Cell Reprogramming: The Many Roads to Success.

Cellular reprogramming experiments from somatic cell types have demonstrated the plasticity of terminally differentiated cell states. Recent efforts in understanding the mechanisms of cellular reprogramming have begun to elucidate the differentiation trajectories along the reprogramming processes. In this review, we focus mainly on direct reprogramming strategies by transcription factors and highlight the variables that contribute to cell fate conversion outcomes. We review key studies that shed light on the cellular and molecular mechanisms by investigating differentiation trajectories and alternative cell states as well as transcription factor regulatory activities during cell fate reprogramming. Finally, we highlight a few concepts that we believe require attention, particularly when measuring the success of cell reprogramming experiments.

The Dynamics of Transcriptional Activation by Hepatic Reprogramming Factors.

Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.

Direct neuronal conversion of microglia/macrophages reinstates neurological function after stroke.

Although generating new neurons in the ischemic injured brain would be an ideal approach to replenish the lost neurons for repairing the damage, the adult mammalian brain retains only limited neurogenic capability. Here, we show that direct conversion of microglia/macrophages into neurons in the brain has great potential as a therapeutic strategy for ischemic brain injury. After transient middle cerebral artery occlusion in adult mice, microglia/macrophages converge at the lesion core of the striatum, where neuronal loss is prominent. Targeted expression of a neurogenic transcription factor, NeuroD1, in microglia/macrophages in the injured striatum enables their conversion into induced neuronal cells that functionally integrate into the existing neuronal circuits. Furthermore, NeuroD1-mediated induced neuronal cell generation significantly improves neurological function in the mouse stroke model, and ablation of these cells abolishes the gained functional recovery. Our findings thus demonstrate that neuronal conversion contributes directly to functional recovery after stroke.

Reprogramming cellular identity in vivo.

Cellular identity is established through complex layers of genetic regulation, forged over a developmental lifetime. An expanding molecular toolbox is allowing us to manipulate these gene regulatory networks in specific cell types in vivo. In principle, if we found the right molecular tricks, we could rewrite cell identity and harness the rich repertoire of possible cellular functions and attributes. Recent work suggests that this rewriting of cell identity is not only possible, but that newly induced cells can mitigate disease phenotypes in animal models of major human diseases. So, is the sky the limit, or do we need to keep our feet on the ground? This Spotlight synthesises key concepts emerging from recent efforts to reprogramme cellular identity in vivo. We provide our perspectives on recent controversies in the field of glia-to-neuron reprogramming and identify important gaps in our understanding that present barriers to progress.

Endothelial reprogramming for vascular regeneration: Past milestones and future directions.

Endothelial cells are critical mediators of health and disease. Regenerative medicine techniques that target the endothelium hold vast promise for improving lifespan and quality of life worldwide. Regenerative therapies via induced pluripotent stem cells (IPSCs) have helped demonstrate disease mechanisms, but so far, concerns regarding their function, malignant potential, and expense have limited therapeutic potential. One alternative approach is direct reprogramming of somatic cells, which avoids the pluripotent state and allows for in vivo reprogramming. Transcription factors from endothelial development have yielded essential transcription factors and small molecules that induce endothelial cell fate. Most direct cell reprogramming strategies targeting endothelial cells use ETV2, a pioneer transcription factor to specify endothelial lineage via histone-modifying enzymes. Many different types of starting cells and strategies, including lentiviral transduction, inducing innate immunity, and small molecule signaling have been leveraged for reprogramming. However, so far therapeutic benefit of these strategies remains unproven. Future research will have to solve scalability, safety, and efficacy hurdles before being ready for the clinic. However, researchers have already discovered meaningful insights into disease mechanisms and development through direct reprogramming.

Lineage tracing identifies in vitro microglia-to-neuron conversion by NeuroD1 expression.

Neuronal regeneration to replenish lost neurons after injury is critical for brain repair. Microglia, brain-resident macrophages that have the propensity to accumulate at the site of injury, can be a potential source for replenishing lost neurons through fate conversion into neurons, induced by forced expression of neuronal lineage-specific transcription factors. However, it has not been strictly demonstrated that microglia, rather than central nervous system-associated macrophages, such as meningeal macrophages, convert into neurons. Here, we show that NeuroD1-transduced microglia can be successfully converted into neurons in vitro using lineage-mapping strategies. We also found that a chemical cocktail treatment further promoted NeuroD1-induced microglia-to-neuron conversion. NeuroD1 with loss-of-function mutation, on the other hand, failed to induce the neuronal conversion. Our results indicate that microglia are indeed reprogrammed into neurons by NeuroD1 with neurogenic transcriptional activity.

