Efficiency Of Cell Reprogramming Research Articles

NELFA and BCL2 induce the 2C-like state in mouse embryonic stem cells in a chemically defined medium.

A minority of mouse embryonic stem cells (ESCs) display totipotent features resembling 2-cell stage embryos and are known as 2-cell-like (2C-like) cells. However, how ESCs transit into this 2C-like state remains largely unknown. Here, we report that the overexpression of negative elongation factor A (Nelfa), a maternally provided factor, enhances the conversion of ESCs into 2C-like cells in chemically defined conditions, while the deletion of endogenous Nelfa does not block this transition. We also demonstrate that Nelfa overexpression significantly enhances somatic cell reprogramming efficiency. Interestingly, we found that the co-overexpression of Nelfa and Bcl2 robustly activates the 2C-like state in ESCs and endows the cells with dual cell fate potential. We further demonstrate that Bcl2 overexpression upregulates endogenous Nelfa expression and can induce the 2C-like state in ESCs even in the absence of Nelfa. Our findings highlight the importance of BCL2 in the regulation of the 2C-like state and provide insights into the mechanism underlying the roles of Nelfa and Bcl2 in the establishment and regulation of the totipotent state in mouse ESCs.

Cell reprogramming in a predictable manner on the superhydrophobic microwell array chip

Reprogramming of somatic cells into the pluripotent state is stochastic and inefficient using the conventional culture plates. Novel micro-culture systems employing precisely controlled biophysical cues can improve the reprogramming efficiencies dramatically. Here we perform iPSC induction on our previously developed superhydrophobic microwell array chip (SMAR-chip) where cells undergo distinctive morphology change, switching from 2D monolayers to 3D clumps, and develop into bona fide colonies in more than 90% of the microwells. The PDMS substrate, together with the microwell structure and the superhydrophobic layer constitute a well-controlled microenvironment favorable for the morphogenesis and pluripotency induction. Investigation of the molecular roadmap demonstrates that the SMAR-chip promotes the transition from the initiation phase to the maturation phase and overcomes the roadblocks for reprogramming. In addition, the SMAR-chip also promotes the reprogramming of human cells, opening our method for translational applications. In summary, our study provides a novel platform for efficient cell reprogramming and emphasizes the advantages of employing the insoluble microenvironmental cues for the precise control of cell fate conversion.

BRD9-containing non-canonical BAF complex maintains somatic cell transcriptome and acts as a barrier to human reprogramming.

Epigenetic reprogramming to pluripotency requires extensive remodeling of chromatin landscapes to silence existing cell-type-specific genes and activate pluripotency genes. ATP-dependent chromatin remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of recently identified Bromodomain-containing protein 9 (BRD9) and the associated non-canonical BRG1-associated factors (ncBAF) complex in reprogramming remains unknown. Here, we show that genetic or chemical inhibition of BRD9, as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase human somatic cell reprogramming efficiency and can replace KLF4 and c-MYC. We find that BRD9 is dispensable for human induced pluripotent stem cells under primed but not under naive conditions. Mechanistically, BRD9 inhibition downregulates fibroblast-related genes and decreases chromatin accessibility at somatic enhancers. BRD9 maintains the expression of transcriptional regulators MN1 and ZBTB38, both of which impede reprogramming. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.

Survival Motor Neuron Enhances Pluripotent Gene Expression and Facilitates Cell Reprogramming.

Survival motor neuron (SMN) plays important roles in snRNP assembly and mRNA splicing. Deficiency of SMN causes spinal muscular atrophy (SMA), a leading genetic disease causing childhood mortality. Previous studies have shown that SMN regulates stem cell self-renewal and pluripotency in Drosophila and mouse and is abundantly expressed in mouse embryonic stem cells. However, whether SMN is required for establishment of pluripotency is unclear. In this study, we show that SMN is gradually upregulated in preimplantation mouse embryos and cultured cells undergoing cell reprogramming. Ectopic expression of SMN increased cell reprogramming efficiency, whereas knockdown of SMN impeded induced pluripotent stem cell (iPSC) colony formation. iPSCs could be derived from SMA model mice, but impairment in differentiation capacity may be present. The ectopic overexpression of SMN in iPSCs can upregulate the expression levels of some pluripotent genes and restore the neuronal differentiation capacity of SMA-iPSCs. Taken together, our findings not only demonstrate the functional relevance of SMN in establishment of cell pluripotency but also propose its potential application in facilitating iPSC derivation.

