Direct cellular reprogramming techniques for cardiovascular regenerative therapeutics.

Cardiovascular diseases remain a leading cause of hospitalization affecting approximately 38 million people worldwide. While pharmacological and revascularization techniques can improve the patient's survival and quality of life, they cannot help reversing myocardial infarction injury and heart failure. Direct reprogramming of somatic cells to cardiomyocyte and cardiac progenitor cells offers a new approach to cellular reprogramming and paves the way for translational regenerative medicine. Direct reprogramming can bypass the pluripotent stage with the potential advantage of non-immunogenic cell products, reduced carcinogenic risk, and no requirement for embryonic tissue. The process of directly reprogramming cardiac cells was first achieved through the overexpression of transcription factors such as GATA4, MEF2C, and TBX5. However, over the past decade, significant work has been focused on enhancing direct reprogramming using a mixture of transcription factors, microRNAs, and small molecules to achieve cardiac cell fate. This review discusses the evolution of direct reprogramming, recent progress in achieving efficient cardiac cell fate conversion, and describes the reprogramming mechanisms at a molecular level. We also explore various viral and non-viral delivery methods currently being used to aid in the delivery of reprogramming factors to improve efficiency. However, further studies will be needed to overcome molecular and epigenetic barriers to successfully achieve translational cardiac regenerative therapeutics.

Reprogramming and multi-lineage transdifferentiation attenuate the tumorigenicity of colorectal cancer cells.

Significant advances have been made in reprogramming various somatic cells into induced pluripotent stem cells (iPSCs) and in multi-lineage differentiation (transdifferentiation) into different tissues. These manipulable transdifferentiating techniques may be applied in cancer therapy. Limited works have been reported that cancer cell malignancy can be switched to benign phenotypes through reprogramming techniques. Here, we reported that two colorectal cancer (CRC) cell lines (DLD1, HT29) could be reprogrammed into iPSCs (D-iPSCs, H-iPSCs). D- and H-iPSCs showed reduced tumorigenesis. Furthermore, we successfully induced D- and H-iPSCs differentiation into terminally differentiated cell types such as cardiomyocyte, neuron, and adipocyte-like cells. Impressively, the differentiated cells exhibited further attenuated tumorigenesis in vitro and in vivo. RNA-Seq further indicated that epigenetic changes occurred after reprogramming and transdifferentiation that caused reduced tumorigenicity. Overall, our study indicated that CRC cells can be reprogrammed and further differentiated into terminally differentiated lineages with attenuation of their malignancy in vitro and in vivo. The current work sheds light on a potential multi-lineage differentiation therapeutic strategy for colorectal cancer.

KERS-Inspired Nanostructured Mineral Coatings Boost IFN-γ mRNA Therapeutic Index for Antitumor Immunotherapy.

Tumor-associated macrophage (TAM) reprogramming is a promising therapeutic approach for cancer immunotherapy; however, its efficacy remains modest due to the low bioactivity of the recombinant cytokines used for TAM reprogramming. mRNA therapeutics are capable of generating fully functional proteins for various therapeutic purposes but accused for its poor sustainability. Inspired by kinetic energy recovery systems (KERS) in hybrid vehicles, a cytokine efficacy recovery system (CERS) is designed to substantially augment the therapeutic index of mRNA-based tumor immunotherapy via a "capture and stabilize" mechanism exerted by a nanostructured mineral coating carrying therapeutic cytokine mRNA. CERS remarkably recycles nearly 40% expressed cytokines by capturing them onto the mineral coating to extend its therapeutic timeframe, further polarizing the macrophages to strengthen their tumoricidal activity and activate adaptive immunity against tumors. Notably, interferon-γ (IFN-γ) produced by CERS exhibits ≈42-fold higher biological activity than recombinant IFN-γ, remarkably decreasing the required IFN-γ dosage for TAM reprogramming. In tumor-bearing mice, IFN-γ cmRNA@CERS effectively polarizes TAMs to inhibit osteosarcoma progression. When combined with the PD-L1 monoclonal antibody, IFN-γ cmRNA@CERS significantly boosts antitumor immune responses, and substantially prevents malignant lung metastases. Thus, CERS-mediated mRNA delivery represents a promising strategy to boost antitumor immunity for tumor treatment.

