also formed capillary-like networks in vitro.ConclusionOur study demonstrates a new simple method to increase the reprogramming efficacy of human fibroblasts to EPCs using ETV2 and hypoxia.

The Effect of Fetal Bovine Serum (FBS) on Efficacy of Cellular Reprogramming for Induced Pluripotent Stem Cell (iPSC) Generation.

Induced pluripotent stem cells (iPSCs) are pivotal to the advancement of regenerative medicine. However, the low efficacy of iPSC generation and insufficient knowledge about the reprogramming mechanisms involved in somatic cell/adult stem cell reversion to a pluripotent phenotype remain critical hurdles to the therapeutic application of iPSCs. The present study investigated whether the concentration of fetal bovine serum (FBS), a widely employed cell culture additive, can influence the cellular reprogramming efficacy (RE) of human adipose-derived stem cells (hADSCs) to generate iPSCs. Compared with the typically employed concentration of FBS (10%), high concentrations (20% and 30%) increased the RE of hADSCs by approximately twofold, whereas a low concentration (5%) decreased the RE by the same extent. Furthermore, cell counting kit-8 (CCK-8), bromodeoxyuridine (BrdU) incorporation, and fluorescence-activated cell sorting (FACS) assays showed that hADSC proliferation during reprogramming was significantly enhanced by FBS at 20% and 30%, whereas quantitative polymerase chain reaction (qPCR) and Western blotting assays revealed a concomitant decrease in p53, p51, and p21 expression. In addition, the efficacy of retrovirus-mediated transduction into hADSCs was increased by approximately 10% at high concentrations of FBS. It was confirmed that platelet-derived growth factor in the FBS enhanced proliferation and reprogramming efficacy. Finally, the generated iPSCs showed a normal karyotype, the same fingerprinting pattern as parental hADSCs, a genome-wide transcriptome pattern similar to that of human embryonic stem cells (hESCs), and in vivo pluripotency. In conclusion, the current investigation demonstrated that high concentrations of FBS can modulate molecular and cellular mechanisms underlying the reprogramming process in hADSCs, thereby augmenting the cellular RE for iPSC generation.

Practical Integration-Free Episomal Methods for Generating Human Induced Pluripotent Stem Cells.

The advent of induced pluripotent stem (iPS) cell technology has revolutionized biomedicine and basic research by yielding cells with embryonic stem (ES) cell-like properties. The use of iPS-derived cells for cell-based therapies and modeling of human disease holds great potential. While the initial description of iPS cells involved overexpression of four transcription factors via viral vectors that integrated within genomic DNA, advances in recent years by our group and others have led to safer and higher quality iPS cells with greater efficiency. Here, we describe commonly practiced methods for non-integrating induced pluripotent stem cell generation using nucleofection of episomal reprogramming plasmids. These methods are adapted from recent studies that demonstrate increased hiPS cell reprogramming efficacy with the application of three powerful episomal hiPS cell reprogramming factor vectors and the inclusion of an accessory vector expressing EBNA1.

Co-Electroporation with Xenopus Laevis Oocytes Reprograms Normal and Cancerous Human Cells to Resemble Reprogramming Normal and Cancerous Human Cells to Resemble induced Human Pluripotent Stem Cells

Objective To investigate reprogramming human cells into induced pluripotent stem cells (iPSc) using co-electroporation with Xenopus laevis oocytes. Methods Human bone marrow stromal cells (BMSC), BJ cells, pre-adipocytes (HPA), CD4+ T-lymphocytes (CD4TLs), buccal mucosa cells, and HeLa and MCF-7 cells were co-electroporated with mature Xenopus laevis oocytes, cultured, and assessed for pluripotency marker expression using fluorescent immunohistochemistry. Results The co-electroporated human cells formed colonies on irradiated mouse embryonic fibroblast cells (all study cells) and StemAdhere TM substrate (assessed for co-electroporated buccal mucosa cells). Cells derived from BMSC, BJ cells, HPA, CD4TLs, and buccal mucosa cells expressed Oct 3/4, Nanog, SOX-2, Rex-1, TRA-1-60, and SSEA-1. Cells derived from co-electroporated HeLa cells expressed Oct 3/4; cells derived from co-electroporated MCF-7 cells expressed Oct 3/4 and Nanog. Reprogramming efficacy for CD4TLs was 23.4 ± 3.5%. Co-electroporated HPA trans-differentiated into neural progenitor cells in culture conditions that foster neural differentiation. Control experiments suggested that the electroporate conveyed a reprogramming factor(s).

Improving the Reprogramming Efficiency of Somatic Cells to Induced Pluripotent Stem Cells.

Reprogramming shifts somatic cells toward a pluripotent state, resulting in cells that are capable of producing any tissue type, which provides a unique system for regenerative medicine. However, the poor efficiency of this process has prevented the use of induced pluripotent stem cells (iPSCs) in clinical application of stem cell biology. Importantly, more recent work has highlighted novel reprogramming factors and new strategies to enhance the reprogramming process. In this review, we focus on the transition of somatic cells to iPSCs, effective routes to improve the reprogramming efficacy, and the potential mechanism underlying this process.