Engineering Critical Residues of SOX9 Discovers a Variant With Potent Capacity to Induce Chondrocytes.

Articular cartilage plays vital roles as a friction minimizer and shock absorber during joint movement but has a poor capacity to self-repair when damaged through trauma or disease. Cartilage tissue engineering is an innovative technique for cartilage regeneration, yet its therapeutic application requires chondrocytes in large numbers. Direct reprogramming of somatic cells to chondrocytes by expressing SOX9, KLF4, and c-MYC offers a promising option to generate chondrocytes in sufficient numbers; however, the low efficiency of the reprogramming system warrants further improvement. Here we referred to structural and functional features of SOX9 and performed alanine-scanning mutagenesis of functionally critical residues in the HMG box and at putative posttranslational modification (PTM) sites. We discovered that a SOX9 variant H131A/K398A, doubly mutated in the HMG box (H131) and at a PTM site (K398), significantly upregulated expression of chondrogenic genes and potently induced chondrocytes from mouse embryonic fibroblasts. The H131A/K398A variant remained unsumoylated in cells and exhibited a stronger DNA-binding activity than wild-type SOX9, especially when complexed with other proteins. Our results show that the novel SOX9 variant may be useful for efficient induction of chondrocytes and illuminate the strategic feasibility of mutating a transcription factor at functionally critical residues to expedite discovery of an optimized reprogramming factor.

MiR-124 synergism with ELAVL3 enhances target gene expression to promote neuronal maturity.

Neuron-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), direct cell fate switching of human fibroblasts to neurons when ectopically expressed by repressing antineurogenic genes. How these miRNAs function after the repression of fibroblast genes for neuronal fate remains unclear. Here, we identified targets of miR-9/9*-124 as reprogramming cells activate the neuronal program and reveal the role of miR-124 that directly promotes the expression of its target genes associated with neuronal development and function. The mode of miR-124 as a positive regulator is determined by the binding of both AGO and a neuron-enriched RNA-binding protein, ELAVL3, to target transcripts. Although existing literature indicates that miRNA-ELAVL family protein interaction can result in either target gene up-regulation or down-regulation in a context-dependent manner, we specifically identified neuronal ELAVL3 as the driver for miR-124 target gene up-regulation in neurons. In primary human neurons, repressing miR-124 and ELAVL3 led to the down-regulation of genes involved in neuronal function and process outgrowth and cellular phenotypes of reduced inward currents and neurite outgrowth. Our results highlight the synergistic role between miR-124 and RNA-binding proteins to promote target gene regulation and neuronal function.

Development of a skeletal muscle sheet with direct reprogramming-induced myoblasts on a nanogel-cross-linked porous freeze-dried gel scaffold in a mouse gastroschisis model.

PURPOSE: In this study, we attempted to create skeletal muscle sheets made of directly converted myoblasts (dMBs) with a nanogel scaffold on a biosheet using a mouse gastroschisis model. METHODS: dMBs were prepared by the co-transfection of MYOD1 and MYCL into human fibroblasts. Silicon tubes were implanted under the skin of NOG/SCID mice, and biosheets were formed. The nanogel was a nanoscale hydrogel based on cholesterol-modified pullulan, and a NanoClip-FD gel was prepared by freeze-drying the nanogel. 7 mm in length was created in the abdominal wall of NOG/SCID mice as a mouse gastroschisis model. Matrigel or NanoCliP-FD gel seeded with dMBs was placed on the biosheet and implanted on the model mice. RESULTS: Fourteen days after surgery, dMBs with Matrigel showed a small amount of coarse aggregations of muscle-like cells. In contrast, dMBs with NanoCliP-FD gel showed multinucleated muscle-like cells, which were expressed as desmin and myogenin by fluorescent immunostaining. CONCLUSION: Nanogels have a porous structure and are useful as scaffolds for tissue regeneration by supplying oxygen and nutrients supply to the cells. Combining dMBs and nanogels on the biosheets resulted in the differentiation and engraftment of skeletal muscle, suggesting the possibility of developing skeletal muscle sheets derived from autologous cells and tissues.

Direct Reprogramming of Somatic Skin Cells from a Patient with Huntington's Disease into Striatal Neurons to Create Models of Pathology.