Efficient Rebalancing of ROS Levels in Plants Links to Temporarily Enhanced Aerobic Fermentation, Distinct Cell Restructuration, and Resilience in Field

Efficient Rebalancing of ROS Levels in Plants Links to Temporarily Enhanced Aerobic Fermentation, Distinct Cell Restructuration, and Resilience in Field

Fibroblast transition to an endothelial \u201ctrans\u201d state improves cell reprogramming efficiency

Fibroblast reprogramming offers the potential for myocardial regeneration via in situ cell transdifferentiation. We explored a novel strategy leveraging endothelial cell plasticity to enhance reprogramming efficiency. Rat cardiac endothelial cells and fibroblasts were treated with Gata4, Mef2c, and Tbx5 (GMT) to assess the cardio-differentiation potential of these cells. The endothelial cell transdifferentiation factor ETV2 was transiently over-expressed in fibroblasts followed by GMT treatment to assess “trans-endothelial” cardio-differentiation. Endothelial cells treated with GMT generated more cTnT+ cells than did cardiac fibroblasts (13% ± 2% vs 4% ± 0.5%, p < 0.01). Cardiac fibroblasts treated with ETV2 demonstrated increased endothelial cell markers, and when then treated with GMT yielded greater prevalence of cells expressing cardiomyocyte markers including cTnT than did fibroblasts treated with GMT or ETV2 (10.3% ± 0.2% vs 1.7% ± 0.06% and 0.6 ± 0.03, p < 0.01). Rat cardiac fibroblasts treated with GMT + ETV2 demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes, whereas cells treated with GMT or ETV2 alone failed to contract in co-culture experiments. Human cardiac fibroblasts treated with ETV2 and then GMT likewise demonstrated greater prevalence of cTnT expression than did cells treated with GMT alone (2.8-fold increase, p < 0.05). Cardiac fibroblast transitioning through a trans-endothelial state appears to enhance cardio-differentiation by enhancing fibroblast plasticity.

Reprogramming Human Cancer Cells into Antigen Presentation

Cancer progression entails close crosstalk between tumor cells and immune system. Recent advances in understanding mutual tumor-immune interactions translated into the development of cancer immunotherapy, changing the cancer treatment paradigm. Nevertheless, despite achieving long-term effect even in advanced malignancies, only a minor subset of patients responds to immunotherapy. The activation of the immune system, as well as good response to the immunotherapy strongly depends on tumor immunogenicity and effective presentation of tumor-associated antigens by conventional dendritic cells type 1 (cDC1). However, downregulation of antigen-presentation machinery and cDC1 exclusion represent major mechanisms of cancer immune evasion, leading to immunotherapy failure. Previously, we identified a combination of 3 transcription factors, PU.1, IRF8, and BATF3 (PIB) that instructed direct reprogramming of fibroblasts into cDC1-like cells endowed with the ability to present and cross-present antigens. Here, we hypothesize that cDC1-direct reprogramming employing PIB transcription factors can impose antigen-presentation directly in human tumor cells, generating functional tumor-antigen presenting cells (tumor-APCs) able to prime antigen-specific T cell responses.Here, we used a large panel of human cell lines (>30) derived from leukemia and multiple solid tumors including glioblastoma, melanoma, breast, pancreatic, ovarian, prostate, head and neck, and lung adenocarcinoma and evaluated dendritic cell reprogramming efficiency. Enforced expression of PIB induced a cDC1-phenotype in tumor cells, as demonstrated by an emerging CD45+HLA-DR+ population and the activation of the cDC1-markers CD11c, CD141, and CLEC9A. Induced cells showed global transcriptional reprogramming towards cDC1 fate imposing a tumor-APC signature in cancer cells at varying efficiencies ranging between 0.2±0.1% to 94.5±7.6% according to the tumor cell of origin. PIB induces rapid transcriptomic and epigenetic rewiring of cancer cells towards cDC1-fate, activating antigen presentation and processing machinery. This reprogramming process is asynchronous and leads to the generation of intermediate populations with active tumor-APC gene expression signatures. Furthermore, tumor-APCs upregulated expression of HLA-ABC and the co-stimulatory molecules CD40, CD80, and CD86, suggesting the acquired ability to prime immune-effectors. Functionally, tumor-APCs secrete pro-inflammatory cytokines including IL12p70, TNFα, CXCL10, and IL-29 upon TLR3/4 stimulation with Poly:IC and LPS, are competent in phagocytosis of dead cells and proteins, and can activate antigen-specific CD8+ T-cells. Importantly, cDC1-cell fate adoption was accompanied by downregulation of proliferation and diminished tumorigenicity, as evidenced by the loss of anchorage-independent growth. Importantly, cDC1-reprogramming was efficient in primary tissues obtained from head and neck, breast, urothelial and pancreatic carcinoma, as well as melanoma patients. Notably, PIB-mediated cDC1 reprogramming of tumor cells is substantially augmented when combined with a histone deacetylase inhibitor, valproic acid.In summary, PIB expression imposes cDC1 fate and function in a broad range of human cancer cells suggesting a capacity to elicit anti-tumor responses by orchestrating innate and adaptive immunity. Our strategy combines cDC1 antigen-processing and presenting abilities with the endogenous generation of tumor-antigens. This approach merges cell fate reprogramming with cancer immunotherapy, paving the way for the development of novel gene therapy for cancer. DisclosuresNo relevant conflicts of interest to declare.