A fast chemical reprogramming system promotes cell identity transition through a diapause-like state.

Cellular reprogramming by only small molecules holds enormous potentials for regenerative medicine. However, chemical reprogramming remains a slow process and labour intensive, hindering its broad applications and the investigation of underlying molecular mechanisms. Here, through screening of over 21,000 conditions, we develop a fast chemical reprogramming (FCR) system, which significantly improves the kinetics of cell identity rewiring. We find that FCR rapidly goes through an interesting route for pluripotent reprogramming, uniquely transitioning through a developmentally diapause-like state. Furthermore, FCR critically enables comprehensive characterizations using multi-omics technologies, and has revealed unexpected important features including key regulatory factors and epigenetic dynamics. Particularly, activation of pluripotency-related endogenous retroviruses via inhibition of heterochromatin significantly enhances reprogramming. Our studies provide critical insights into how only environmental cues are sufficient to rapidly reinstate pluripotency in somatic cells, and make notable technical and conceptual advances for solving the puzzle of regeneration.

Reprogramming tumour-associated macrophages to outcompete cancer cells.

In metazoan organisms, cell competition acts as a quality control mechanism to eliminate unfit cells in favour of their more robust neighbours1,2. This mechanism has the potential to be maladapted, promoting the selection of aggressive cancer cells3-6. Tumours are metabolically active and are populated by stroma cells7,8, but how environmental factors affect cancer cell competition remains largely unknown. Here we show that tumour-associated macrophages (TAMs) can be dietarily or genetically reprogrammed to outcompete MYC-overexpressing cancer cells. In a mouse model of breast cancer, MYC overexpression resulted in an mTORC1-dependent 'winner' cancer cell state. A low-protein diet inhibited mTORC1 signalling in cancer cells and reduced tumour growth, owing unexpectedly to activation of the transcription factors TFEB and TFE3 and mTORC1 in TAMs. Diet-derived cytosolic amino acids are sensed by Rag GTPases through the GTPase-activating proteins GATOR1 and FLCN to control Rag GTPase effectors including TFEB and TFE39-14. Depletion of GATOR1 in TAMs suppressed the activation of TFEB, TFE3 and mTORC1 under the low-protein diet condition, causing accelerated tumour growth; conversely, depletion of FLCN or Rag GTPases in TAMs activated TFEB, TFE3 and mTORC1 under the normal protein diet condition, causing decelerated tumour growth. Furthermore, mTORC1 hyperactivation in TAMs and cancer cells and their competitive fitness were dependent on the endolysosomal engulfment regulator PIKfyve. Thus, noncanonical engulfment-mediated Rag GTPase-independent mTORC1 signalling in TAMs controls competition between TAMs and cancer cells, which defines a novel innate immune tumour suppression pathway that could be targeted for cancer therapy.

Direct Reprogramming of Resident Non-Myocyte Cells and Its Potential for In Vivo Cardiac Regeneration.

Cardiac diseases are the foremost cause of morbidity and mortality worldwide. The heart has limited regenerative potential; therefore, lost cardiac tissue cannot be replenished after cardiac injury. Conventional therapies are unable to restore functional cardiac tissue. In recent decades, much attention has been paid to regenerative medicine to overcome this issue. Direct reprogramming is a promising therapeutic approach in regenerative cardiac medicine that has the potential to provide in situ cardiac regeneration. It consists of direct cell fate conversion of one cell type into another, avoiding transition through an intermediary pluripotent state. In injured cardiac tissue, this strategy directs transdifferentiation of resident non-myocyte cells (NMCs) into mature functional cardiac cells that help to restore the native tissue. Over the years, developments in reprogramming methods have suggested that regulation of several intrinsic factors in NMCs can help to achieve in situ direct cardiac reprogramming. Among NMCs, endogenous cardiac fibroblasts have been studied for their potential to be directly reprogrammed into both induced cardiomyocytes and induced cardiac progenitor cells, while pericytes can transdifferentiate towards endothelial cells and smooth muscle cells. This strategy has been indicated to improve heart function and reduce fibrosis after cardiac injury in preclinical models. This review summarizes the recent updates and progress in direct cardiac reprogramming of resident NMCs for in situ cardiac regeneration.

