Partial or transient cellular reprogramming is defined by the limited induction of pluripotency factors without full dedifferentiation of cells to a pluripotent state. Comparing in vitro and in vivo mouse studies, and in vitro studies in humans, supported by visualizations of data interconnections, we show consistent patterns in how such reprogramming modulates key biological processes. Generally, partial reprogramming drives dynamic chromatin remodelling, involving histone modifications that regulate accessibility and facilitate pluripotency gene activation while silencing somatic identity. These changes are accompanied by modifications in stress response programs, such as inflammation, autophagy, and cellular senescence, as well as improved mitochondrial activity and dysregulation of extracellular matrix pathways. We also underscore the challenges in evaluating complex processes like aging and cellular senescence, given the variability in biomarkers used across studies. Overall, we highlight biological processes consistently influenced by reprogramming while noting that some effects are context-dependent, varying according to cell type, species, sex, recovery time, and the reprogramming method employed. These insights inform future research and potential therapeutic applications in aging and regenerative medicine.

Cell plasticity enables the dynamic changes in cell identities necessary for normal development and tissue repair. Induced cell reprogramming, which leverages this plasticity, holds great promise for regenerative medicine and personalized therapies. However, the success of cell reprogramming is often impeded by various molecular barriers, such as epigenetic marks, cell senescence, and the activation of alternative or refractory routes. In this review, we examine the cell reprogramming events that occur within or between germ layers and adult stem cell lineages and propose that the overall similarity in the pre-existing chromatin accessibility landscape is a major determinant of reprogramming efficiency from one cell type to another. A better understanding of the regulation and control of chromatin accessibility should facilitate the development of new methods and strategies to improve cell reprogramming efficiency and advance translational research.

Slow wound healing in the elderly has attracted much attention recently due to the associated infection risks and decreased longevity. The "brain-skin axis" theory suggests that abnormalities in the brain and nervous system can lead to skin degeneration because abnormal mental states, like chronic stress, can have negative physiological and functional effects on the skin through a variety of processes, resulting in delayed wound healing and accelerated skin aging. However, it remains unclear whether maintaining a youthful brain has beneficial effects on aged skin healing. In light of this, we identified youthful brain-derived extracellular vesicles (YBEVs) and created a composite GelMA hydrogel material that encourages scarless wound healing in aged skin. We found that YBEVs reduce the expression of senescence, senescence-associated secretory phenotypes, and inflammation-associated proteins, and even restore dysfunction in senescent cells. Furthermore, by encouraging collagen deposition, angiogenesis, epidermal and dermal regeneration, and folliculogenesis, we demonstrated that YBEV-containing composite hydrogels accelerated scarless wound healing in skin wounds of aged rats. The pro-repairing speed and effect of this composite hydrogel even matched that of young rats. Subsequent proteomic analysis revealed the presence of numerous proteins within YBEVs, some of which may play a role in the regulation of skin energy intake, particularly through oxidative phosphorylation and mitochondrial function. In conclusion, the findings suggest that maintaining a youthful brain could potentially alleviate skin aging, and the proposed YBEVs-GelMA hydrogel emerges as a promising strategy for addressing age-related impairments in skin healing.

Cell reprogramming represents a powerful approach to achieve the conversion cells of one type into cells of another type of interest, which has substantially changed the landscape in the field of developmental biology, regenerative medicine, disease modeling, drug discovery and cancer immunotherapy. Cell reprogramming is a complex and ordered process that involves the coordination of transcriptional, epigenetic, translational and metabolic changes. Over the past two decades, a range of questions regarding the facilitators/barriers, the trajectories, and the mechanisms of cell reprogramming have been extensively investigated. This review summarizes the recent advances in cell reprogramming mediated by transcription factors or chemical molecules, followed by elaborating on the important roles of biophysical cues in cell reprogramming. Additionally, this review will detail our current understanding of the mechanisms that govern cell reprogramming, including the involvement of the recently discovered biomolecular condensates. Finally, the review discusses the broad applications and future directions of cell reprogramming in developmental biology, disease modeling, drug development, regenerative/rejuvenation therapy, and cancer immunotherapy.