Vertically Aligned Nanostructured Topographies for Human Neural Stem Cell Differentiation and Neuronal Cell Interrogation

AbstractNeurodegenerative disorders are a widespread global health concern caused by aging, disease, and trauma, for which there are limited treatment options. Stem cell therapies, tissue engineering, and nanobiotechnologies offer hope for improved therapeutic delivery approaches, as well as tissue repair and regenerative medicine interventions. The complexity of the human brain, coupled with its limited availability for research, makes human neural lineage cells and their precursor stem cells integral to the further understanding of brain functions in health, development, and disease.Engineered nanomaterials provide highly specialized microenvironments, enabling precise interrogation of the impact of external and spatial stimuli on human neural cells in vitro, greatly advancing the knowledge of human neural function. Interacting with neural cells at the nanoscale, vertically aligned nanostructured (VA‐NS) arrays can influence cell fate and aid in more efficient cell reprogramming, and lend themselves to the development of highly targeted, sensitive signal transducer platforms suitable for in vivo monitoring of neural cell health and activity.This perspective highlights the current state of stem cell nanoneurobiology, specifically focusing on interdisciplinary advances made by VA‐NS arrays to manipulate human neural stem cells in translatable research applications. Current challenges and identify are discussed underexplored and emerging future research areas.

Molecular signature and colony morphology affect in vitro pluripotency of porcine induced pluripotent stem cells.

Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n=10) or loose (n=10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p<.05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line.

Hydrogen sulfide facilitates reprogramming and trans-differentiation in 3D dermal fibroblast.

The efficiency of cell reprogramming in two-dimensional (2D) cultures is limited. Given that cellular stemness is intimately related to microenvironmental changes, 3D cell cultures have the potential of overcoming this limited capacity by allowing cells to self-organize by aggregation. In 3D space, cells interact more efficiently, modify their cellular topology, gene expression, signaling, and metabolism. It is yet not clear as how 3D culture environments modify the reprogramming potential of fibroblasts. We demonstrate that 3D spheroids from dermal fibroblasts formed under ultra-low attachment conditions showed increased lactate production. This is a requisite for cell reprogramming, increase their expression of pluripotency genes, such as OCT4, NANOG and SOX2, and display upregulated cystathionine-β-synthase (CBS) and hydrogen sulfide (H2S) production. Knockdown of CBS by RNAi suppresses lactic acid and H2S production and concomitantly decreases the expression of OCT4 and NANOG. On the contrary, H2S donors, NaHS and garlic-derived diallyl trisulfide (DATS), promote the expression of OCT4, and support osteogenic trans-differentiation of fibroblasts. These results demonstrate that CBS mediated release of H2S regulates the reprogramming of dermal fibroblasts grown in 3D cultures and supports their trans-differentiation.

Non-viral, direct neuronal reprogramming from human fibroblast using a polymer-functionalized nanodot

Among various strategies to treat neurodegenerative disorders, cell replacement therapies have drawn much attention recently. Such a trend led to the increase in demand for the rare and specialized cells, followed by the outburst development of various cell reprogramming strategies. However, several limitations on these conventional methods remain to be solved, including the genetic instability of the viral vectors and the high cytotoxicity or poor performance of the non-viral carriers. Therefore, non-viral methods need to be developed to ensure safe and efficient cell reprogramming. Here, we introduce a polymer-modified nano-reagent (Polymer-functionalized Nanodot, PolyN) for the safe and efficient, non-viral direct cell reprogramming. PolyN facilitated the highly efficient contemporary overexpression of the transgene compared to the conventional reagent. With our nano-reagent, we demonstrated the SOX2-mediated cell reprogramming and successfully generated the neuron-like cell from the human fibroblast.

