Cell fate conversion prediction by group sparse optimization method utilizing single-cell and bulk OMICs data.

Cell fate conversion by overexpressing defined factors is a powerful tool in regenerative medicine. However, identifying key factors for cell fate conversion requires laborious experimental efforts; thus, many of such conversions have not been achieved yet. Nevertheless, cell fate conversions found in many published studies were incomplete as the expression of important gene sets could not be manipulated thoroughly. Therefore, the identification of master transcription factors for complete and efficient conversion is crucial to render this technology more applicable clinically. In the past decade, systematic analyses on various single-cell and bulk OMICs data have uncovered numerous gene regulatory mechanisms, and made it possible to predict master gene regulators during cell fate conversion. By virtue of the sparse structure of master transcription factors and the group structure of their simultaneous regulatory effects on the cell fate conversion process, this study introduces a novel computational method predicting master transcription factors based on group sparse optimization technique integrating data from multi-OMICs levels, which can be applicable to both single-cell and bulk OMICs data with a high tolerance of data sparsity. When it is compared with current prediction methods by cross-referencing published and validated master transcription factors, it possesses superior performance. In short, this method facilitates fast identification of key regulators, give raise to the possibility of higher successful conversion rate and in the hope of reducing experimental cost.

Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering.

Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.

Somatic cell conversion to a germ cell lineage: A violation or a revelation?

The germline is unique and immortal (or at least its genome is). It is able to perform unique jobs (meiosis) and is selected for genetic changes. Part of being this special also means that entry into the germline club is restricted and cells of the soma are always left out. However, the recent evidence from multiple animals now suggests that somatic cells may join the club and become germline cells in an animal when the original germline is removed. This "violation" may have garnered acceptance by the observation that iPScells, originating experimentally from somatic cells of an adult, can form reproductively successful eggs and sperm, all in vitro. Each of the genes and their functions used to induce pluripotentiality are found normally in the cell and the in vitro conditions to direct germline commitment replicate conditions in vivo. Here, we discuss evidence from three different animals: an ascidian, a segmented worm, and a sea urchin; and that the cells of a somatic cell lineage can convert into the germline in vivo. We discuss the consequences of such transitions and provide thoughts as how this process may have equal precision to the original germline formation of an embryo.

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 cell-fate conversion of somatic cells: Toward regenerative medicine and industries.

Cells of multicellular organisms have diverse characteristics despite having the same genetic identity. The distinctive phenotype of each cell is determined by molecular mechanisms such as epigenetic changes that occur throughout the lifetime of an individual. Recently, technologies that enable modification of the fate of somatic cells have been developed, and the number of studies using these technologies has increased drastically in the last decade. Various cell types, including neuronal cells, cardiomyocytes, and hepatocytes, have been generated using these technologies. Although most direct reprogramming methods employ forced transduction of a defined sets of transcription factors to reprogram cells in a manner similar to induced pluripotent cell technology, many other strategies, such as methods utilizing chemical compounds and microRNAs to change the fate of somatic cells, have also been developed. In this review, we summarize transcription factor-based reprogramming and various other reprogramming methods. Additionally, we describe the various industrial applications of direct reprogramming technologies.

Transcription factor-mediated reprogramming toward hematopoietic stem cells.

De novo generation of human hematopoietic stem cells (HSCs) from renewable cell types has been a long sought-after but elusive goal in regenerative medicine. Paralleling efforts to guide pluripotent stem cell differentiation by manipulating developmental cues, substantial progress has been made recently toward HSC generation via combinatorial transcription factor (TF)-mediated fate conversion, a paradigm established by Yamanaka's induction of pluripotency in somatic cells by mere four TFs. This review will integrate the recently reported strategies to directly convert a variety of starting cell types toward HSCs in the context of hematopoietic transcriptional regulation and discuss how these findings could be further developed toward the ultimate generation of therapeutic human HSCs.

Engineering new neurons: in vivo reprogramming in mammalian brain and spinal cord.