A new in vitro model of Huntington's disease (HD) was developed via a direct reprogramming of dermal fibroblasts from HD patients into striatal neurons. A reprogramming into induced pluripotent stem (iPS) cells is obviated in the case of direct reprogramming, which thus yields neurons that preserve the epigenetic information inherent in cells of a particular donor and, consequently, the age-associated disease phenotype. A main histopathological feature of HD was reproduced in the new model; i.e., aggregates of mutant huntingtin accumulated in striatal neurons derived from a patient's fibroblasts. Experiments with cultured neurons obtained via direct reprogramming make it possible to individually assess the progression of neuropathology and to implement a personalized approach to choosing the treatment strategy and drugs for therapy. The in vitro model of HD can be used in preclinical drug studies.

A novel transcription factor combination for direct reprogramming to a spontaneously contracting human cardiomyocyte-like state.

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.

Endothelial cell direct reprogramming: Past, present, and future.

Ischemic cardiovascular disease still remains as a leading cause of morbidity and mortality despite various medical, surgical, and interventional therapy. As such, cell therapy has emerged as an attractive option because it tackles underlying problem of the diseases by inducing neovascularization in ischemic tissue. After overall failure of adult stem or progenitor cells, studies attempted to generate endothelial cells (ECs) from pluripotent stem cells (PSCs). While endothelial cells (ECs) differentiated from PSCs successfully induced vascular regeneration, differentiating volatility and tumorigenic potential is a concern for their clinical applications. Alternatively, direct reprogramming strategies employ lineage-specific factors to change cell fate without achieving pluripotency. ECs have been successfully reprogrammed via ectopic expression of transcription factors (TFs) from endothelial lineage. The reprogrammed ECs induced neovascularization in vitro and in vivo and thus demonstrated their therapeutic value in animal models of vascular insufficiency. Methods of delivering reprogramming factors include lentiviral or retroviral vectors and more clinically relevant, non-integrative adenoviral and episomal vectors. Most studies made use of fibroblast as a source cell for reprogramming, but reprogrammability of other clinically relevant source cell types has to be evaluated. Specific mechanisms and small molecules that are involved in the aforementioned processes tackles challenges associated with direct reprogramming efficiency and maintenance of reprogrammed EC characteristics. After all, this review provides summary of past and contemporary methods of direct endothelial reprogramming and discusses the future direction to overcome these challenges to acquire clinically applicable reprogrammed ECs.

Probing cell identity hierarchies by fate titration and collision during direct reprogramming.

Despite the therapeutic promise of direct reprogramming, basic principles concerning fate erasure and the mechanisms to resolve cell identity conflicts remain unclear. To tackle these fundamental questions, we established a single-cell protocol for the simultaneous analysis of multiple cell fate conversion events based on combinatorial and traceable reprogramming factor expression: Collide-seq. Collide-seq revealed the lack of a common mechanism through which fibroblast-specific gene expression loss is initiated. Moreover, we found that the transcriptome of converting cells abruptly changes when a critical level of each reprogramming factor is attained, with higher or lower levels not contributing to major changes. By simultaneously inducing multiple competing reprogramming factors, we also found a deterministic system, in which titration of fates against each other yields dominant or colliding fates. By investigating one collision in detail, we show that reprogramming factors can disturb cell identity programs independent of their ability to bind their target genes. Taken together, Collide-seq has shed light on several fundamental principles of fate conversion that may aid in improving current reprogramming paradigms.

Direct cardiac reprogramming: basics and future challenges.

BACKGROUND: Heart failure is the leading cause of morbidity and mortality worldwide and is characterized by reduced cardiac function. Currently, cardiac transplantation therapy is applied for end-stage heart failure, but it is limited by the number of available donors. METHODS AND RESULTS: Following an assessment of available literature, a narrative review was conducted to summarizes the current status and challenges of cardiac reprogramming for clinical application. Scientists have developed different regenerative treatment strategies for curing heart failure, including progenitor cell delivery and pluripotent cell delivery. Recently, a novel strategy has emerged that directly reprograms cardiac fibroblast into a functional cardiomyocyte. In this treatment, transcription factors are first identified to reprogram fibroblast into a cardiomyocyte. After that, microRNA and small molecules show great potential to optimize the reprogramming process. Some challenges regarding cell reprogramming in humans are conversion efficiency, virus utilization, immature and heterogenous induced cardiomyocytes, technical reproducibility issues, and physiological effects of depleted fibroblasts on myocardial tissue. CONCLUSION: Several strategies have shown positive results in direct cardiac reprogramming. However, direct cardiac reprogramming still needs improvement if it is used as a mainstay therapy in humans, and challenges need to be overcome before cardiac reprogramming can be considered a viable therapeutic strategy. Further advances in cardiac reprogramming studies are needed in cardiac regenerative therapy.

