Customized Proteinaceous Nanoformulation for In Vivo Chemical Reprogramming.

Sweat gland (SwG) regeneration is crucial for the functional rehabilitation of burn patients. In vivo chemical reprogramming that harnessing the patient's own cells in damaged tissue is of substantial interest to regenerate organs endogenously by pharmacological manipulation, which could compensate for tissue loss in devastating diseases and injuries, for example, burns. However, achieving in vivo chemical reprogramming is challenging due to the low reprogramming efficiency and an unfavorable tissue environment. Herein, this work has developed a functionalized proteinaceous nanoformulation delivery system containing prefabricated epidermal growth factor structure for on-demand delivery of a cocktail of seven SwG reprogramming components to the dermal site. Such a chemical reprogramming system can efficiently induce the conversion of epidermal keratinocytes into SwG myoepithelial cells, resulting in successful in situ regeneration of functional SwGs. Notably, in vivo chemical reprogramming of SwGs is achieved for the first time with an impressive efficiency of 30.6%, surpassing previously reported efficiencies. Overall, this proteinaceous nanoformulation provides a platform for coordinating the target delivery of multiple pharmacological agents and facilitating in vivo SwG reprogramming by chemicals. This advancement greatly improves the clinical accessibility of in vivo reprogramming and offers a non-surgical, non-viral, and cell-free strategy for in situ SwG regeneration.

Chemical reprogramming for cell fate manipulation: Methods, applications, and perspectives.

Chemical reprogramming offers an unprecedented opportunity to control somatic cell fate and generate desired cell types including pluripotent stem cells for applications in biomedicine in a precise, flexible, and controllable manner. Recent success in the chemical reprogramming of human somatic cells by activating a regeneration-like program provides an alternative way of producing stem cells for clinical translation. Likewise, chemical manipulation enables the capture of multiple (stem) cell states, ranging from totipotency to the stabilization of somatic fates in vitro. Here, we review progress in using chemical approaches for cell fate manipulation in addition to future opportunities in this promising field.

Chemical-induced epigenome resetting for regeneration program activation in human cells.

Human somatic cells can be reprogrammed to pluripotent stem cells by small molecules through an intermediate stage with a regeneration signature, but how this regeneration state is induced remains largely unknown. Here, through integrated single-cell analysis of transcriptome, we demonstrate that the pathway of human chemical reprogramming with regeneration state is distinct from that of transcription-factor-mediated reprogramming. Time-course construction of chromatin landscapes unveils hierarchical histone modification remodeling underlying the regeneration program, which involved sequential enhancer recommissioning and mirrored the reversal process of regeneration potential lost in organisms as they mature. In addition, LEF1 is identified as a key upstream regulator for regeneration gene program activation. Furthermore, we reveal that regeneration program activation requires sequential enhancer silencing of somatic and proinflammatory programs. Altogether, chemical reprogramming resets the epigenome through reversal of the loss of natural regeneration, representing a distinct concept for cellular reprogramming and advancing the development of regenerative therapeutic strategies.

Harnessing endogenous transcription factors directly by small molecules for chemically induced pluripotency inception.

Chemistry-alone approach has recently been applied for incepting pluripotency in somatic cells, representing a breakthrough in biology. However, chemical reprogramming is hampered by low efficiency, and the underlying molecular mechanisms remain unclear. Particularly, chemical compounds do not have specific DNA-recognition domains or transcription regulatory domains, and then how do small molecules work as a driving force for reinstating pluripotency in somatic cells? Furthermore, how to efficiently clear materials and structures of an old cell to prepare the rebuilding of a new one? Here, we show that small molecule CD3254 activates endogenous existing transcription factor RXRα to significantly promote mouse chemical reprogramming. Mechanistically, CD3254-RXRα axis can directly activate all the 11 RNA exosome component genes (Exosc1-10 and Dis3) at transcriptional level. Unexpectedly, rather than degrading mRNAs as its substrates, RNA exosome mainly modulates the degradation of transposable element (TE)-associated RNAs, particularly MMVL30, which is identified as a new barrier for cell-fate determination. In turn, MMVL30-mediated inflammation (IFN-γ and TNF-α pathways) is reduced, contributing to the promotion of successful reprogramming. Collectively, our study provides conceptual advances for translating environmental cues into pluripotency inception, particularly, identifies that CD3254-RXRα-RNA exosome axis can promote chemical reprogramming, and suggests modulation of TE-mediated inflammation via CD3254-inducible RNA exosome as important opportunities for controlling cell fates and regenerative medicine.

Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming.

We recently demonstrated the chemical reprogramming of human somatic cells to pluripotent stem cells (hCiPSCs), which provides a robust approach for cell fate manipulation. However, the utility of this chemical approach is currently hampered by slow kinetics. Here, by screening for small molecule boosters and systematically optimizing the original condition, we have established a robust, chemically defined reprogramming protocol, which greatly shortens the induction time from ∼50 days to a minimum of 16 days and enables highly reproducible and efficient generation of hCiPSCs from all 17 tested donors. We found that this optimized protocol enabled a more direct reprogramming process by promoting cell proliferation and oxidative phosphorylation metabolic activities at the early stage. Our results highlight a distinct chemical reprogramming pathway that leads to a shortcut for the generation of human pluripotent stem cells, which represents a powerful strategy for human cell fate manipulation.

Homogeneity of XEN Cells Is Critical for Generation of Chemically Induced Pluripotent Stem Cells.

In induced pluripotent stem cells (iPSCs), pluripotency is induced artificially by introducing the transcription factors Oct4, Sox2, Klf4, and c-Myc. When a transgene is introduced using a viral vector, the transgene may be integrated into the host genome and cause a mutation and cancer. No integration occurs when an episomal vector is used, but this method has a limitation in that remnants of the virus or vector remain in the cell, which limits the use of such iPSCs in therapeutic applications. Chemical reprogramming, which relies on treatment with small-molecule compounds to induce pluripotency, can overcome this problem. In this method, reprogramming is induced according to the gene expression pattern of extra-embryonic endoderm (XEN) cells, which are used as an intermediate stage in pluripotency induction. Therefore, iPSCs can be induced only from established XEN cells. We induced XEN cells using small molecules that modulate a signaling pathway and affect epigenetic modifications, and devised a culture method in which can be produced homogeneous XEN cells. At least 4 passages were required to establish morphologically homogeneous chemically induced XEN (CiXEN) cells, whose properties were similar to those of XEN cells, as revealed through cellular and molecular characterization. Chemically iPSCs derived from CiXEN cells showed characteristics similar to those of mouse embryonic stem cells. Our results show that the homogeneity of CiXEN cells is critical for the efficient induction of pluripotency by chemicals.

Global Transcriptional and Epigenetic Reconfiguration during Chemical Reprogramming of Human Retinal Pigment Epithelial Cells into Photoreceptor-like Cells.

Retinal degenerative diseases are frequently caused by the loss of retinal neural cells such as photoreceptors. Cell replacement is regarded as one of the most promising therapies. Multiple types of stem and somatic cells have been tested for photoreceptor conversion. However, current induction efficiencies are still low and the molecular mechanisms underlying reprogramming remain to be clarified. In this work, by combining treatment with small molecules, we directly reprogrammed human fetal retinal pigment epithelial (RPE) cells into chemically induced photoreceptor-like cells (CiPCs) in vitro. Bulk and single-cell RNA sequencing, as well as methylation sequencing, were performed to understand the transcriptional and epigenetic changes during CiPCs conversion. A multi-omics analysis showed that the direct reprogramming process partly resembled events of early retina development. We also found that the efficiency of CiPCs conversion from RPE is much better than that from human dermal fibroblasts (HDF). The small molecules effectively induced RPE cells into CiPCs via suppression of the epithelial-to-mesenchymal transition (EMT). Among the signaling pathways involved in CiPCs conversion, glutamate receptor activation is prominent. In summary, RPE cells can be efficiently reprogrammed into photoreceptor-like cells through defined pharmacological modulations, providing a useful cell source for photoreceptor generation in cell replacement therapy for retinal degenerative diseases.

Direct chemical reprogramming of human cord blood erythroblasts to induced megakaryocytes that produce platelets.

