Proline restores mitochondrial function and reverses aging hallmarks in senescent cells.

Mitochondrial dysfunction is a hallmark of cellular senescence, with the loss of mitochondrial function identified as a potential causal factor contributing to senescence-associated decline in cellular functions. Our recent findings revealed that ectopic expression of the pluripotency transcription factor NANOG rejuvenates dysfunctional mitochondria of senescent cells by rewiring metabolic pathways. In this study, we report that NANOG restores the expression of key enzymes, PYCR1 and PYCR2, in the proline biosynthesis pathway. Additionally, senescent mesenchymal stem cells manifest severe mitochondrial respiratory impairment, which is alleviated through proline supplementation. Proline induces mitophagy by activating AMP-activated protein kinase α and upregulating Parkin expression, enhancing mitochondrial clearance and ultimately restoring cell metabolism. Notably, proline treatment also mitigates several aging hallmarks, including DNA damage, senescence-associated ß-galactosidase, inflammatory cytokine expressions, and impaired myogenic differentiation capacity. Overall, this study highlights the role of proline in mitophagy and its potential in reversing senescence-associated mitochondrial dysfunction and aging hallmarks.

Fibrin hydrogels fortified with FGF-7/10 and laminin-1 peptides promote regeneration of irradiated salivary glands.

Ionizing radiation, commonly used for head and neck cancer treatment, typically damages the salivary glands, resulting in hyposalivation. The development of treatments to restore this lost function is crucial for improving the quality of life for patients suffering from this condition. To address this clinical need, we have developed an innovative hydrogel by chemically conjugating laminin-1 peptides (A99 and YIGSR) and growth factors, FGF-7 and FGF-10, to fibrin hydrogels. Our results demonstrate that FGF-7/10 and laminin-1 peptides fortified fibrin hydrogel [enhanced laminin-1 peptides fibrin hydrogel (Ep-FH)] promotes salivary gland regeneration and functionality by improving epithelial tissue organization, establishing a healthy network of blood vessels and nerves, while reducing fibrosis in a head and neck irradiated mouse model. These results indicate that fibrin hydrogel-based implantable scaffolds containing pro-regenerative signals promote sustained secretory function of irradiated salivary glands, offering a potential alternative treatment for hyposalivation in head and neck cancer patients undergoing radiation treatment. These unique findings emphasize the potential of fibrin hydrogel-based implantable scaffolds enriched with pro-regenerative signals in sustaining the secretory function of irradiated salivary glands and offer a promising alternative treatment for addressing hyposalivation in head and neck cancer patients undergoing radiation therapy. STATEMENT OF SIGNIFICANCE: Radiation therapies used to treat head and neck cancers often result in damaged salivary gland, leading to severe dryness of the oral cavity. In this study, we engineered FGF-7 and FGF-10 and immobilized them into L1p-FH. The resulting hydrogel, Ep-FH, restored irradiated salivary gland functionality by enhancing epithelial tissue organization, promoting the development of a healthy network of blood vessels and nerves as well as reduction of fibrosis.

Cadherin-11 increases tumor cell proliferation and metastatic potential via Wnt pathway activation.

During epithelial-mesenchymal transition (EMT) in cancer progression, tumor cells switch cadherin profile from E-cadherin to cadherin-11 (CDH11), which is accompanied by increased invasiveness and metastatic activity. However, the mechanism through which CDH11 may affect tumor growth and metastasis remains elusive. Here, we report that CDH11 was highly expressed in multiple human tumors and was localized on the membrane, in the cytoplasm and, surprisingly, also in the nucleus. Interestingly, ß-catenin remained bound to carboxy-terminal fragments (CTFs) of CDH11, the products of CDH11 cleavage, and co-localized with CTFs in the nucleus in the majority of breast cancer samples. Binding of ß-catenin to CTFs preserved ß-catenin activity, whereas inhibiting CDH11 cleavage led to ß-catenin phosphorylation and diminished Wnt signaling, similar to CDH11 knockout. Our data elucidate a previously unknown role of CDH11, which serves to stabilize ß-catenin in the cytoplasm and facilitates its translocation to the nucleus, resulting in activation of Wnt signaling, with subsequent increased proliferation, migration and invasion potential.

Wnt/BMP Mediated Metabolic Reprogramming Preserves Multipotency of Neural Crest-Like Stem Cells.