Lactate preconditioning promotes a HIF-1\u03b1-mediated metabolic shift from OXPHOS to glycolysis in normal human diploid fibroblasts

Recent evidence has emerged that cancer cells can use various metabolites as fuel sources. Restricting cultured cancer cells to sole metabolite fuel sources can promote metabolic changes leading to enhanced glycolysis or mitochondrial OXPHOS. However, the effect of metabolite-restriction on non-transformed cells remains largely unexplored. Here we examined the effect of restricting media fuel sources, including glucose, pyruvate or lactate, on the metabolic state of cultured human dermal fibroblasts. Fibroblasts cultured in lactate-only medium exhibited reduced PDH phosphorylation, indicative of OXPHOS, and a concurrent elevation of ROS. Lactate exposure primed fibroblasts to switch to glycolysis by increasing transcript abundance of genes encoding glycolytic enzymes and, upon exposure to glucose, increasing glycolytic enzyme levels. Furthermore, lactate treatment stabilized HIF-1α, a master regulator of glycolysis, in a manner attenuated by antioxidant exposure. Our findings indicate that lactate preconditioning primes fibroblasts to switch from OXPHOS to glycolysis metabolism, in part, through ROS-mediated HIF-1α stabilization. Interestingly, we found that lactate preconditioning results in increased transcript abundance of MYC and SNAI1, key facilitators of early somatic cell reprogramming. Defined metabolite treatment may represent a novel approach to increasing somatic cell reprogramming efficiency by amplifying a critical metabolic switch that occurs during iPSC generation.

Acceleration of somatic cell reprogramming into the induced pluripotent stem cell using a mycosporine-like amino acid, Porphyra 334

Porphyra 334 (P334), a mycosporine-like amino acid (MAA), is a secondary metabolite found in diverse marine and terrestrial organisms and has several beneficial effects on fibroblast proliferation, wound healing, and antioxidant activity. Here, we report that P334 accelerates the cell reprogramming process of mouse tail-tip fibroblasts (TTFs) and human dermal papilla (HDP) cells into induced pluripotent stem cells (iPSCs). We found that P334 significantly improved the cell reprogramming efficiency by activating the tri-methylation of histone 3 lysine 4 (H3K4me3), which controls mesenchymal to epithelial transition (MET) during the reprogramming process. Thus, we found that P334 directly regulates epigenetic changes, providing an efficient approach for natural compound-based cell reprogramming.

Mitochondrial Akt Signaling Modulated Reprogramming of Somatic Cells

The signaling mechanisms controlling somatic cell reprogramming are not fully understood. In this study, we report a novel role for mitochondrial Akt1 signaling that enhanced somatic cell reprogramming efficiency. The role of mitochondrial Akt1 in somatic cell reprogramming was investigated by transducing fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, c-Myc) in conjunction with Mito-Akt1, Mito-dnAkt1, or control virus. Mito-Akt1 enhanced reprogramming efficiency whereas Mito-dnAkt1 inhibited reprogramming. The resulting iPSCs formed embryoid bodies in vitro and teratomas in vivo. Moreover, Oct4 and Nanog promoter methylation was reduced in the iPSCs generated in the presence of Mito-Akt1. Akt1 was activated and translocated into mitochondria after growth factor stimulation in embryonic stem cells (ESCs). To study the effect of mitochondrial Akt in ESCs, a mitochondria-targeting constitutively active Akt1 (Mito-Akt1) was expressed in ESCs. Gene expression profiling showed upregulation of genes that promote stem cell proliferation and survival and down-regulation of genes that promote differentiation. Analysis of cellular respiration indicated similar metabolic profile in the resulting iPSCs and ESCs, suggesting comparable bioenergetics. These findings showed that activation of mitochondrial Akt1 signaling was required during somatic cell reprogramming.