Impaired insulin secretion contributes to the pathogenesis of type 1 diabetes mellitus through autoimmune destruction of pancreatic β-cells and the pathogenesis of severe forms of type 2 diabetes mellitus through β-cell dedifferentiation and other mechanisms. Replenishment of malfunctioning β-cells via islet transplantation has the potential to induce long-term glycemic control in the body. However, this treatment option cannot widely be implemented in clinical due to healthy islet donor shortage. Emerging β-cell replacement with human-induced pluripotent stem cell (iPSC) provides high remedial therapy hopes. Thus, tremendous progress has been made in developing β-cell differentiation protocols in vitro; however, most of the differentiated iPSC-derived β-cells showed immature phenotypes associated with low efficiency depending on the iPSC lines used, creating a crucial barrier for their clinical implementation. Multiple mechanisms including differences in genetic, cell cycle patterns, and mitochondrial dysfunction underlie the defective differentiation propensity of iPSC into insulin-producing β-cells. Accumulating evidence recently indicated that, following the reprogramming, epigenetic memory inherited from parental cells substantially affects the differentiation capacity of many iPSC lines. Therefore, differences in epigenetic signature are likely to be essential contributing factors influencing the propensity of iPSC differentiation. In this review, we will document the impact of the epigenome on the reprogramming efficacy and differentiation potential of iPSCs and how targeting the epigenetic residual memory could be an additional strategy to improve the differentiation efficiency of existing protocols to generate fully functional hPSC-derived pancreatic β-cells for diabetes therapy and drug screening.

Preclinical models of breast cancer that better predict patient-specific drug responses are critical for expanding the clinical utility of targeted therapies, including for inhibitors of poly(ADP-ribose) polymerase (PARP). Reprogramming primary cancer cells into human induced pluripotent stem cells (hiPSCs) recently emerged as a powerful tool to model drug response phenotypes, but its use to date has been limited to hematopoietic malignancies. We designed an optimized reprogramming methodology to generate breast cancer-derived hiPSCs (BC-hiPSCs) from nine patients representing all major subtypes of breast cancer. BC-hiPSCs retain patient-specific oncogenic variants, including variants unique to individual tumor subclones. Additionally, we developed a protocol to differentiate BC-hiPSCs into mammary epithelial cells and mammary-like organoids for in vitro disease modeling, including drug response phenotyping. Using these tools, we demonstrated that BC-hiPSCs can be used to screen for differential sensitivity to PARP inhibitors and mechanistically investigated the causal genetic variant driving drug sensitivity in one patient.

Chromosomal aneuploidies are a major cause of developmental failure and pregnancy loss. To investigate the possible consequences of aneuploidy on early embryonic development in vitro, we focused on primed pluripotent stem cells that are relatable to the epiblast of post-implantation embryos in vivo. We used human induced pluripotent stem cells (iPSCs) as an epiblast model and altered chromosome numbers by treating with reversine, a small-molecule inhibitor of monopolar spindle 1 kinase (MSP1) that inactivates the spindle assembly checkpoint, which has been strongly implicated in chromosome mis-segregation and aneuploidy generation. Upon reversine treatment, we obtained cells with varied chromosomal content that retained pluripotency and potential to differentiate into cells of three germ lineages. However, these cells displayed lagging chromosomes, increased micronuclei content, high p53 expression and excessive apoptotic activity. Cell proliferation was not affected. Prolonged in vitro culture of these cells resulted in a selective pool of cells with supernumerary chromosomes, which exhibited cellular hypertrophy, enlarged nuclei, and overproduction of total RNAs and proteins. We conclude that increased DNA damage responses, apoptosis, and improper cellular mass and functions are possible mechanisms that contribute to abnormal epiblast development.

Somatic cells can be reprogrammed into pluripotent stem cells (iPSCs) by overexpressing defined transcription factors. Specifically, overexpression of OCT4 alone has been demonstrated to reprogram mouse fibroblasts into iPSCs. However, it remains unclear whether any other single factor can induce iPSCs formation. Here, we report that SALL4 alone, under an optimized reprogramming medium iCD4, is capable of reprogramming mouse fibroblasts into iPSCs. Mechanistically, SALL4 facilitates reprogramming by inhibiting somatic genes and activating pluripotent genes, such as Esrrb and Tfap2c. Furthermore, we demonstrate that co-overexpressing SALL4 and OCT4 synergistically enhances reprogramming efficiency. Specifically, the activation of Rsk1/Esrrb/Tfap2c by SALL4, alongside OCT4's activation of Sox2 and the suppression of Mndal by SALL4 and Sbsn by OCT4, cooperate to facilitate SALL4+OCT4-mediated reprogramming. Overall, our study not only establishes an efficient method for iPSCs induction using the SALL4 single factor but also provides insights into the synergistic effects of SALL4 and OCT4 in reprogramming.