Stem cell factors reverse signs of aging in mice.

Will reprogramming technique one day help people?

Advances in Cellular Reprogramming-Based Approaches for Heart Regenerative Repair.

Continuous loss of cardiomyocytes (CMs) is one of the fundamental characteristics of many heart diseases, which eventually can lead to heart failure. Due to the limited proliferation ability of human adult CMs, treatment efficacy has been limited in terms of fully repairing damaged hearts. It has been shown that cell lineage conversion can be achieved by using cell reprogramming approaches, including human induced pluripotent stem cells (hiPSCs), providing a promising therapeutic for regenerative heart medicine. Recent studies using advanced cellular reprogramming-based techniques have also contributed some new strategies for regenerative heart repair. In this review, hiPSC-derived cell therapeutic methods are introduced, and the clinical setting challenges (maturation, engraftment, immune response, scalability, and tumorigenicity), with potential solutions, are discussed. Inspired by the iPSC reprogramming, the approaches of direct cell lineage conversion are merging, such as induced cardiomyocyte-like cells (iCMs) and induced cardiac progenitor cells (iCPCs) derived from fibroblasts, without induction of pluripotency. The studies of cellular and molecular pathways also reveal that epigenetic resetting is the essential mechanism of reprogramming and lineage conversion. Therefore, CRISPR techniques that can be repurposed for genomic or epigenetic editing become attractive approaches for cellular reprogramming. In addition, viral and non-viral delivery strategies that are utilized to achieve CM reprogramming will be introduced, and the therapeutic effects of iCMs or iCPCs on myocardial infarction will be compared. After the improvement of reprogramming efficiency by developing new techniques, reprogrammed iCPCs or iCMs will provide an alternative to hiPSC-based approaches for regenerative heart therapies, heart disease modeling, and new drug screening.

Macrophage Reprogramming with Anti-miR223-Loaded Artificial Protocells Enhances In Vivo Cancer Therapeutic Potential.

Several immune cell-expressed miRNAs (miRs) are associated with altered prognostic outcome in cancer patients, suggesting that they may be potential targets for development of cancer therapies. Here, translucent zebrafish (Danio rerio) is utilized to demonstrate that genetic knockout or knockdown of one such miR, microRNA-223 (miR223), globally or specifically in leukocytes, does indeed lead to reduced cancer progression. As a first step toward potential translation to a clinical therapy, a novel strategy is described for reprogramming neutrophils and macrophages utilizing miniature artificial protocells (PCs) to deliver anti-miRs against the anti-inflammatory miR223. Using genetic and live imaging approaches, it is shown that phagocytic uptake of anti-miR223-loaded PCs by leukocytes in zebrafish (and by human macrophages in vitro) effectively prolongs their pro-inflammatory state by blocking the suppression of pro-inflammatory cytokines, which, in turn, drives altered immune cell-cancer cell interactions and ultimately leads to a reduced cancer burden by driving reduced proliferation and increased cell death of tumor cells. This PC cargo delivery strategy for reprogramming leukocytes toward beneficial phenotypes has implications also for treating other systemic or local immune-mediated pathologies.

In Vitro Differentiation of Human Amniotic Epithelial Cells into Hepatocyte-like Cells.

Human amniotic epithelial cells (hAECs) represent an interesting clinical alternative to human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells in regenerative medicine. The potential of hAECs can be enhanced ex vivo by their partial pre-differentiation. The aim of this study was to evaluate the effectiveness of 18-day differentiation of hAECs into endodermal cells, hepatic precursor cells, and cells showing functional features of hepatocytes using culture media supplemented with high (100 ng/mL) concentrations of EGF or HGF. The cells obtained after differentiation showed changes in morphology and increased expression of AFP, ALB, CYP3A4, CYP3A7, and GSTP1 genes. HGF was more effective than EGF in increasing the expression of liver-specific genes in hAECs. However, EGF stimulated the differentiation process more efficiently and yielded more hepatocyte-like cells capable of synthesizing α-fetoprotein during differentiation. Additionally, after 18 days, GST transferases, albumin, and CYP P450s, which proved their partial functionality, were expressed. In summary, HGF and EGF at a dose of 100 ng/mL can be successfully used to obtain hepatocyte-like cells between days 7 and 18 of hAEC differentiation. However, the effectiveness of this process is lower compared with hiPSC differentiation; therefore, optimization of the composition of the medium requires further research.