Reprogramming somatic cells into megakaryocytes (MKs) would provide a promising source of platelets. However, using a pharmacological approach to generate human MKs from somatic cells remains an unmet challenge. Here, we report that a combination of four small molecules (4M) successfully converted human cord blood erythroblasts (EBs) into induced MKs (iMKs). The iMKs could produce proplatelets and release functional platelets, functionally resembling natural MKs. Reprogramming trajectory analysis revealed an efficient cell fate conversion of EBs into iMKs by 4M via the intermediate state of bipotent precursors. 4M induced chromatin remodeling and drove the transition of transcription factor (TF) regulatory network from key erythroid TFs to essential TFs for megakaryopoiesis, including FLI1 and MEIS1. These results demonstrate that the chemical reprogramming of cord blood EBs into iMKs provides a simple and efficient approach to generate MKs and platelets for clinical applications.

Chemical Pretreatment Activated a Plastic State Amenable to Direct Lineage Reprogramming.

Somatic cells can be chemically reprogrammed into a pluripotent stem cell (CiPSC) state, mediated by an extraembryonic endoderm- (XEN-) like state. We found that the chemical cocktail applied in CiPSC generation initially activated a plastic state in mouse fibroblasts before transitioning into XEN-like cells. The plastic state was characterized by broadly activated expression of development-associated transcription factors (TFs), such as Sox17, Ascl1, Tbx3, and Nkx6-1, with a more accessible chromatin state indicating an enhanced capability of cell fate conversion. Intriguingly, introducing such a plastic state remarkably improved the efficiency of chemical reprogramming from fibroblasts to functional neuron-like cells with electrophysiological activity or beating skeletal muscles. Furthermore, the generation of chemically induced neuron-like cells or skeletal muscles from mouse fibroblasts was independent of the intermediate XEN-like state or the pluripotency state. In summary, our findings revealed a plastic chemically activated multi-lineage priming (CaMP) state at the onset of chemical reprogramming. This state enhanced the cells' potential to adapt to other cell fates. It provides a general approach to empowering chemical reprogramming methods to obtain functional cell types bypassing inducing pluripotent stem cells.

Small molecules mediate cellular reprogramming across two kingdoms.

The fertilized egg is the single totipotent cell from which multicellular organisms arise through the processes of cell division and differentiation. While animals typically lose their capacity to redifferentiate cells that are already fully differentiated, plant cells are thought to remain totipotent (Su et al., 2020). Every gardener knows well that plants can regenerate a full array of plant tissues from already differentiated organs. This also seems to be true for single plant cells such as protoplasts, which, under proper in vitro culture conditions, served as the initial source for generation of transgenic plants (Skoog and Miller, 1957; Birnbaum and Sánchez Alvarado, 2008). However, the mechanisms behind the totipotency of plant cells remain elusive, with the exception of the knowledge that the developmental fate of regenerating tissues can be directed by the ratio of two plant hormones, auxin and cytokinin (Skoog and Miller, 1957).

Direct Reprogramming of Fibroblasts to Astrocytes Using Small Molecules.

Astrocytes play important roles in neurodevelopment and diseases. Previous studies described ways to derive astrocytes from somatic cells by going through iPSC or iNSC/iNPC intermediates. Here we describe a method to directly convert mouse fibroblasts into functional astrocytes using small molecules without transgenes or viral transduction. The direct chemical reprogramming method described in this study provides a more rapid way to derive astrocytes from fibroblasts.

Conversion of stem cells from apical papilla into endothelial cells by small molecules and growth factors.

OBJECTIVES: Recently, a new strategy has been developed to directly reprogram one cell type towards another targeted cell type using small molecule compounds. Human fibroblasts have been chemically reprogrammed into neuronal cells, Schwann cells and cardiomyocyte-like cells by different small molecule combinations. This study aimed to explore whether stem cells from apical papilla (SCAP) could be reprogrammed into endothelial cells (ECs) using the same strategy. MATERIALS AND METHODS: The expression level of endothelial-specific genes and proteins after chemical induction of SCAP was assessed by RT-PCR, western blotting, flow cytometry and immunofluorescence. The in vitro functions of SCAP-derived chemical-induced endothelial cells (SCAP-ECs) were evaluated by tube-like structure formation assay, acetylated low-density lipoprotein (ac-LDL) uptake and NO secretion detection. The proliferation and the migration ability of SCAP-ECs were evaluated by CCK-8 and Transwell assay. LPS stimulation was used to mimic the inflammatory environment in demonstrating the ability of SCAP-ECs to express adhesion molecules. The in vivo Matrigel plug angiogenesis assay was performed to assess the function of SCAP-ECs in generating vascular structures using the immune-deficient mouse model. RESULTS: SCAP-ECs expressed upregulated endothelial-specific genes and proteins; displayed endothelial transcriptional networks; exhibited the ability to form functional tubular-like structures, uptake ac-LDL and secrete NO in vitro; and contributed to generate blood vessels in vivo. The SCAP-ECs could also express adhesion molecules in the pro-inflammatory environment and have a similar migration and proliferation ability as HUVECs. CONCLUSIONS: Our study demonstrates that the set of small molecules and growth factors could significantly promote endothelial transdifferentiation of SCAP, which provides a promising candidate cell source for vascular engineering and treatment of ischemic diseases.

Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2 S production.

Chemical compounds have recently been introduced as alternative and non-integrating inducers of pluripotent stem cell fate. However, chemical reprogramming is hampered by low efficiency and the molecular mechanisms remain poorly characterized. Here, we show that inhibition of spleen tyrosine kinase (Syk) by R406 significantly promotes mouse chemical reprogramming. Mechanistically, R406 alleviates Syk / calcineurin (Cn) / nuclear factor of activated T cells (NFAT) signaling-mediated suppression of glycine, serine, and threonine metabolic genes and dependent metabolites. Syk inhibition upregulates glycine level and downstream transsulfuration cysteine biosynthesis, promoting cysteine metabolism and cellular hydrogen sulfide (H2 S) production. This metabolic rewiring decreased oxidative phosphorylation and ROS levels, enhancing chemical reprogramming. In sum, our study identifies Syk-Cn-NFAT signaling axis as a new barrier of chemical reprogramming and suggests metabolic rewiring and redox homeostasis as important opportunities for controlling cell fates.

Chemical induction of neurogenic properties in mammalian Müller glia.

Müller glia (MG), cells that maintain homeostasis in the retina, are dormant stem cells that can regenerate neurons upon injury. However, the regenerative property of MG, which is reproducibly displayed in the lower vertebrates, is not readily observed in the mammals even upon forced expression of regulatory genes or exposure to growth factors. Here, we demonstrate a reproducible unmasking of the neurogenic properties of enriched rodent MG by serial exposure to different combinations of small molecules. The enriched MG, in response to changing culture conditions, silenced glia-specific genes and acquired transcriptional signature of neurons, accompanied by upregulation of genes known to regulate neuronal potential of MG. The MG-derived neurons expressed immunoreactivities corresponding to neuronal proteins and displayed electrophysiological features of immature neurons. Our study presents a proof of principle of pharmacological activation of neurogenic properties of mammalian MG, which may be utilized for therapeutic regeneration.

Fish Pluripotent Stem-Like Cell Line Induced by Small-Molecule Compounds From Caudal Fin and its Developmental Potentiality.

The technique of induced pluripotent stem cells has significant application value in breeding and preserving the genetic integrity of fish species. However, it is still unclear whether the chemically induced pluripotent stem cells can be induced from non-mammalian cells or not. In this article, we first verify that fibroblasts of fish can be chemically reprogrammed into pluripotent stem cells. These induced pluripotent stem-like cells possess features of colony morphology, expression of pluripotent marker genes, formation of embryoid bodies, teratoma formation, and the potential to differentiate into germ cell-like cells in vitro. Our findings will offer a new way to generate induced pluripotent stem cells in teleost fish and a unique opportunity to breed commercial fish and even save endangered fish species.

High-Resolution Dissection of Chemical Reprogramming from Mouse Embryonic Fibroblasts into Fibrocartilaginous Cells.

Articular cartilage injury and degeneration causing pain and loss of quality-of-life has become a serious problem for increasingly aged populations. Given the poor self-renewal of adult human chondrocytes, alternative functional cell sources are needed. Direct reprogramming by small molecules potentially offers an oncogene-free and cost-effective approach to generate chondrocytes, but has yet to be investigated. Here, we directly reprogrammed mouse embryonic fibroblasts into PRG4+ chondrocytes using a 3D system with a chemical cocktail, VCRTc (valproic acid, CHIR98014, Repsox, TTNPB, and celecoxib). Using single-cell transcriptomics, we revealed the inhibition of fibroblast features and activation of chondrogenesis pathways in early reprograming, and the intermediate cellular process resembling cartilage development. The in vivo implantation of chemical-induced chondrocytes at defective articular surfaces promoted defect healing and rescued 63.4% of mechanical function loss. Our approach directly converts fibroblasts into functional cartilaginous cells, and also provides insights into potential pharmacological strategies for future cartilage regeneration.