Oncogenic transformation and Oct4, Sox2, Klf4 and c-Myc (OSKM)-mediated induction of pluripotency are two independent and incompatible cellular fates. While continuous expression of OSKM can convert normal somatic cells into teratogenic pluripotent cells, it remains speculative what is the impact of transient OSKM expression in cancer cells. Here, we find that OSKM expression limits the growth of transformed lung cells by inducing apoptosis and senescence. We identify Oct4 and Klf4 as the main individual reprogramming factors responsible for this effect. Mechanistically, the induction of cell cycle inhibitor p21 downstream of the reprogramming factors acts as mediator of cell death and senescence. Using a variety of in vivo systems, including allografts, orthotopic transplantation and KRAS-driven lung cancer mouse models, we demonstrate that transient reprogramming by OSKM expression in cancer cells impairs tumor growth and reduces tumor burden. Altogether, our results show that the induction of transient reprogramming in cancer cells is antitumorigenic opening novel potential therapeutic avenues in oncology.

Cellular plasticity in adult multicellular organisms is a protective mechanism that allows certain tissues to regenerate in response to injury. Considering that aging involves exposure to repeated injuries over a lifetime, it is conceivable that cell identity itself is more malleable-and potentially erroneous-with age. In this review, we summarize and critically discuss the available evidence that cells undergo age-related shifts in identity, with an emphasis on those that contribute to age-associated pathologies, including neurodegeneration and cancer. Specifically, we focus on reported instances of programs associated with dedifferentiation, biased differentiation, acquisition of features from alternative lineages, and entry into a preneoplastic state. As some of the most promising approaches to rejuvenate cells reportedly also elicit transient changes to cell identity, we further discuss whether cell state change and rejuvenation can be uncoupled to yield more tractable therapeutic strategies.

During aging and in some contexts, like embryonic development, wound healing, and diseases such as cancer, senescent cells accumulate and play a key role in different pathophysiological functions. A long-held belief was that cellular senescence decreased normal cell functions, given the loss of proliferation of senescent cells. This view radically changed following the discovery of the senescence-associated secretory phenotype (SASP), factors released by senescent cells into their microenvironment. There is now accumulating evidence that cellular senescence also promotes gain-of-function effects by establishing, reinforcing, or changing cell identity, which can have a beneficial or deleterious impact on pathophysiology. These effects may involve both proliferation arrest and autocrine SASP production, although they largely remain to be defined. Here, we provide a historical overview of the first studies on senescence and an insight into emerging trends regarding the effects of senescence on cell identity.

Cellular senescence is the state in which cells undergo irreversible cell cycle arrest and acquire diverse phenotypes. It has been linked to chronic inflammation and fibrosis in various organs as well as to individual aging. Therefore, eliminating senescent cells has emerged as a potential target for extending healthy lifespans. Cellular senescence plays a beneficial role in many biological processes, including embryonic development, wound healing, and tissue regeneration, which is mediated by the activation of stem cells. Therefore, a comprehensive understanding of cellular senescence, including both its beneficial and detrimental effects, is critical for developing safe and effective treatment strategies to target senescent cells. This review provides an overview of the biological and pathological roles of cellular senescence, with a particular focus on its beneficial or detrimental functions among its various roles.

The risk of aberrant growth of induced pluripotent stem cell (iPSC)-derived cells in response to DNA damage is a potential concern as the tumor suppressor genes TP53 and CDKN2A are transiently inactivated during reprogramming. Herein, we evaluate the integrity of cellular senescence pathways and DNA double-strand break (DSB) repair in Sendai virus reprogrammed iPSC-derived human fibroblasts (i-HF) compared to their parental skin fibroblasts (HF). Using transcriptomics analysis and a variety of functional assays, we show that the capacity of i-HF to enter senescence and repair DSB is not compromised after damage induced by ionizing radiation (IR) or the overexpression of H-RASV12. Still, i-HF lines are transcriptionally different from their parental lines, showing enhanced metabolic activity and higher expression of p53-related effector genes. As a result, i-HF lines generally exhibit increased sensitivity to various stresses, have an elevated senescence-associated secretory phenotype (SASP), and cannot be immortalized unless p53 expression is knocked down. In conclusion, while our results suggest that i-HF are not at a greater risk of transformation, their overall hyperactivation of senescence pathways may impede their function as a cell therapy product.