Direct Reprogramming of Somatic Cells into Induced ß-Cells: An Overview.

The persistent shortage of insulin-producing islet mass or ß-cells for transplantation in the ever-growing diabetic population worldwide is a matter of concern. To date, permanent cure to this medical complication is not available and soon after the establishment of lineage-specific reprogramming, direct ß-cell reprogramming became a viable alternative for ß-cell regeneration. Direct reprogramming is a straightforward and powerful technique that can provide an unlimited supply of cells by transdifferentiating terminally differentiated cells toward the desired cell type. This approach has been extensively used by multiple groups to reprogram non-ß-cells toward insulin-producing ß-cells. The ß-cell identity has been achieved by various studies via ectopic expression of one or more pancreatic-specific transcription factors in somatic cells, bypassing the pluripotent state. This work highlights the importance of the direct reprogramming approaches (both integrative and non-integrative) in generating autologous ß-cells for various applications. An in-depth understanding of the strategies and cell sources could prove beneficial for the efficient generation of integration-free functional insulin-producing ß-cells for diabetic patients lacking endogenous ß-cells.

Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons.

The modeling of neuropathology on induced neurons obtained by cell reprogramming technologies can fill a gap between clinical trials and studies on model organisms for the development of treatment strategies for neurodegenerative diseases. Patient-specific models based on patients' cells play an important role in such studies. There are two ways to obtain induced neuronal cells. One is based on induced pluripotent stem cells. The other is based on direct reprogramming, which allows us to obtain mature neuronal cells from adult somatic cells, such as dermal fibroblasts. Moreover, the latter method makes it possible to better preserve the age-related aspects of neuropathology, which is valuable for diseases that occur with age. However, direct methods of reprogramming have a significant drawback associated with low cell viability during procedures. Furthermore, the number of reprogrammable neurons available for morphological and functional studies is limited by the initial number of somatic cells. In this article, we propose modifications of a previously developed direct reprogramming method, based on the combination of microRNA and transcription factors, which allowed us to obtain a population of functionally active induced striatal neurons (iSNs) with a high efficiency. We also overcame the problem of the presence of multinucleated neurons associated with the cellular division of starting fibroblasts. Synchronization cells in the G1 phase increased the homogeneity of the fibroblast population, increased the survival rate of induced neurons, and eliminated the presence of multinucleated cells at the end of the reprogramming procedure. We have demonstrated that iSNs are functionally active and able to form synaptic connections in co-cultures with mouse cortical neurons. The proposed modifications can also be used to obtain a population of other induced neuronal types, such as motor and dopaminergic ones, by selecting transcription factors that determine differentiation into a region-specific neuron.

The heart of cardiac reprogramming: The cardiac fibroblasts.

Cardiovascular disease is the leading cause of death worldwide, outpacing pulmonary disease, infectious disease, and all forms of cancer. Myocardial infarction (MI) dominates cardiovascular disease, contributing to four out of five cardiovascular related deaths. Following MI, patients suffer adverse and irreversible myocardial remodeling associated with cardiomyocyte loss and infiltration of fibrotic scar tissue. Current therapies following MI only mitigate the cardiac physiological decline rather than restore damaged myocardium function. Direct cardiac reprogramming is one strategy that has promise in repairing injured cardiac tissue by generating new, functional cardiomyocytes from cardiac fibroblasts (CFs). With the ectopic expression of transcription factors, microRNAs, and small molecules, CFs can be reprogrammed into cardiomyocyte-like cells (iCMs) that display molecular signatures, structures, and contraction abilities similar to endogenous cardiomyocytes. The in vivo induction of iCMs following MI leads to significant reduction in fibrotic cardiac remodeling and improved heart function, indicating reprogramming is a viable option for repairing damaged heart tissue. Recent work has illustrated different methods to understand the mechanisms driving reprogramming, in an effort to improve the efficiency of iCM generation and create an approach translational into clinic. This review will provide an overview of CFs and describe different in vivo reprogramming methods.