Neural crest-like stem cells resembling embryonic neural crest cells (NCs) can be derived from adult human tissues such as the epidermis. However, these cells lose their multipotency rapidly in culture limiting their expansion for clinical use. Here, we show that the multipotency of keratinocyte-derived NCs (KC-NCs) can be preserved by activating the Wnt and BMP signaling axis, promoting expression of key NC-specifier genes and ultimately enhancing their differentiation potential. We also show that transcriptional changes leading to multipotency are linked to metabolic reprogramming of KC-NCs to a highly glycolytic state. Specifically, KC-NCs treated with CHIR and BMP2 rely almost exclusively on glycolysis for their energy needs, as seen by increased lactate production, glucose uptake, and glycolytic enzyme activities. This was accompanied by mitochondrial depolarization and decreased mitochondrial ATP production. Interestingly, the glycolytic end-product lactate stabilized ß-catenin and further augmented NC-gene expression. Taken together, our study shows that activation of the Wnt/BMP signaling coordinates the metabolic demands of neural crest-like stem cells governing decisions regarding multipotency and differentiation, with possible implications for regenerative medicine.

Inhibition of glutaminolysis restores mitochondrial function in senescent stem cells.

Mitochondrial dysfunction, a hallmark of aging, has been associated with the onset of aging phenotypes and age-related diseases. Here, we report that impaired mitochondrial function is associated with increased glutamine catabolism in senescent human mesenchymal stem cells (MSCs) and myofibroblasts derived from patients suffering from Hutchinson-Gilford progeria syndrome. Increased glutaminase (GLS1) activity accompanied by loss of urea transporter SLC14A1 induces urea accumulation, mitochondrial dysfunction, and DNA damage. Conversely, blocking GLS1 activity restores mitochondrial function and leads to amelioration of aging hallmarks. Interestingly, GLS1 expression is regulated through the JNK pathway, as demonstrated by chemical and genetic inhibition. In agreement with our in vitro findings, tissues isolated from aged or progeria mice display increased urea accumulation and GLS1 activity, concomitant with declined mitochondrial function. Inhibition of glutaminolysis in progeria mice improves mitochondrial respiratory chain activity, suggesting that targeting glutaminolysis may be a promising strategy for restoring age-associated loss of mitochondrial function.

Laminin-1 Peptides Conjugated to Fibrin Hydrogels Promote Salivary Gland Regeneration in Irradiated Mouse Submandibular Glands.

Previous studies demonstrated that salivary gland morphogenesis and differentiation are enhanced by modification of fibrin hydrogels chemically conjugated to Laminin-1 peptides. Specifically, Laminin-1 peptides (A99: CGGALRGDN-amide and YIGSR: CGGADPGYIGSRGAA-amide) chemically conjugated to fibrin promoted formation of newly organized salivary epithelium both in vitro (e.g., using organoids) and in vivo (e.g., in a wounded mouse model). While these studies were successful, the model's usefulness for inducing regenerative patterns after radiation therapy remains unknown. Therefore, the goal of the current study was to determine whether transdermal injection with the Laminin-1 peptides A99 and YIGSR chemically conjugated to fibrin hydrogels promotes tissue regeneration in irradiated salivary glands. Results indicate that A99 and YIGSR chemically conjugated to fibrin hydrogels promote formation of functional salivary tissue when transdermally injected to irradiated salivary glands. In contrast, when left untreated, irradiated salivary glands display a loss in structure and functionality. Together, these studies indicate that fibrin hydrogel-based implantable scaffolds containing Laminin-1 peptides promote secretory function of irradiated salivary glands.

Ameliorating the hallmarks of cellular senescence in skeletal muscle myogenic progenitors in vitro and in vivo.

Senescence of myogenic progenitors impedes skeletal muscle regeneration. Here, we show that overexpression of the transcription factor NANOG in senescent myoblasts can overcome the effects of cellular senescence and confer a youthful phenotype to senescent cells. NANOG ameliorated primary hallmarks of cellular senescence including genomic instability, loss of proteostasis, and mitochondrial dysfunction. The rejuvenating effects of NANOG included restoration of DNA damage response via up-regulation of DNA repair proteins, recovery of heterochromatin marks via up-regulation of histones, and reactivation of autophagy and mitochondrial energetics via up-regulation of AMP-activated protein kinase (AMPK). Expression of NANOG in the skeletal muscle of a mouse model of premature aging restored the number of myogenic progenitors and induced formation of eMyHC+ myofibers. This work demonstrates the feasibility of reversing the effects of cellular senescence in vitro and in vivo, with no need for reprogramming to the pluripotent state.

Bioengineered Skeletal Muscle as a Model of Muscle Aging and Regeneration.