Induced pluripotent stem cells (iPSCs) have wide applications in disease modeling, personalized medicine, and tissue engineering. The generation of iPSCs from somatic cells via transcriptional-factor- or chemical molecule-based approaches are time-consuming and inefficient. Here, a cell-reprogramming-inspired dynamically responsive hydrogel is fabricated via a synthetic-biology-based strategy. Human and mouse somatic cells (including senescent cells) are efficiently reprogrammed into iPSCs that exhibit key features of embryonic stem cells. The cell-reprogramming-responsive hydrogel possesses dynamic bioresponsiveness, and it faithfully senses metabolic remodeling and extracellular acidification during cell reprogramming, responding by changing its mechanical properties accordingly. Mechanistic study demonstrates that the autonomous change of the mechanical properties of the cell-reprogramming-responsive hydrogel elicits the formation of Yes-associated protein (YAP) biomolecular condensates with the appropriate timing during cell reprogramming, ensuring a faster and more efficient generation of iPSCs than conventional cell reprogramming approach. Taken together, this study reveals the robust induction of pluripotency by coordination of cell-reprogramming-inspired dynamically responsive hydrogel and phase-separated biomolecular condensates.

Cellular senescence is a critical stress response program implicated in embryonic development, wound healing, aging, and immunity, and it backs up apoptosis as an ultimate cell-cycle exit mechanism. In analogy to replicative exhaustion of telomere-eroded cells, premature types of senescence-referring to oncogene-, therapy-, or virus-induced senescence-are widely considered irreversible growth arrest states as well. We discuss here that entry into full-featured senescence is not necessarily a permanent endpoint, but dependent on essential maintenance components, potentially transient. Unlike a binary state switch, we view senescence with its extensive epigenomic reorganization, profound cytomorphological remodeling, and distinctive metabolic rewiring rather as a journey toward a full-featured arrest condition of variable strength and depth. Senescence-underlying maintenance-essential molecular mechanisms may allow cell-cycle reentry if not continuously provided. Importantly, senescent cells that resumed proliferation fundamentally differ from those that never entered senescence, and hence would not reflect a reversion but a dynamic progression to a post-senescent state that comes with distinct functional and clinically relevant ramifications.

Cancer is a leading cause of death worldwide. Targeted therapies aimed at key oncogenic driver mutations in combination with chemotherapy and radiotherapy as well as immunotherapy have benefited cancer patients considerably. Tumor protein p53 (TP53), a crucial tumor suppressor gene encoding p53, regulates numerous downstream genes and cellular phenotypes in response to various stressors. The affected genes are involved in diverse processes, including cell cycle arrest, DNA repair, cellular senescence, metabolic homeostasis, apoptosis, and autophagy. However, accumulating recent studies have continued to reveal novel and unexpected functions of p53 in governing the fate of tumors, for example, functions in ferroptosis, immunity, the tumor microenvironment and microbiome metabolism. Among the possibilities, the evolutionary plasticity of p53 is the most controversial, partially due to the dizzying array of biological functions that have been attributed to different regulatory mechanisms of p53 signaling. Nearly 40 years after its discovery, this key tumor suppressor remains somewhat enigmatic. The intricate and diverse functions of p53 in regulating cell fate during cancer treatment are only the tip of the iceberg with respect to its equally complicated structural biology, which has been painstakingly revealed. Additionally, TP53 mutation is one of the most significant genetic alterations in cancer, contributing to rapid cancer cell growth and tumor progression. Here, we summarized recent advances that implicate altered p53 in modulating the response to various cancer therapies, including chemotherapy, radiotherapy, and immunotherapy. Furthermore, we also discussed potential strategies for targeting p53 as a therapeutic option for cancer.