Directly reprogrammed Huntington's disease neural precursor cells generate striatal neurons exhibiting aggregates and impaired neuronal maturation.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by the progressive loss of striatal medium spiny neurons. Using a highly efficient protocol for direct reprogramming of adult human fibroblasts with chemically modified mRNA, we report the first generation of HD induced neural precursor cells (iNPs) expressing striatal lineage markers that differentiated into DARPP32+ neurons from individuals with adult-onset HD (41-57 CAG). While no transcriptional differences between normal and HD reprogrammed neurons were detected by NanoString nCounter analysis, a subpopulation of HD reprogrammed neurons contained ubiquitinated polyglutamine aggregates. Importantly, reprogrammed HD neurons exhibited impaired neuronal maturation, displaying altered neurite morphology and more depolarized resting membrane potentials. Reduced BDNF protein expression in reprogrammed HD neurons correlated with increased CAG repeat lengths and earlier symptom onset. This model represents a platform for investigating impaired neuronal maturation and screening for neuronal maturation modifiers to treat HD.

Combination of Melatonin and Small Molecules Improved Reprogramming Neural Cell Fates via Autophagy Activation.

Reprogramming cell fates towards mature cell types are a promising cell supply for treating degenerative diseases. Recently, transcription factors and some small molecules have turned into impressive modulating elements for reprogramming cell fates. Melatonin, a pineal hormone, has neuroprotective functions including neural stem cell (NSC) proliferative and differentiative modulation in both embryonic and adult brain. We developed a protocol that could be implemented in the direct reprogramming of human skin fibroblast towards neural cells by using histone deacetylase (HDAC) inhibitor, glycogen synthase kinase-3 (GSK3) inhibitor (CHIR99021), c-Jun N-terminal kinase (JNK) inhibitor, rho-associated protein kinase inhibitor (Y-27632), cAMP activator, and melatonin treatment. We found that melatonin enhanced neural-transcription factor genes expressions, including brain-specific homeobox/POU domain protein 2 (BRN2), Achaete-Scute Family BHLH transcription Factor 1 (ASCL1), and Myelin Transcription Factor 1 Like (MYT1L). Melatonin also increased the expression of different neural-specific proteins such as doublecortin (DCX), Sex determining region Y-box 2 (Sox2), and neuronal nuclei (NeuN) compared with other five small molecules (valproic acid (VPA), CHIR99021, Forskolin, 1,9 pyrazoloanthrone (SP600125), and Y-27632) combination in the presence and absence of melatonin. A noticeable upregulation of autophagy proteins (microtubule-associated protein 1A/1B-light chain 3 (LC3) and Beclin-1) were seen in the melatonin treatment during the induction period while these were reverted in the presence of L-leucine, an autophagy inhibitor. In addition, the expression of NeuN was also significantly reduced by L-leucine. Collectively, our findings revealed an activation of autophagy during neural induction; melatonin enhanced reprogramming efficiency for neuron induction through the modulation of autophagy activation.

Lipoplexes for effective in vitro delivery of microRNAs to adult human cardiac fibroblasts for perspective direct cardiac cell reprogramming.

Design of nanocarriers for efficient miRNA delivery can significantly improve miRNA-based therapies. Lipoplexes based on helper lipid, dioleoyl phosphatidylethanolamine (DOPE) and cationic lipid [2-(2,3-didodecyloxypropyl)-hydroxyethyl] ammonium bromide (DE) were formulated to efficiently deliver miR-1 or a combination of four microRNAs (miRcombo) to adult human cardiac fibroblasts (AHCFs). Lipoplexes with amino-to-phosphate groups ratio of 3 (N/P 3) showed nanometric hydrodynamic size (372 nm), positive Z-potential (40 mV) and high stability under storage conditions. Compared to commercial DharmaFECT1 (DF), DE-DOPE/miRNA lipoplexes showed superior miRNA loading efficiency (99 % vs. 64 %), and faster miRNA release (99 % vs. 82 % at 48 h). DE-DOPE/miR-1 lipoplexes showed superior viability (80-100 % vs. 50 %) in AHCFs, a 2-fold higher miR-1 expression and Twinfilin-1 (TWF-1) mRNA downregulation. DE-DOPE/miRcombo lipoplexes significantly enhanced AHCFs reprogramming into induced cardiomyocytes (iCMs), as shown by increased expression of CM markers compared to DF/miRcombo.