Direct Lineage Reprogramming Research Articles

Genetic conversion of proliferative astroglia into neurons after cerebral ischemia: a new therapeutic tool for the aged brain?

Ischemic stroke represents the 2nd leading cause of death worldwide and the leading cause for long-term disabilities, for which no cure exists. After stroke, neurons are frequently lost in the infarct core. On the other hand, other cells such as astrocytes become reactive and proliferative, disrupting the neurovascular unit in the lesioned area, especially in the aged brain. Therefore, restoring the balance between neurons and nonneuronal cells within the perilesional area is crucial for post stroke recovery. In addition, the aged post stroke brain mounts a fulminant proliferative astroglial response leading to the buildup of gliotic scars that prevent neural regeneration. Therefore, "melting" glial scars has been attempted for decades, albeit with little success. Alternative strategies include transforming inhibitory gliotic tissue into an environment conducive to neuronal regeneration and axonal growth by genetic conversion of astrocytes into neurons. The latter idea has gained momentum following the discovery that in vivo direct lineage reprogramming in the adult mammalian brain is a feasible strategy for reprogramming nonneuronal cells into neurons. This exciting new technology emerged as a new approach to circumvent cell transplantation for stroke therapy. However, the potential of this new methodology has not been yet tested to improve restoration of structure and function in the hostile environment caused by the fulminant inflammatory reaction in the brains of aged animals.

Somatic MIWI2 Hinders Direct Lineage Reprogramming From Fibroblast to Hepatocyte.

Remodeling of the gene regulatory network in cells is believed to be a prerequisite for their lineage reprogramming. However, its key regulatory factors are not yet elucidated. In this article, we investigate the role of PIWI proteins and provide evidence that one of them, MIWI2, is elicited during transdifferentiation of fibroblasts into hepatocyte‐like cells. In coincidence with the peak expression of MIWI2, we identified the appearance of a unique intermediate epigenetic state characterized by a specific Piwi‐interacting RNA (piRNA) profile consisting of 219 novel sequences. Knockout of MIWI2 greatly improved the formation of the induced hepatocytes, whereas overexpression of exogenous MIWI2 completely abolished the stimulated effect. A bioinformatics analysis of piRNA interaction network, followed by experimental validation, revealed the Notch signaling pathway as one of the immediate effectors of MIWI2. Altogether, our results show for the first time that temporal expression of MIWI2 contributes negatively to cell plasticity not only in germline, but also in developed cells, such as mouse fibroblasts. stem cells 2019;37:803–812

Direct and selective lineage conversion of human fibroblasts to dopaminergic precursors

Transplantation of dopaminergic precursors (DPs) is a promising therapeutic strategy of Parkinson’s disease (PD). However, limited cell source for dopaminergic precursors has become a major obstacle for transplantation therapy. Our group demonstrated previously that mouse fibroblasts can be reprogrammed into induced dopaminergic precursors (iDPs) with high differentiation efficiency. In the current study, we hypothesized that a similar strategy can be applied to generate human iDPs for future cell therapy of PD. We overexpressed transcription factors Brn2, Sox2, and Foxa2 in human fibroblasts and observed formation of neurospheres. Subsequent characterization of the precursor colonies confirmed the generation of human induced dopaminergic precursors (hiDPs). These hiDPs were capable of self-renewal, proliferation, and differentiation. The hiDPs demonstrated high immunoreactivity for neural progenitor markers and high levels of gene expression for ventral mesencephalon-related neural progenitor markers such as Lmx1a, NIKX6.1, Corin, Otx2 and Mash1. Furthermore, the hiDPs could be differentiated into dopaminergic neurons with ˜80% efficiency, which significantly increased major functionally relevant proteins such as TH, DAT, AADC, Lmx1B, and VMAT2 compared to hiDPs. Additionally, hiDPs are more dopaminergic progenitor-restricted compare to those hiDP-like cells reprogrammed only by Brn2 and Sox2. Together, these results suggest that hiDPs with high differentiation efficiency can be generated by direct lineage reprogramming of fibroblasts with transcription factors Brn2, Sox2, and Foxa2. These hiDPs may serve as a safe and effective cell source for transplantation treatment of PD.

Direct Lineage Reprogramming in the CNS.