Investigating the role of Sirtuins in cell reprogramming

Cell reprogramming has been considered a powerful technique in the regenerative medicine field. In addition to diverse its strengths, cell reprogramming technology also has several drawbacks generated during the process of reprogramming. Telomere shortening caused by the cell reprogramming process impedes the efficiency of cell reprogramming. Transcription factors used for reprogramming alter genomic contents and result in genetic mutations. Additionally, defective mitochondria functioning such as excessive mitochondrial fission leads to the limitation of pluripotency and ultimately reduces the efficiency of reprogramming. These problems including genomic instability and impaired mitochondrial dynamics should be resolved to apply cell reprograming in clinical research and to address efficiency and safety concerns. Sirtuin (NAD+-dependent histone deacetylase) has been known to control the chromatin state of the telomere and influence mitochondria function in cells. Recently, several studies reported that Sirtuins could control for genomic instability in cell reprogramming. Here, we review recent findings regarding the role of Sirtuins in cell reprogramming. And we propose that the manipulation of Sirtuins may improve defects that result from the steps of cell reprogramming.

Tgfbr2 inactivation facilitates cellular plasticity and development of Pten-null prostate cancer.

Mutations in tumors can create a state of increased cellular plasticity that promotes resistance to treatment. Thus, there is an urgent need to develop novel strategies for identifying key factors that regulate cellular plasticity in order to combat resistance to chemotherapy and radiation treatment. Here we report that prostate epithelial cell reprogramming could be exploited to identify key factors required for promoting prostate cancer tumorigenesis and cellular plasticity. Deletion of phosphatase and tensin homolog (Pten) and transforming growth factor-beta receptor type 2 (Tgfbr2) may increase prostate epithelial cell reprogramming efficiency in vitro and cause rapid tumor development and early mortality in vivo. Tgfbr2 ablation abolished TGF-β signaling but increased the bone morphogenetic protein (BMP) signaling pathway through the negative regulator Tmeff1. Furthermore, increased BMP signaling promotes expression of the tumor marker genes ID1, Oct4, Nanog, and Sox2; ID1/STAT3/NANOG expression was inversely correlated with patient survival. Thus, our findings provide information about the molecular mechanisms by which BMP signaling pathways render stemness capacity to prostate tumor cells.

Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4

The development of human induced pluripotent stem cells (iPSCs) holds great promise for regenerative medicine. However the iPSC induction efficiency is still very low and with lengthy reprogramming process. We utilized the highly potent transactivation domain (TAD) of MYC protein to engineer the human OCT4 fusion proteins. Applying the MYC-TAD-OCT4 fusion proteins in mouse iPSC generation leads to shorter reprogramming dynamics, with earlier activation of pluripotent markers in reprogrammed cells than wild type OCT4 (wt-OCT4). Dramatic enhancement of iPSC colony induction efficiency and shortened reprogramming dynamics were observed when these MYC-TAD-OCT4 fusion proteins were used to reprogram primary human cells. The OCT4 fusion proteins induced human iPSCs are pluripotent. We further show that the MYC Box I (MBI) is dispensable while both MBII and the linking region between MBI/II are essential for the enhanced reprogramming activity of MYC-TAD-OCT4 fusion protein. Consistent with an enhanced transcription activity, the engineered OCT4 significantly stimulated the expression of genes specifically targeted by OCT4-alone, OCT4/SOX2, and OCT4/SOX2/KLF4 during human iPSC induction, compared with the wt-OCT4. The MYC-TAD-OCT4 fusion proteins we generated will be valuable tools for studying the reprogramming mechanisms and for efficient iPSC generation for humans as well as for other species.

Degradation rate defines cell reprogramming efficiency of the active chitosan implants

Background: Local delivery of drugs is an effective way to reach efficient concentration without general toxicity. With advances of gene therapy the need for implantable local delivery scaffolds is growing rapidly. Chitosan is an inexpensive material with many valuable features for developing biomedical applications. It is biocompatible, biodegradable, easy modifiable, and positively charged.

Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity.

Comparison of Reprogramming Methods for Generation of Induced-Oligodendrocyte Precursor Cells.

Direct conversion by trans-differentiation is of growing interest in cell therapy for incurable diseases. The efficiency of cell reprogramming and functionality of converted cells are important considerations in cell transplantation therapy. Here, we compared two representative protocols for the generation of induced-oligodendrocyte progenitor cells (iOPCs) from mouse and rat fibroblasts. Then, we showed that induction of Nkx6.2, Olig2, and Sox10 (NOS) was more effective in mouse fibroblasts and that induction of Olig2, Sox10, and Zfp536 (OSZ) was more effective at reprogramming iOPCs from rat fibroblasts. However, OSZ-iOPCs did not show greater proliferation than NOS-induced cells. Because the efficiency of iOPCs generation appears to differ between cell species depending on transcription factors and culture conditions, it is important to select appropriate methods for efficient reprogramming.