With age, adult skeletal muscle (SkM) is known to decrease in muscle mass, strength, and functional capacity, a state known as sarcopenia. Here we developed an in vitro three-dimensional (3D) bioengineered senescent SkM tissue using primary human myoblasts. These tissues exhibited the characteristics of atrophied muscle, including expression of senescent genes, decreased number of satellite cells, reduced number and size of myofibers, and compromised metabolism and calcium flux. As a result, senescent SkM tissues showed impaired ability to generate force in response to electrical stimulation compared with young tissues. Furthermore, in contrast to young SkM tissues, senescent tissues failed to regenerate in response to injury, possibly as a result of persistent apoptosis and failure to initiate a proliferation program. Our findings suggest that 3D senescent SkM may provide a powerful model for studying aging and a platform for drug testing and discovery of therapeutic compounds to improve the function of sarcopenic muscle. Impact statement Skeletal muscle (SkM) plays important physiological roles and has significant regenerative capacity. However, aged SkM lose their functionality and regeneration ability. In this article, we present a senescent human bioengineering SkM tissue model that can be used to investigate senescence, metabolic or genetic diseases that inflict SkM, and to test various strategies including novel small molecules that restore muscle function and promote regeneration. One key limitation of two-dimensional cell culture system is the detachment of contractile myotubes from the surface over time, thereby limiting the evaluation of myogenic function. Here we use primary human myoblasts, which exhibit all major hallmarks of aging to mimic the organization and function of native muscle. Using this system, we were able to measure the contractile function, calcium transients, and regeneration capacity of SkM tissues. We also evaluated the response of senescent SkM tissues to injury and their ability to regenerate and recover, compared with "young" tissues. Our results suggest that three-dimensional constructs enable organization of contractile units including myosin and actin filaments, thereby providing a powerful platform for the quantitative assessment of muscle myotubes in response to injury, genetic or metabolic disorders, or pharmacological testing.

Sex-dependent Regeneration Patterns in Mouse Submandibular Glands.

Our previous studies indicated that YIGSR-A99 peptides chemically conjugated to fibrin hydrogel (FH) and applied to wounded submandibular gland (SMG) in vivo, formed new organized salivary tissue, whereas wounded SMG treated with FH alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. While these studies indicated that damaged SMG grow and differentiate when treated with FH containing L1 peptide, they were performed only in female mice. However, there is a well-established sexual dimorphism present in mouse SMG (e.g., males develop well-differentiated granular convoluted tubules, but these structures are poorly developed in females) and little is known about how these sex differences influence wound healing events. Therefore, the goal of this study was to conduct comparative analyses of regeneration patterns in male and female mice using L1p-FH in a wounded SMG mouse model. Particularly, we focused on sex-dependent wound healing events such as macrophage polarization, vascularization, tissue organization, and collagen deposition, and how these events affect salivary gland functioning.

PEGylated Amine-Functionalized Poly(ε-caprolactone) for the Delivery of Plasmid DNA.

As a promising strategy for the treatment of various diseases, gene therapy has attracted increasing attention over the past decade. Among various gene delivery approaches, non-viral vectors made of synthetic biomaterials have shown significant potential. Due to their synthetic nature, non-viral vectors can have tunable structures and properties by using various building units. In particular, they can offer advantages over viral vectors with respect to biosafety and cytotoxicity. In this study, a well-defined poly(ethylene glycol)-block-poly(α-(propylthio-N,N-diethylethanamine hydrochloride)-ε-caprolactone) diblock polymer (PEG-b-CPCL) with one poly(ethylene glycol) (PEG) block and one tertiary amine-functionalized cationic poly(ε-caprolactone) (CPCL) block, as a novel non-viral vector in the delivery of plasmid DNA (pDNA), was synthesized and studied. Despite having a degradable polymeric structure, the polymer showed remarkable hydrolytic stability over multiple weeks. The optimal ratio of the polymer to pDNA for nanocomplex formation, pDNA release from the nanocomplex with the presence of heparin, and serum stability of the nanocomplex were probed through gel electrophoresis. Nanostructure of the nanocomplexes was characterized by DLS and TEM imaging. Relative to CPCL homopolymers, PEG-b-CPCL led to better solubility over a wide range of pH. Overall, this work demonstrates that PEG-b-CPCL possesses a range of valuable properties as a promising synthetic vector for pDNA delivery.

Engineering the mode of morphogenetic signal presentation to promote branching from salivary gland spheroids in 3D hydrogels.

Previously we developed a fibrin hydrogel (FH) decorated with laminin-111 peptides (L1p-FH) and supports three-dimensional (3D) gland microstructures containing polarized acinar cells. Here we expand on these results and show that co-culture of rat parotid Par-C10 cells with mesenchymal stem cells produces migrating branches of gland cells into the L1p-FH and we identify FGF-7 as the principal morphogenetic signal responsible for branching. On the other hand, another FGF family member and gland morphogen, FGF-10 increased proliferation but did not promote migration and therefore, limited the number and length of branched structures grown into the gel. By controlling the mode of growth factor presentation and delivery, we can control the length and cellularity of branches as well as formation of new nodes/clusters within the hydrogel. Such spatial delivery of two or more morphogens may facilitate engineering of anatomically complex tissues/mini organs such as salivary glands that can be used to address developmental questions or as platforms for drug discovery. STATEMENT OF SIGNIFICANCE: Hyposalivation leads to the development of a host of oral diseases. Current treatments only provide temporary relief. Tissue engineering may provide promising permanent solutions. Yet current models are limited to salivary spheroids with no branching networks. Branching structures are vital to an effective functioning gland as they increase the surface area/glandular volume ratio of the tissue, allowing a higher output from the small-sized gland. We describe a strategy that controls branch network formation in salivary glands that is a key in advancing the field of salivary gland tissue engineering.