Direct lineage reprogramming is the conversion of one specialized cell type to another without the need for a pluripotent intermediate. To date, a wide variety of cell types have been successfully generated using direct reprogramming, both in vitro and in vivo. These newly converted cells have the potential to replace cells that are lost to disease and/or injury. In this chapter, we will focus on direct reprogramming in the central nervous system. We will review current progress in the field with regards to all the major neural cell types and explore how cellular heterogeneity, both in the starter cell and target cell population, may have implications for direct reprogramming. Finally, we will discuss new technologies that will improve our understanding of the reprogramming process and aid the development of more specific and efficient future CNS-based reprogramming strategies.

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Single-cell mapping of lineage and identity in direct reprogramming.

Direct lineage reprogramming involves the remarkable conversion of cellular identity. Single-cell technologies aid in deconstructing the considerable heterogeneity that emerges during lineage conversion. However, lineage relationships are typically lost during cell processing, complicating trajectory reconstruction. Here, we present ‘CellTagging’, a combinatorial cell indexing methodology, permitting the parallel capture of clonal history and cell identity, where sequential rounds of cell labelling enable the construction of multi-level lineage trees. CellTagging and longitudinal tracking of fibroblast to induced endoderm progenitor (iEP) reprogramming reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a ‘dead-end’ state, paths determined in the earliest reprogramming stages. We find that expression of a putative methyltransferase, Mettl7a1, is associated with the successful reprogramming trajectory, where its addition to the reprogramming cocktail increases the yield of iEPs. Together, these results demonstrate the utility of our lineage tracing method to reveal dynamics of direct reprogramming.

A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1A4V). Moreover, we found that the administration of TUDCA in an hSOD1G93A mouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.

Progress in Dopaminergic Cell Replacement and Regenerative Strategies for Parkinson's Disease.

Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder symptomatically characterized by resting tremor, rigidity, bradykinesia, and gait impairment. These motor deficits suffered by PD patients primarily result from selective dysfunction or loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Most of the existing therapies for PD are based on the replacement of dopamine, which is symptomatically effective in the early stage but becomes increasingly less effective and is accompanied by serious side effects in the advanced stages of the disease. Currently, there are no strategies to slow neuronal degeneration or prevent the progression of PD. Thus, the prospect of regenerating functional dopaminergic neurons is very attractive. Over the last few decades, significant progress has been made in the development of dopaminergic regenerative strategies for curing PD. The most promising approach seems to be cell-replacement therapy (CRT) using human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), which are unlimitedly available and have gained much success in preclinical trials. Despite the challenges, stem cell-based CRT will make significant steps toward the clinic in the coming decade. Alternatively, direct lineage reprogramming, especially in situ direct conversion of glia cells to induced neurons, which exhibits some advantages including no ethical concerns, no risk of tumor formation, and even no need for transplantation, has gained much attention recently. Evoking the endogenous regeneration ability of neural stem cells (NSCs) is an idyllic method of dopaminergic neuroregeneration which remains highly controversial. Here, we review many of these advances, highlighting areas and strategies that might be particularly suited to the development of regenerative approaches that restore dopaminergic function in PD.

Cell Aggregation Culture Induces Functional Differentiation of Induced Hepatocyte-like Cells through Activation of Hippo Signaling

Recent progress in direct lineage reprogramming has enabled the generation of induced hepatocyte-like (iHep) cells and revealed their potential as an alternative to hepatocytes for medical applications. However, the hepatic functions of iHep cells are insufficient compared with those of primary hepatocytes. Here, we show that cell-aggregate formation can rapidly induce growth arrest and hepatic maturationof iHep cells through activation of Hippo signaling. During formation of iHep cell aggregates, Yap inactivation is induced by actin reorganization and intercellular adhesion, leading to upregulation of Hnf1α expression in the absence of the Yap/Tead/Chd4 transcriptional repressor unit. Hnf1α then acts as a central transcription factor that regulates liver-enriched gene expression in iHep cell aggregates and induces functional differentiation of iHep cells. Moreover, iHep cell aggregates efficiently reconstitute injured liver tissues and support hepatic function after transplantation. Thus, iHep cell aggregates may provide insights into basic research and potential therapies for liver diseases.

Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells

Currently, much effort is directed towards the development of new cell sources for clinical therapy using cell fate conversion by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by 2 small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of the fate conversion process. Based on functional evidence, activation of the HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through the activation of the ERK pathway. hCdHs expressed hepatic progenitor markers and could self-renew for at least 10 passages while retaining a normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. Gene expression profiling using RNAseq confirmed the transcriptional reprogramming of hCdHs towards a progenitor state and the suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation in several models of therapeutic liver repopulation, hCdHs effectively repopulated the damaged parenchyma. Our study is the first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a novel tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and the repair of diseased livers. Human primary hepatocytes were reprogrammed towards hepatic progenitor cells by a combined treatment with 2 small molecules, A83-01 and CHIR99021, and HGF. Chemically derived hepatic progenitors exhibited a high proliferation potential and the ability to differentiate into hepatocytes and biliary epithelial cells both in vitro and in vivo. This approach enables the generation of patient-specific hepatic progenitors and provides a platform for personal and stem cell-based regenerative medicine.

Directed Evolution of Reprogramming Factors by Cell Selection andSequencing.

SummaryDirected biomolecular evolution is widely used to tailor and enhance enzymes, fluorescent proteins, and antibodies but has hitherto not been applied in the reprogramming of mammalian cells. Here, we describe a method termed directed evolution of reprogramming factors by cell selection and sequencing (DERBY-seq) to identify artificially enhanced and evolved reprogramming transcription factors. DERBY-seq entails pooled screens with libraries of positionally randomised genes, cell selection based on phenotypic readouts, and genotyping by amplicon sequencing for candidate identification. We benchmark this approach using pluripotency reprogramming with libraries based on the reprogramming factor SOX2 and the reprogramming incompetent endodermal factor SOX17. We identified several SOX2 variants outperforming the wild-type protein in three- and four-factor cocktails. The most effective variants were discovered from the SOX17 library, demonstrating that this factor can be converted into a highly potent inducer of pluripotency with a range of diverse modifications. We propose DERBY-seq as a broad-based approach to discover reprogramming factors for any donor/target cell combination applicable to direct lineage reprogramming in vitro and in vivo.

Long-Term Correction of Diabetes in Mice by InVivo Reprogramming of Pancreatic Ducts.

Direct lineage reprogramming can convert readily available cells in the body into desired cell types for cell replacement therapy. This is usually achieved through forced activation or repression of lineage-defining factors or pathways. In particular, reprogramming toward the pancreatic β cell fate has been of great interest in the search for new diabetes therapies. It has been suggested that cells from various endodermal lineages can be converted to β-like cells. However, it is unclear how closely induced cells resemble endogenous pancreatic β cells and whether different cell types have the same reprogramming potential. Here, we report invivo reprogramming of pancreatic ductal cells through intra-ductal delivery of an adenoviral vector expressing the transcription factors Pdx1, Neurog3, and Mafa. Induced β-like cells are mono-hormonal, express genes essential for βcell function, and correct hyperglycemia in both chemically and genetically induced diabetes models. Compared with intrahepatic ducts and hepatocytes treated with the same vector, pancreatic ducts demonstrated more rapid activation of β cell transcripts and repression of donorcell markers. This approach could be readily adapted to humans through a commonly performed procedure, endoscopic retrograde cholangiopancreatography (ERCP), and provides potential for cell replacement therapy in type 1 diabetes patients.

The multipotency-to-commitment transition in Caenorhabditis elegans-implications for reprogramming from cells to organs.

In animal embryos, cells transition from a multipotential state, with the capacity to adopt multiple fates, into an irreversible, committed state of differentiation. This multipotency-to-commitment transition (MCT) is evident from experiments in which cell fate is reprogrammed by transcription factors for cell type-specific differentiation, as has been observed extensively in Caenorhabditis elegans. Although factors that direct differentiation into each of the three germ layer types cannot generally reprogram cells after the MCT in this animal, transcription factors for endoderm development are able to do so in multiple differentiated cell types. In one case, these factors can redirect the development of an entire organ in the process of "transorganogenesis". Natural transdifferentiation also occurs in a small number of differentiated cells during normal C. elegans development. We review these reprogramming and transdifferentiation events, highlighting the cellular and developmental contexts in which they occur, and discuss common themes underlying direct cell lineage reprogramming. Although certain aspects may be unique to the model system, growing evidence suggests that some mechanisms are evolutionarily conserved and may shed light on cellular plasticity and disease in humans.

Culture and Nucleofection of Postnatal Day 7 Cortical and Cerebellar Mouse Astroglial Cells.