Cellular senescence is a stress response mechanism that induces proliferative arrest. Hypoxia can bypass senescence and extend the lifespan of primary cells, mainly by decreasing oxidative damage. However, how hypoxia promotes these effects prior to malignant transformation is unknown. Here we observed that the lifespan of mouse embryonic fibroblasts (MEFs) is increased when they are cultured in hypoxia by reducing the expression of p16INK4a, p15INK4b and p21Cip1. We found that proliferating MEFs in hypoxia overexpress Tfcp2l1, which is a main regulator of pluripotency and self-renewal in embryonic stem cells, as well as stemness genes including Oct3/4, Sox2 and Nanog. Tfcp2l1 expression is lost during culture in normoxia, and its expression in hypoxia is regulated by Hif1α. Consistently, its overexpression in hypoxic levels increases the lifespan of MEFs and promotes the overexpression of stemness genes. ATAC-seq and Chip-seq experiments showed that Tfcp2l1 regulates genes that control proliferation and stemness such as Sox2, Sox9, Jarid2 and Ezh2. Additionally, Tfcp2l1 can replicate the hypoxic effect of increasing cellular reprogramming. Altogether, our data suggest that the activation of Tfcp2l1 by hypoxia contributes to immortalization prior to malignant transformation, facilitating tumorigenesis and dedifferentiation by regulating Sox2, Sox9, and Jarid2.

Pluripotency can be induced in somatic cells by the expression of OCT4, KLF4, SOX2, and MYC. Usually only a rare subset of cells reprogram, and the molecular characteristics of this subset remain unknown. We apply retrospective clone tracing to identify and characterize the rare human fibroblasts primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis increased the reprogramming efficiency. We provide evidence for a unified model in which cells can move into and out of the primed state over time, explaining how reprogramming appears deterministic at short timescales and stochastic at long timescales. Furthermore, inhibiting the activity of LSD1 enlarged the pool of cells that were primed for reprogramming. Thus, even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.

Pluripotent stem cells can be generated from somatic cells by the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc.

Mouse embryonic fibroblasts (MEFs) were transduced with the Yamanaka factors and generation of induced pluripotent stem cells (iPSCs) was assessed by formation of alkaline phosphatase positive colonies, pluripotency gene expression and embryod bodies formation.

The thyroid hormone triiodothyronine (T3) enhances MEFs reprogramming. T3-induced iPSCs resemble embryonic stem cells in terms of the expression profile and DNA methylation pattern of pluripotency genes, and of their potential for embryod body formation and differentiation into the three major germ layers. T3 induces reprogramming even though it increases expression of the cyclin kinase inhibitors p21 and p27, which are known to oppose acquisition of pluripotency. The actions of T3 on reprogramming are mainly mediated by the thyroid hormone receptor beta and T3 can enhance iPSC generation in the absence of c-Myc. The hormone cannot replace Oct4 on reprogramming, but in the presence of T3 is possible to obtain iPSCs, although with low efficiency, without exogenous Klf4. Furthermore, depletion of the corepressor NCoR (or Nuclear Receptor Corepressor 1) reduces MEFs reprogramming in the absence of the hormone and strongly decreases iPSC generation by T3 and also by 9cis-retinoic acid, a well-known inducer of reprogramming. NCoR depletion also markedly antagonizes induction of pluripotency gene expression by both ligands.

Inclusion of T3 on reprogramming strategies has a potential use in enhancing the generation of functional iPSCs for studies of cell plasticity, disease and regenerative medicine.

Pluripotent stem cells (PSCs) are a promising source of stem cells for regenerative therapies. Stem cell function depends on telomere maintenance mechanisms that provide them with the proliferative capacity and genome stability necessary to multiply and regenerate tissues. We show here that established human embryonic stem cells (hESCs) have stable telomere length that is dependent on telomerase but not on alternative mechanisms based on homologous recombination pathways. Here, we show that human-induced pluripotent stem cells (hiPSCs) reprogrammed from somatic cells show progressive telomere lengthening until reaching a length similar to ESCs. hiPSCs also acquire telomeric chromatin marks of ESCs including decreased abundance of tri-methylated histone H3K9 and H4K20 and HP1 heterochromatic marks, as well as of the shelterin component TRF2. These chromatin features are accompanied with increased abundance of telomere transcripts or TERRAs. We also found that telomeres of both hESCs and hiPSCs are well protected from DNA damage during telomere elongation and once full telomere length is achieved, and exhibit stable genomes. Collectively, this study highlights that hiPSCs acquire ESC features during reprogramming and reveals the telomere biology in human pluripotent stem cells (hPSCs).

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.