Pancreatic Transdifferentiation Using ß-Cell Transcription Factors for Type 1 Diabetes Treatment.

Type 1 diabetes is a chronic illness in which the native beta (ß)-cell population responsible for insulin release has been the subject of autoimmune destruction. This condition requires patients to frequently measure their blood glucose concentration and administer multiple daily exogenous insulin injections accordingly. Current treatments fail to effectively treat the disease without significant side effects, and this has led to the exploration of different approaches for its treatment. Gene therapy and the use of viral vectors has been explored extensively and has been successful in treating a range of diseases. The use of viral vectors to deliver ß-cell transcription factors has been researched in the context of type 1 diabetes to induce the pancreatic transdifferentiation of cells to replace the ß-cell population destroyed in patients. Studies have used various combinations of pancreatic and ß-cell transcription factors in order to induce pancreatic transdifferentiation and have achieved varying levels of success. This review will outline why pancreatic transcription factors have been utilised and how their application can allow the development of insulin-producing cells from non ß-cells and potentially act as a cure for type 1 diabetes.

PI3Kδ/γ inhibition promotes human CART cell epigenetic and metabolic reprogramming to enhance antitumor cytotoxicity.

Current limitations in using chimeric antigen receptor T(CART) cells to treat patients with hematological cancers include limited expansion and persistence in vivo that contribute to cancer relapse. Patients with chronic lymphocytic leukemia (CLL) have terminally differentiated T cells with an exhausted phenotype and experience low complete response rates after autologous CART therapy. Because PI3K inhibitor therapy is associated with the development of T-cell-mediated autoimmunity, we studied the effects of inhibiting the PI3Kδ and PI3Kγ isoforms during the manufacture of CART cells prepared from patients with CLL. Dual PI3Kδ/γ inhibition normalized CD4/CD8 ratios and maximized the number of CD8+ T-stem cell memory, naive, and central memory T-cells with dose-dependent decreases in expression of the TIM-3 exhaustion marker. CART cells manufactured with duvelisib (Duv-CART cells) showed significantly increased in vitro cytotoxicity against CD19+ CLL targets caused by increased frequencies of CD8+ CART cells. Duv-CART cells had increased expression of the mitochondrial fusion protein MFN2, with an associated increase in the relative content of mitochondria. Duv-CART cells exhibited increased SIRT1 and TCF1/7 expression, which correlated with epigenetic reprograming of Duv-CART cells toward stem-like properties. After transfer to NOG mice engrafted with a human CLL cell line, Duv-CART cells expressing either a CD28 or 41BB costimulatory domain demonstrated significantly increased in vivo expansion of CD8+ CART cells, faster elimination of CLL, and longer persistence. Duv-CART cells significantly enhanced survival of CLL-bearing mice compared with conventionally manufactured CART cells. In summary, exposure of CART to a PI3Kδ/γ inhibitor during manufacturing enriched the CART product for CD8+ CART cells with stem-like qualities and enhanced efficacy in eliminating CLL in vivo.

Comprehensive analyses of the inotropic compound omecamtiv mecarbil in rat and human cardiac preparations.