Neurons are postmitotic. Once lost because of injury or degeneration, they do not regenerate in most regions of the mammalian central nervous system. Recent advancements nevertheless clearly reveal that new neurons can be reprogrammed from non-neuronal cells, especially glial cells, in the adult mammalian brain and spinal cord. Here, we give a brief overview concerning cell fate reprogramming in vivo and then focus on the underlying molecular and cellular mechanisms. Specifically, we critically review the cellular sources and the reprogramming factors for in vivo neuronal conversion. Influences of environmental cues and the challenges ahead are also discussed. The ability of inducing new neurons from an abundant and broadly distributed non-neuronal cell source brings new perspectives regarding regeneration-based therapies for traumatic brain and spinal cord injuries and degenerative diseases.

Induction of human cardiomyocyte-like cells from fibroblasts by defined factors.

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca(2+) oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

The role of pioneer transcription factors in the induction of direct cellular reprogramming.

Regenerative medicine is a highly advanced medical field that aims to restore tissues and organs lost due to diseases and injury using a person's own cells or those of others. Direct cellular reprogramming is a promising technology that can directly induce cell-fate conversion from terminally differentiated cells to other cell types and is expected to play a pivotal role in applications in regenerative medicine. The induction of direct cellular reprogramming requires one or more master transcription factors with the potential to reconstitute cell type-specific transcription factor networks. The set of master transcription factors may contain unique transcription factors called pioneer factors that can open compacted chromatin structures and drive the transcriptional activation of target genes. Therefore, pioneer factors may play a central role in direct cellular reprogramming. However, our understanding of the molecular mechanisms by which pioneer factors induce cell-fate conversion is still limited. This review briefly summarizes the outcomes of recent findings and discusses future perspectives, focusing on the role of pioneer factors in direct cellular reprogramming.

Gene Therapy Using Efficient Direct Lineage Reprogramming Technology for Neurological Diseases.

Gene therapy is an innovative approach in the field of regenerative medicine. This therapy entails the transfer of genetic material into a patient's cells to treat diseases. In particular, gene therapy for neurological diseases has recently achieved significant progress, with numerous studies investigating the use of adeno-associated viruses for the targeted delivery of therapeutic genetic fragments. This approach has potential applications for treating incurable diseases, including paralysis and motor impairment caused by spinal cord injury and Parkinson's disease, and it is characterized by dopaminergic neuron degeneration. Recently, several studies have explored the potential of direct lineage reprogramming (DLR) for treating incurable diseases, and highlighted the advantages of DLR over conventional stem cell therapy. However, application of DLR technology in clinical practice is hindered by its low efficiency compared with cell therapy using stem cell differentiation. To overcome this limitation, researchers have explored various strategies such as the efficiency of DLR. In this study, we focused on innovative strategies, including the use of a nanoporous particle-based gene delivery system to improve the reprogramming efficiency of DLR-induced neurons. We believe that discussing these approaches can facilitate the development of more effective gene therapies for neurological disorders.

Cross-lineage potential of Ascl1 uncovered by comparing diverse reprogramming regulatomes.

Direct reprogramming has revolutionized the fields of stem cell biology and regenerative medicine. However, the common mechanisms governing how reprogramming cells undergo transcriptome and epigenome remodeling (i.e., regulatome remodeling) have not been investigated. Here, by characterizing early changes in the regulatome of three different types of direct reprogramming, we identify lineage-specific features as well as common regulatory transcription factors. Of particular interest, we discover that the neuronal factor Ascl1 possesses cross-lineage potential; together with Mef2c, it drives efficient cardiac reprogramming toward a mature and induced cardiomyocyte phenotype. Through ChIP-seq and RNA-seq, we find that MEF2C drives the shift in ASCL1 binding away from neuronal genes toward cardiac genes, guiding their co-operative epigenetic and transcription activities. Together, these findings demonstrate the existence of common regulators of different direct reprogramming and argue against the premise that transcription factors possess only lineage-specific capabilities for altering cell fate - the basic premise used to develop direct reprogramming approaches.

Isolation and Neuronal Reprogramming of Mouse Embryonic Fibroblasts.

Forced expression of specific neuronal transcription factors in mouse embryonic fibroblasts (MEFs) can lead to their direct conversion into functional neurons. Direct neuronal reprogramming has become a powerful tool to characterize individual factors and molecular mechanisms involved in forced and normal neurogenesis and to generate neuronal cell types for in vitro studies. Here we provide a detailed protocol for the isolation of MEFs devoid of neural tissue and their direct reprogramming into functional neurons by overexpression of neuronal reprogramming factors (Ascl1, Brn2, and Myt1l) using lentiviral vectors. This method enables quick and efficient generation of mouse neurons in vitro for versatile functional and mechanistic characterization.