CYLD is a tumor suppressor gene coding for a deubiquitinating enzyme that has a critical regulatory function in a variety of signaling pathways and biological processes involved in cancer development and progression, many of which are also key modulators of somatic cell reprogramming. Nevertheless, the potential role of CYLD in this process has not been studied. With the dual aim of investigating the involvement of CYLD in reprogramming and developing a better understanding of the intricate regulatory system governing this process, we reprogrammed control (CYLDWT/WT) and CYLD DUB-deficient (CYLDΔ9/Δ9) mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) through ectopic overexpression of the Yamanaka factors (Oct3/4, Sox2, Klf4, c-myc). CYLD DUB deficiency led to significantly reduced reprogramming efficiency and slower early reprogramming kinetics. The introduction of WT CYLD to CYLDΔ9/Δ9 MEFs rescued the phenotype. Nevertheless, CYLD DUB-deficient cells were capable of establishing induced pluripotent colonies with full spontaneous differentiation potential of the three germ layers. Whole proteome analysis (Data are available via ProteomeXchange with identifier PXD044220) revealed that the mesenchymal-to-epithelial transition (MET) during the early reprogramming stages was disrupted in CYLDΔ9/Δ9 MEFs. Interestingly, differentially enriched pathways revealed that the primary processes affected by CYLD DUB deficiency were associated with the organization of the extracellular matrix and several metabolic pathways. Our findings not only establish for the first time CYLD's significance as a regulatory component of early reprogramming but also highlight its role as an extracellular matrix regulator, which has profound implications in cancer research.

Despite advances in four-factor (4F)-induced reprogramming (4FR) in vitro and in vivo, how 4FR interconnects with senescence remains largely under investigated. Here, using genetic and chemical approaches to manipulate senescent cells, we show that removal of p16High cells resulted in the 4FR of somatic cells into totipotent-like stem cells. These cells expressed markers of both pluripotency and the two-cell embryonic state, readily formed implantation-competent blastoids and, following morula aggregation, contributed to embryonic and extraembryonic lineages. We identified senescence-dependent regulation of nicotinamide N-methyltransferase as a key mechanism controlling the S-adenosyl-L-methionine levels during 4FR that was required for expression of the two-cell genes and acquisition of an extraembryonic potential. Importantly, a partial 4F epigenetic reprogramming in old mice was able to reverse several markers of liver aging only in conjunction with the depletion of p16High cells. Our results show that the presence of p16High senescent cells limits cell plasticity, whereas their depletion can promote a totipotent-like state and histopathological tissue rejuvenation during 4F reprogramming.

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers.

We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation. See related commentary by Doulatov, p. 252. This article is highlighted in the In This Issue feature, p. 247.

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.

Induced pluripotent stem cells (iPSCs) are promising cell sources for regenerative medicine and disease modeling. iPSCs are commonly established by introducing the defined reprogramming factors Oct4, Sox2, Klf4, and c-Myc. However, iPSC reprogramming efficiency remains low. Although recent studies have identified microRNAs that contribute to efficient reprogramming, the underlying molecular mechanisms are not completely understood. miR-17-92 is highly expressed in embryonic stem cells and may play an important role in regulating stem cell properties. Therefore, we examined the role of miR-17-92 in the induction of mouse iPSC production. c-Myc-mediated miR-17-92 upregulation increased reprogramming efficiency, whereas CRISPR/Cas9-based deletion of the miR-17-92 cluster decreased reprogramming efficiency. A combination of in silico and microarray analyses revealed that Pten and cyclin-dependent kinase inhibitor 1 (known as p21) are common target genes of miR-17 and miR-20a, which are transcribed from the miR-17-92 cluster. Moreover, miR-17-92 downregulated p21 in the early phase and PTEN in the mid-to-late phase of reprogramming. These downregulations were perturbed by introducing the 3' UTR of PTEN and p21, respectively, suggesting that PTEN and p21 mRNAs are competing endogenous RNAs (ceRNA) against miR-17-92. Collectively, we propose that the c-Myc-mediated expression of miR-17-92 is involved in iPSC reprogramming through the phase-dependent inhibition of PTEN and p21 in a ceRNA manner, thus elucidating an underlying mechanism of iPSC reprogramming.