Omecamtiv mecarbil (OM), a myosin activator, was reported to induce complex concentration- and species-dependent effects on contractile function, and clinical studies indicated a low therapeutic index with diastolic dysfunction at concentrations above 1 µM. To further characterize effects of OM in a human context and under different preload conditions, we constructed a setup that allows isometric contractility analysis of human induced pluripotent stem cell (hiPSC)-derived engineered heart tissues (EHTs). The results were compared with effects of OM on the very same EHTs measured under auxotonic conditions. OM induced a sustained, concentration-dependent increase in time to peak under all conditions (maximally two- to threefold). Peak force, in contrast, was increased by OM only in human, but not rat EHTs and only under isometric conditions, varied between hiPSC lines and showed a biphasic concentration dependency with maximal effects at 1 µM. Relaxation time tended to fall under auxotonic and strongly increased under isometric conditions, again with biphasic concentration dependency. Diastolic tension concentration dependently increased under all conditions. The latter was reduced by an inhibitor of the mitochondrial sodium calcium exchanger (CGP-37157). OM induced increases in mitochondrial oxidation in isolated cardiomyocytes, indicating that OM, an inotrope that does not increase intracellular and mitochondrial Ca2+, can induce mismatch between an increase in ATP and ROS production and unstimulated mitochondrial redox capacity. Taken together, we developed a novel setup well suitable for isometric measurements of EHTs. The effects of OM on contractility and diastolic tension are complex with concentration-, time-, species- and loading-dependent differences. Effects on mitochondrial function require further studies.NEW & NOTEWORTHY We developed a novel setup allowing precise control of preload of EHT and characterized effects of the myosin activator OM. OM not only exerted contraction-slowing and positive inotropic effects but also increased diastolic tension. Effect size and direction varied between species, auxotonic and isometric conditions, concentration, and time. We also observed OM-induced increase of mitochondrial ROS, which has not been observed before and may explain part of the effects on contractility.

Reprogramming of Chinese hamster ovary cells towards enhanced protein secretion.

The deficient secretory phenotype of Chinese hamster ovary (CHO) cells is a major limitation for high-level production of biopharmaceuticals, particularly for those with complex molecular architectures and post-translational modifications. To improve CHO cell secretory capacity, we recently engineered CHO cell hosts to overexpress BLIMP1 (CHOB), in a cell engineering strategy that transformed the cellular machinery and led to significantly higher product yields and cell-specific productivities for different rproteins. Here, as a follow-up to our previous study, we developed new CHO cell hosts that co-overexpress BLIMP1 and XBP1s ( CHOBX ), two transcription factors that together drive the professional secretory function of antibody-producing plasma cells. We found that the CHOBX cells presented an improved performance over that of CHOB cells, with better product yields and cell-specific productivities for a recombinant IgG1 and a 'difficult-to-express' EPO-Fc fusion protein. These improvements in the CHOBX-derived cell lines resulted from a series of physiological and metabolic changes due to the synergetic co-expression of BLIMP1 and XBP1s. Firstly, cells presented an inhibited cell growth and arrested cell cycle in G1/G0 phase, features that were directly linked to BLIMP1 expression levels. Secondly, cells increased protein translation (both overall and recombinant protein), expanded the endoplasmic reticulum and improved their capacity to secrete protein more effectively. Lastly, cells showed a metabolic profile favouring energy production, with a pronounced lactate switch and increased consumption of amino acids. This study highlights the value of transcription factors for reprogramming CHO cells towards a desired phenotype, offering the potential to engineer cells with new functionalities for enhanced manufacturing of recombinant therapeutic proteins.

Early reactivation of clustered genes on the inactive X chromosome during somatic cell reprogramming.

Reprogramming of murine female somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by X chromosome reactivation (XCR), by which the inactive X chromosome (Xi) in female somatic cells becomes reactivated. However, how Xi initiates reactivation during reprogramming remains poorly defined. Here, we used a Sendai virus-based reprogramming system to generate partially reprogrammed iPSCs that appear to be undergoing the initial phase of XCR. Allele-specific RNA-seq of these iPSCs revealed that XCR initiates at a subset of genes clustered near the centromere region. The initial phase of XCR occurs when the cells transit through mesenchymal-epithelial transition (MET) before complete shutoff of Xist expression. Moreover, regulatory regions of these genes display dynamic changes in lysine-demethylase 1a (KDM1A) occupancy. Our results identified clustered genes on the Xi that show reactivation in the initial phase of XCR during reprogramming and suggest a possible role for histone demethylation in this process.