Combining Cell Fate Reprogramming and Protein Engineering to Study Transcription Factor Functions.

Gene expression regulation by transcription factors plays a central role in determining and maintaining cell fate during normal development as well as induced cell fate reprogramming. Induction of cell identity-determining gene regulatory networks by reprogramming factors that act as transcriptional activators is key to induce desired cell fates. Conversely, repression of unwanted genetic programs by transcriptional repressors is equally important to ensure cell fate fidelity. Here we describe engineering techniques to create fusion proteins that allow exploration of the major transcriptional contribution (activation or repression) of specific neuronal reprogramming factors during direct cell fate conversion. This method can be extended to every reprogramming regime to enable the functional categorization of any transcription factor.

Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors.

Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on antigen cross-presentation, a critical step for inducing CD8+ T cell cytotoxic responses. The rarity of naturally occurring cDC1s and lack of in vitro methodologies for the generation of pure cDC1 populations strongly hinders the study of cDC1 lineage specification and function. Here, we describe a protocol for the generation of induced DCs (iDCs) by lentiviral-mediated expression of the transcription factors PU.1, IRF8 and BATF3 in mouse embryonic fibroblasts. iDCs acquire DC morphology, cDC1 phenotype and transcriptional signatures within 9 days. iDCs generated with this protocol acquire functional ability to respond to inflammatory stimuli, engulf dead cells, process and cross-present antigens to CD8+ T cells. DC reprogramming provides a simple and tractable system to generate high numbers of cDC1-like cells for high content screening, opening new avenues to better understand cDC1 specification and function. In the future, faithful induction of cDC1 fate in fibroblasts may lead to the generation of patient-specific DCs for vaccination.

Transcription Factor-Based Fate Specification and Forward Programming for Neural Regeneration.

Traditionally, in vitro generation of donor cells for brain repair has been dominated by the application of extrinsic growth factors and morphogens. Recent advances in cell engineering strategies such as reprogramming of somatic cells into induced pluripotent stem cells and direct cell fate conversion have impressively demonstrated the feasibility to manipulate cell identities by the overexpression of cell fate-determining transcription factors. These strategies are now increasingly implemented for transcription factor-guided differentiation of neural precursors and forward programming of pluripotent stem cells toward specific neural subtypes. This review covers major achievements, pros and cons, as well as future prospects of transcription factor-based cell fate specification and the applicability of these approaches for the generation of donor cells for brain repair.

Choice of factors and medium impinge on success of ESC to TSC conversion.

INTRODUCTION: The first lineage separation in mammalian development occurs when totipotent cells of the zygote give rise to the inner cell mass and the trophectoderm. The lineages are strictly separated by an epigenetic barrier. In vitro derivatives of these lineages embryonic stem cells (ESC) and trophoblast stem cells (TSC) are used to study the requirements needed to overcome the barrier in ESC to TSC conversion approaches. METHODS: Different combinations of TSC transcription factors were induced in ESC for three days. Cells were kept in TS medium with fetal bovine serum (FBS) or the chemically defined TX medium. Obtained cells were analysed for OCT4 levels, TSC surface marker levels, expression of TSC markers and methylation status of Elf5, Oct4 and Nanog promoters. Further, long-term culture stability and in vitro and in vivo differentiation was tested. RESULTS: Overexpression of Gata3, Eomes, Tfap2c, Ets2 and Cdx2 in ESC resulted in induction of TSC fate. Overexpression of Cdx2 or four factors (Gata3, Eomes, Tfap2c and Ets2) resulted in complete conversion only when cells were cultured in TX medium. The obtained induced TSC (iTSC) display characteristics of bona fide TSC in terms of marker expression and promoter methylation patterns. The generated converted cells were shown to display self-renewal and to be capable to differentiate into TSC derivatives in vitro and in vivo. CONCLUSION: Gata3, Eomes, Tfap2c, Ets2 and Cdx2 overexpression in ESC resulted in stable iTSC fate independent of culture conditions. For four factors or Cdx2 alone, TX medium is required for complete TSC conversion.