Tumor-infiltrating lymphocytes (TIL) that can recognize and kill tumor cells have curative potential in subsets of patients treated with adoptive cell transfer (ACT). However, lack of TIL therapeutic efficacy in many patients may be due in large part to a paucity of tumor-reactive T cells in TIL and the exhausted and terminally differentiated status of those tumor-reactive T cells. We sought to reprogram exhausted TIL that possess T-cell receptors (TCR) specific for tumor antigens into induced pluripotent stem cells (iPSC) to rejuvenate them for more potent ACT. We first attempted to reprogram tumor neoantigen-specific TIL by αCD3 Ab prestimulation which resulted in failure of establishing tumor-reactive TIL-iPSCs, instead, T cell-derived iPSCs from bystander T cells were established. To selectively activate and enrich tumor-reactive T cells from the heterogenous TIL population, CD8⁺ PD-1⁺ 4-1BB⁺ TIL population were isolated after coculture with autologous tumor cells, followed by direct reprogramming into iPSCs. TCR sequencing analysis of the resulting iPSC clones revealed that reprogrammed TIL-iPSCs encoded TCRs that were identical to the pre-identified tumor-reactive TCRs found in minimally cultured TIL. Moreover, reprogrammed TIL-iPSCs contained rare tumor antigen-specific TCRs, which were not detectable by TCR sequencing of the starting cell population. Thus, reprogramming of PD-1⁺ 4-1BB⁺ TIL after coculture with autologous tumor cells selectively generates tumor antigen-specific TIL-iPSCs, and is a distinctive method to enrich and identify tumor antigen-specific TCRs of low frequency from TIL.

Reprogramming of TIL into iPSC holds great promise for the future treatment of cancer due to their rejuvenated nature and the retention of tumor-specific TCRs. One limitation is the lack of selective and efficient methods for reprogramming tumor-specific T cells from polyclonal TIL. Here we addressed this limitation and present a method to efficiently reprogram TIL into iPSC colonies carrying diverse tumor antigen reactive TCR recombination.

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.

Pluripotency can be induced in somatic cells by the expression of the four "Yamanaka" factors OCT4, KLF4, SOX2, and MYC. However, even in homogeneous conditions, usually only a rare subset of cells admit reprogramming, and the molecular characteristics of this subset remain unknown. Here, we apply retrospective clone tracing to identify and characterize the individual human fibroblast cells that are primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis led to increased reprogramming efficiency, identifying it as a barrier to reprogramming. Changing the frequency of reprogramming by inhibiting the activity of LSD1 led to an enlarging of the pool of cells that were primed for reprogramming. Our results show that even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.

Induced pluripotent stem cell (iPSC) reprogramming is inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 24 reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhances murine iPSC generation in several reprogramming settings, emerging as the most robust roadblock. We show that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2, and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with co-activator domains converts ZFP266 from an inhibitor to a potent facilitator of iPSC reprogramming. We propose that the SINE-KRAB-ZFP interaction is a critical regulator of chromatin accessibility at regulatory elements required for efficient cellular identity changes. In addition, this work serves as a resource to further illuminate molecular mechanisms hindering reprogramming.

Aging is driven by hallmarks fulfilling the following three premises: (1) their age-associated manifestation, (2) the acceleration of aging by experimentally accentuating them, and (3) the opportunity to decelerate, stop, or reverse aging by therapeutic interventions on them. We propose the following twelve hallmarks of aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis. These hallmarks are interconnected among each other, as well as to the recently proposed hallmarks of health, which include organizational features of spatial compartmentalization, maintenance of homeostasis, and adequate responses to stress.

Lung adenocarcinoma (LUAD) is the most prevalent lung cancer and one of the leading causes of death. Previous research found a link between LUAD and Aldehyde Dehydrogenase 2 (ALDH2), a member of aldehyde dehydrogenase gene (ALDH) superfamily. In this study, we identified additional useful prognostic markers for early LUAD identification and targeting LUAD therapy by analyzing the expression level, epigenetic mechanism, and signaling activities of ALDH2 in LUAD patients. The obtained results demonstrated that ALDH2 gene and protein expression significantly downregulated in LUAD patient samples. Furthermore, The American Joint Committee on Cancer (AJCC) reported that diminished ALDH2 expression was closely linked to worse overall survival (OS) in different stages of LUAD. Considerably, ALDH2 showed aberrant DNA methylation status in LUAD cancer. ALDH2 was found to be downregulated in the proteomic expression profile of several cell biology signaling pathways, particularly stem cell-related pathways. Finally, the relationship of ALDH2 activity with stem cell-related factors and immune system were reported. In conclusion, the downregulation of ALDH2, abnormal DNA methylation, and the consequent deficit of stemness signaling pathways are relevant prognostic and therapeutic markers in LUAD.