Lineage reprogramming of astroglial cells isolated from different brain regions leads to the generation of different neuronal subtypes. This protocol describes the isolation and culture of neocortical and cerebellar astrocytes from postnatal mice. We also present a comprehensive description of the main steps towards successful gene delivery in these cells using nucleofection. Neocortex and cerebellum astrocyte cultures obtained with these methods are suitable for the study of molecular and cellular mechanisms involved in direct cell lineage reprogramming into induced neurons (iNs).

Generation of Mouse and Human Organoid-Forming Intestinal Progenitor Cells by Direct Lineage Reprogramming

Intestinal organoids hold great promise as a valuable tool for studying and treating intestinal diseases. The currently available sources of human intestinal organoids, tissue fragments or pluripotent stem cells, involve invasive procedures or complex differentiation protocols, respectively. Here, we show that a set of four transcription factors, Hnf4α, Foxa3, Gata6, and Cdx2, can directly reprogram mouse fibroblasts to acquire the identity of fetal intestine-derived progenitor cells (FIPCs). These induced FIPCs (iFIPCs) form spherical organoids that develop into adult-type budding organoids containing cells with intestinal stem cell properties. The resulting stem cells produce all intestinal epithelial cell lineages and undergo self-renewing cell divisions. After transplantation, the induced spherical and budding organoids can reconstitute colonic and intestinal epithelia, respectively. The same combination of four defined transcription factors can also induce human iFIPCs. This alternative approach for producing intestinal organoids may well facilitate application for disease analysis and therapy development.

Electromagnetized gold nanoparticles mediate direct lineage reprogramming into induced dopamine neurons in vivo for Parkinson's disease therapy

Electromagnetic fields (EMF) are physical energy fields generated by electrically charged objects, and specific ranges of EMF can influence numerous biological processes, which include the control of cell fate and plasticity. In this study, we show that electromagnetized gold nanoparticles (AuNPs) in the presence of specific EMF conditions facilitate an efficient direct lineage reprogramming to induced dopamine neurons in vitro and in vivo. Remarkably, electromagnetic stimulation leads to a specific activation of the histone acetyltransferase Brd2, which results in histone H3K27 acetylation and a robust activation of neuron-specific genes. In vivo dopaminergic neuron reprogramming by EMF stimulation of AuNPs efficiently and non-invasively alleviated symptoms in mouse Parkinson's disease models. This study provides a proof of principle for EMF-based in vivo lineage conversion as a potentially viable and safe therapeutic strategy for the treatment of neurodegenerative disorders.

Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State

Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprograminto functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state.

Systemic evaluation of cellular reprogramming processes exploiting a novel R-tool: eegc.

MotivationCells derived by cellular engineering, i.e. differentiation of induced pluripotent stem cells and direct lineage reprogramming, carry a tremendous potential for medical applications and in particular for regenerative therapies. These approaches consist in the definition of lineage-specific experimental protocols that, by manipulation of a limited number of biological cues—niche mimicking factors, (in)activation of transcription factors, to name a few—enforce the final expression of cell-specific (marker) molecules. To date, given the intricate complexity of biological pathways, these approaches still present imperfect reprogramming fidelity, with uncertain consequences on the functional properties of the resulting cells.ResultsWe propose a novel tool eegc to evaluate cellular engineering processes, in a systemic rather than marker-based fashion, by integrating transcriptome profiling and functional analysis. Our method clusters genes into categories representing different states of (trans)differentiation and further performs functional and gene regulatory network analyses for each of the categories of the engineered cells, thus offering practical indications on the potential lack of the reprogramming protocol.Availability and Implementation eegc R package is released under the GNU General Public License within the Bioconductor project, freely available at https://bioconductor.org/packages/eegc/.Supplementary information Supplementary data are available at Bioinformatics online.

Efficient Direct Lineage Reprogramming of Fibroblasts into Induced Cardiomyocytes Using Nanotopographical Cues.

Induced cardiomyocytes (iCMs) generated via direct lineage reprogramming offer a novel therapeutic target for the study and treatment of cardiac diseases. However, the efficiency of iCM generation is significantly low for therapeutic applications. Here, we show an efficient direct conversion of somatic fibroblasts into iCMs using nanotopographic cues. Compared with flat substrates, the direct conversion of fibroblasts into iCMs on nanopatterned substrates resulted in a dramatic increase in the reprogramming efficiency and maturation of iCM phenotypes. Additionally, enhanced reprogramming by substrate nanotopography was due to changes in the activation of focal adhesion kinase and specific histone modifications. Taken together, these results suggest that nanotopographic cues can serve as an efficient stimulant for direct lineage reprogramming into iCMs.