Functional Oocytes Derived from Granulosa Cells.

The generation of genomically stable and functional oocytes has great potential for preserving fertility and restoring ovarian function. It remains elusive whether functional oocytes can be generated from adult female somatic cells through reprogramming to germline-competent pluripotent stem cells (gPSCs) by chemical treatment alone. Here, we show that somatic granulosa cells isolated from adult mouse ovaries can be robustly induced to generate gPSCs by a purely chemical approach, with additional Rock inhibition and critical reprogramming facilitated by crotonic sodium or acid. These gPSCs acquired high germline competency and could consistently be directed to differentiate into primordial-germ-cell-like cells and form functional oocytes that produce fertile mice. Moreover, gPSCs promoted by crotonylation and the derived germ cells exhibited longer telomeres and high genomic stability like PGCs in vivo, providing additional evidence supporting the safety and effectiveness of chemical induction, which is particularly important for germ cells in genetic inheritance.

Chemical Reprogramming of Somatic Cells in Neural Direction: Myth or Reality?

In in vitro experiments on cultures of human multipotent stem cells from the human bonearrow and dental pulp, we studied direct reprogramming towards neuro-glial lineage cells using a cocktail of small molecules. Reprogramming by the previously published protocol (with a cocktail containing ß-mercaptoethanol, LIF, VPA, CHIR99021, and RepSox) and by the optimized protocol (VPA, RG108, А83-01, dorsomorphin, thiazovivin, CHIR99021, forskolin, and Isx9) allows obtaining cells with immunophenotypic and genetic signs of neural stem cells. However, neither the former, nor the optimized protocols allowed preparing neural progenitors capable of adequate terminal differentiation from both bone marrow-derived mesenchymal stem cells and nestin-positive neural crest-derived mesenchymal stem cells. Real-time PCR demonstrated the expression of some neurogenesis markers, but neural stem cell-specific expression pattern was not observed. The findings lead us to a conclusion that reprogramming with small molecules without additional factors modifying gene expression does not allow reproducible production of human neural stem cell-like progenitors that can be used as the source of neural tissue for the regenerative therapy.

Transcriptome Analysis of Small Molecule-Mediated Astrocyte-to-Neuron Reprogramming.

Chemical reprogramming of astrocytes into neurons represents a promising approach to regenerate new neurons for brain repair, but the underlying mechanisms driving this trans-differentiation process are not well understood. We have recently identified four small molecules - CHIR99021, DAPT, LDN193189, and SB431542 - that can efficiently reprogram cultured human fetal astrocytes into functional neurons. Here we employ the next generation of RNA-sequencing technology to investigate the transcriptome changes during the astrocyte-to-neuron (AtN) conversion process. We found that the four small molecules can rapidly activate the hedgehog signaling pathway while downregulating many glial genes such as FN1 and MYL9 within 24 h of treatment. Chemical reprogramming is mediated by several waves of differential gene expression, including upregulation of hedgehog, Wnt/ß-catenin, and Notch signaling pathways, together with downregulation of TGF-ß and JAK/STAT signaling pathways. Our gene network analyses reveal many well-connected hub genes such as repulsive guidance molecule A (RGMA), neuronatin (NNAT), neurogenin 2 (NEUROG2), NPTX2, MOXD1, JAG1, and GAP43, which may coordinate the chemical reprogramming process. Together, these findings provide critical insights into the molecular cascades triggered by a combination of small molecules that eventually leads to chemical conversion of astrocytes into neurons.

CRISPR-Knockout Screen Identifies Dmap1 as a Regulator of Chemically Induced Reprogramming and Differentiation of Cardiac Progenitors.

Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome-wide knockout screen with clustered regularly interspaced short palindromic repeats-guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5, and enhanced the retention of self-renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958-972.