The histone lysine demethylase 2 (KDM2) family of α-Ketoglutarate-Fe++-dependent dioxygenases were the first Jumonji-domain-containing proteins reported to harbor demethylase activity. This landmark discovery paved the way for the characterization of more than 25 enzymes capable of demethylating lysine residues on histones-an epigenetic modification previously thought to be irreversible. The KDM2 family is comprised of KDM2A and KDM2B which share significant structural similarities and demethylate lysine 36 on histone H3. However, they exert distinct cellular functions and are frequently deregulated in a broad spectrum of human cancers. With the advent of next generation sequencing and development of genetically engineered mouse models, it was shown that KDM2A and KDM2B play critical roles in stem cell biology, somatic cell reprograming, and organismal development by regulating cell fate and lineage commitment decisions. Thus, understanding the biochemistry and elucidating the context-dependent function of these enzymes is an emerging new frontier for the development of small molecule inhibitors to treat cancer and other diseases.

A feasible method to detect somatic copy number deletion (SCND) of genes is still absent to date.

Interstitial base-resolution deletion/fusion coordinates for CDKN2A were extracted from published articles and our whole genome sequencing (WGS) datasets. The copy number of the CDKN2A gene was measured with a quantitative multiplex PCR assay P16-Light and confirmed with whole genome sequencing (WGS).

Estimated common deletion regions (CDRs) were observed in many tumor suppressor genes, such as ATM, CDKN2A, FAT1, miR31HG, PTEN, and RB1, in the SNP array-based COSMIC datasets. A 5.1 kb base-resolution CDR could be identified in >90% of cancer samples with CDKN2A deletion by sequencing. The CDKN2A CDR covers exon-2, which is essential for P16INK4A and P14ARF synthesis. Using the true CDKN2A CDR as a PCR target, a quantitative multiplex PCR assay P16-Light was programmed to detect CDKN2A gene copy number. P16-Light was further confirmed with WGS as the gold standard among cancer tissue samples from 139 patients.

The 5.1 kb CDKN2A CDR was found in >90% of cancers containing CDKN2A deletion. The CDKN2A CDR was used as a potential target for developing the P16-Light assay to detect CDKN2A SCND and amplification for routine clinical practices.

Cellular senescence, a stable form of cell cycle arrest, accompanied by pronounced secretory activity, has functional roles in both physiological and pathological conditions. Although senescence has been linked for a long time with cancer and ageing, recent studies have revealed a functional role of senescence in development, regeneration and reprogramming. Notably, the transient presence of senescent cells may be beneficial, in contrast to the potential deleterious effects of persistent senescence in aged or chronically damaged tissues. We will discuss how senescence contributes to embryonic development, cell plasticity and tissue regeneration, as a highly coordinated and programmed cellular state.

Embryonic stem cells (ESCs) have a significantly lower mutation load compared to somatic cells, but the mechanisms that guard genomic integrity in ESCs remain largely unknown. Here we show that BNIP3-dependent mitophagy protects genomic integrity in mouse ESCs. Deletion of Bnip3 increases cellular reactive oxygen species (ROS) and decreases ATP generation. Increased ROS in Bnip3^-/- ESCs compromised self-renewal and were partially rescued by either NAC treatment or p53 depletion. The decreased cellular ATP in Bnip3^-/- ESCs induced AMPK activation and deteriorated homologous recombination, leading to elevated mutation load during long-term propagation. Whereas activation of AMPK in X-ray-treated Bnip3^+/+ ESCs dramatically ascended mutation rates, inactivation of AMPK in Bnip3^-/- ESCs under X-ray stress remarkably decreased the mutation load. In addition, enhancement of BNIP3-dependent mitophagy during reprogramming markedly decreased mutation accumulation in established iPSCs. In conclusion, we demonstrated a novel pathway in which BNIP3-dependent mitophagy safeguards ESC genomic stability, and that could potentially be targeted to improve pluripotent stem cell genomic integrity for regenerative medicine.

Recent advances in cell reprogramming showed that OSKM induction is able to improve cell physiology in vitro and in vivo. Here, we show that a single short reprogramming induction is sufficient to prevent musculoskeletal functions deterioration of mice, when applied in early life. In addition, in old age, treated mice have improved tissue structures in kidney, spleen, skin, and lung, with an increased lifespan of 15% associated with organ-specific differential age-related DNA methylation signatures rejuvenated by the treatment. Altogether, our results indicate that a single short reprogramming early in life might initiate and propagate an epigenetically related mechanism to promote a healthy lifespan.