A Switch in Tissue Stem Cell Identity Causes Neuroendocrine Tumors in Drosophila Gut.

Intestinal stem cells (ISCs) are able to generate gut-specific enterocytes, as well as neural-like enteroendocrine cells. It is unclear how the tissue identity of the ISC lineage is regulated to confer cell-lineage fidelity. Here, we show that, in adult Drosophila midgut, loss of the transcriptional repressor Tramtrack in ISCs causes a self-renewal program switch to neural stem cell (NSC)-like, and that switch drives neuroendocrine tumor development. In Tramtrack-depleted ISCs, the ectopically expressed Deadpan acts as a major self-renewal factor for cell propagation, and Sequoia acts as a differentiation factor for the neuroendocrine phenotype. In addition, the expression of Sequoia renders NSC-specific self-renewal genes responsive to Notch in ISCs, thus inverting the differentiation-promoting function of Notch into a self-renewal role as in normal NSCs. These results suggest an active maintenance mechanism for the gut identity of ISCs, whose disruption may lead to an improper acquisition of NSC-like traits and tumorigenesis.

Induction of Human Trophoblast Stem Cells from Somatic Cells and Pluripotent Stem Cells.

Human trophoblast stem cells (hTSCs) derived from blastocysts and first-trimester cytotrophoblasts offer an unprecedented opportunity to study the placenta. However, access to human embryos and first-trimester placentas is limited, thus preventing the establishment of hTSCs from diverse genetic backgrounds associated with placental disorders. Here, we show that hTSCs can be generated from numerous genetic backgrounds using post-natal cells via two alternative methods: (1) somatic cell reprogramming of adult fibroblasts with OCT4, SOX2, KLF4, MYC (OSKM) and (2) cell fate conversion of naive and extended pluripotent stem cells. The resulting induced/converted hTSCs recapitulated hallmarks of hTSCs including long-term self-renewal, expression of specific transcription factors, transcriptomic signature, and the potential to differentiate into syncytiotrophoblast and extravillous trophoblast cells. We also clarified the developmental stage of hTSCs and show that these cells resemble day 8 cytotrophoblasts. Altogether, hTSC lines of diverse genetic origins open the possibility to model both placental development and diseases in a dish.

Direct Lineage Reprogramming for Brain Repair: Breakthroughs and Challenges.

Injury to the human central nervous system (CNS) is devastating because our adult mammalian brain lacks intrinsic regenerative capacity to replace lost neurons and induce functional recovery. An emerging approach towards brain repair is to instruct fate conversion of brain-resident non-neuronal cells into induced neurons (iNs) by direct lineage reprogramming. Considerable progress has been made in converting various source cell types of mouse and human origin into clinically relevant iNs. Recent achievements using transcriptomics and epigenetics have shed light on the molecular mechanisms underpinning neuronal reprogramming, while the potential capability of iNs in promoting functional recovery in pathological contexts has started to be evaluated. Although future challenges need to be overcome before clinical translation, lineage reprogramming holds promise for effective cell-replacement therapy in regenerative medicine.

Conversion of mouse fibroblasts into oligodendrocyte progenitor-like cells through a chemical approach.

Transplantation of oligodendrocyte progenitor cells (OPCs) is a promising way for treating demyelinating diseases. However, generation of scalable and autologous sources of OPCs has proven difficult. We previously established a chemical condition M9 that could specifically initiate neural program in mouse embryonic fibroblasts. Here we found that M9 could induce the formation of colonies that undergo mesenchymal-to-epithelial transition at the early stage of reprogramming. These colonies may represent unstable and neural lineage-restricted intermediates that have not established a neural stem cell identity. By modulating the culture signaling recapitulating the principle of OPC development, these intermediate cells could be reprogrammed towards OPC fate. The chemical-induced OPC-like cells (ciOPLCs) resemble primary neural stem cell-derived OPCs in terms of their morphology, gene expression, and the ability of self-renewal. Upon differentiation, ciOPLCs could produce functional oligodendrocytes and myelinate the neuron axons in vitro, validating their OPC identity molecularly and functionally. Therefore, our study provides a non-integrating approach to OPC reprogramming that may ultimately provide an avenue to patient-specific cell-based or in situ regenerative therapy.