Network Analysis Identifies Regulators of Basal-Like Breast Cancer Reprogramming and Endocrine Therapy Vulnerability.

Basal-like breast cancer is the most aggressive breast cancer subtype with the worst prognosis. Despite its high recurrence rate, chemotherapy is the only treatment for basal-like breast cancer, which lacks expression of hormone receptors. In contrast, luminal A tumors express ERα and can undergo endocrine therapy for treatment. Previous studies have tried to develop effective treatments for basal-like patients using various therapeutics but failed due to the complex and dynamic nature of the disease. In this study, we performed a transcriptomic analysis of patients with breast cancer to construct a simplified but essential molecular regulatory network model. Network control analysis identified potential targets and elucidated the underlying mechanisms of reprogramming basal-like cancer cells into luminal A cells. Inhibition of BCL11A and HDAC1/2 effectively drove basal-like cells to transition to luminal A cells and increased ERα expression, leading to increased tamoxifen sensitivity. High expression of BCL11A and HDAC1/2 correlated with poor prognosis in patients with breast cancer. These findings identify mechanisms regulating breast cancer phenotypes and suggest the potential to reprogram basal-like breast cancer cells to enhance their targetability. SIGNIFICANCE: A network model enables investigation of mechanisms regulating the basal-to-luminal transition in breast cancer, identifying BCL11A and HDAC1/2 as optimal targets that can induce basal-like breast cancer reprogramming and endocrine therapy sensitivity.

Tumor reversion and embryo morphogenetic factors.

Several studies have shown that cancer cells can be "phenotypically reversed", thus achieving a "tumor reversion", by losing malignant hallmarks as migrating and invasive capabilities. These findings suggest that genome activity can switch to assume a different functional configuration, i.e. a different Gene Regulatory Network pattern. Indeed, once "destabilized", cancer cells enter into a critical transition phase that can be adequately "oriented" by yet unidentified morphogenetic factors - acting on both cells and their microenvironment - that trigger an orchestrated array of structural and epigenetic changes. Such process can bypass genetic abnormalities, through rerouting cells toward a benign phenotype. Oocytes and embryonic tissues, obtained by animals and humans, display such "reprogramming" capability, as a number of yet scarcely identified embryo-derived factors can revert the malignant phenotype of several types of tumors. Mechanisms involved in the reversion process include the modification of cell-microenvironment cross talk (mostly through cytoskeleton reshaping), chromatin opening, demethylation, and epigenetic changes, modulation of biochemical pathways, comprising TCTP-p53, PI3K-AKT, FGF, Wnt, and TGF-ß-dependent cascades. Results herein discussed promise to open new perspectives not only in the comprehension of cancer biology but also toward different therapeutic options, as suggested by a few preliminary clinical studies.

Programming cell growth into different cluster shapes using diffusible signals.

Advances in genetic engineering technologies have allowed the construction of artificial genetic circuits, which have been used to generate spatial patterns of differential gene expression. However, the question of how cells can be programmed, and how complex the rules need to be, to achieve a desired tissue morphology has received less attention. Here, we address these questions by developing a mathematical model to study how cells can collectively grow into clusters with different structural morphologies by secreting diffusible signals that can influence cellular growth rates. We formulate how growth regulators can be used to control the formation of cellular protrusions and how the range of achievable structures scales with the number of distinct signals. We show that a single growth inhibitor is insufficient for the formation of multiple protrusions but may be achieved with multiple growth inhibitors, and that other types of signals can regulate the shape of protrusion tips. These examples illustrate how our approach could potentially be used to guide the design of regulatory circuits for achieving a desired target structure.

Fabrication and use of silicon hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo.

Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In <1 s, these silicon hollow-needle arrays can be used to deliver plasmids to a predetermined specific depth in murine skin in response to pulsed nanoporation. Tissue nanotransfection eliminates the need to use viral vectors, minimizing the risk of genomic integration or cell transformation. The TNT chip fabrication process typically takes 5-6 d, and in vivo TNT takes 30 min. This protocol does not require specific expertise beyond a clean room equipped for basic nanofabrication processes.