Cadherin-11 binds to PDGFRß and enhances cell proliferation and tissue regeneration via the PDGFR-AKT signaling axis.

Intercellular adhesion through homotypic interaction between cadherins regulates multiple cellular processes including cytoskeletal organization, proliferation, and survival. In this paper, we provide evidence that cadherin-11 (CDH11) binds to and promotes cell proliferation both in vitro and in vivo in synergy with the platelet-derived growth factor receptor beta (PDGFRß). Engagement of CDH11 increased the sensitivity of cells to PDGF-BB by 10- to 100-fold, resulting in rapid and sustained phosphorylation of AKT, ultimately promoting and cell proliferation and tissue regeneration. Indeed, wound healing experiments showed that healing was severely compromised in Cdh11-/- mice, as evidenced by significantly decreased proliferation, AKT phosphorylation, and extracellular matrix (ECM) synthesis of dermal cells. Our results shed light into understanding how intercellular adhesion can promote cell proliferation and may have implications for tissue regeneration and cancer progression.

Neural crest stem cells from human epidermis of aged donors maintain their multipotency in vitro and in vivo.

Neural crest (NC) cells are multipotent stem cells that arise from the embryonic ectoderm, delaminate from the neural tube in early vertebrate development and migrate throughout the developing embryo, where they differentiate into various cell lineages. Here we show that multipotent and functional NC cells can be derived by induction with a growth factor cocktail containing FGF2 and IGF1 from cultures of human inter-follicular keratinocytes (KC) isolated from elderly donors. Adult NC cells exhibited longer doubling times as compared to neonatal NC cells, but showed limited signs of cellular senescence despite the advanced age of the donors and exhibited significantly younger epigenetic age as compared to KC. They also maintained their multipotency, as evidenced by their ability to differentiate into all NC-specific lineages including neurons, Schwann cells, melanocytes, and smooth muscle cells (SMC). Notably, upon implantation into chick embryos, adult NC cells behaved similar to their embryonic counterparts, migrated along stereotypical pathways and contributed to multiple NC derivatives in ovo. These results suggest that KC-derived NC cells may provide an easily accessible, autologous source of stem cells that can be used for treatment of neurodegenerative diseases or as a model system for studying disease pathophysiology and drug development.

Synergistic effects of laminin-1 peptides, VEGF and FGF9 on salivary gland regeneration.

Hyposalivation is associated with radiation therapy, Sjögren's syndrome and/or aging, and is a significant clinical problem that decreases oral health and overall health in many patients and currently lacks effective treatment. Hence, methods to regenerate salivary glands and restore saliva secretion are urgently needed. To this end, this study describes the modification of fibrin hydrogels with a combination of laminin-1 peptides (YIGSR and A99) and human growth factors (vascular endothelial growth factor and fibroblast growth factor 9) to enhance regeneration in a salivary gland injury mouse model. Our results indicate that these fortified hydrogels enhanced angiogenesis and neurogenesis while promoting formation of acinar structures, thereby leading to enhanced saliva secretion. Such functional recovery indicates salivary gland regeneration and suggests that our technology may be useful in promoting gland regeneration and reversing hyposalivation in a clinical setting. STATEMENT OF SIGNIFICANCE: We engineered Fibrin Hydrogels (FH) to contain multiple regenerative cues including laminin-1 peptides (L1p) and growth factors (GFs). L1p and GF modified FH were used to induce salivary gland regeneration in a wounded mouse model. Treatment with L1p and GF modified FH promoted salivary epithelial tissue regeneration, vascularization, neurogenesis and healing as compared to L1p-FH or FH alone. Results indicate that L1p and GF modified FH can be used for future therapeutic applications.

Derivation of neural crest stem cells from human epidermal keratinocytes requires FGF-2, IGF-1, and inhibition of TGF-ß1.

Neural crest (NC) cells play a central role in forming the peripheral nervous system, the craniofacial skeleton, and the pigmentation of the skin during development due to their broad multilineage differentiation potential into neurons, Schwann cells, melanocytes, and mesenchymal stem cells. Recently, we identified an easily accessible source of pluripotent NC stem cells from human inter-follicular keratinocyte (KC) cultures (KC-NC). In this work, we examined specific conditions for the derivation of NC from KC cultures. More specifically, we examined the role of two growth factors, FGF2 and IGF1, in NC proliferation and in expression of two potent NC transcription factors, Sox10 and FoxD3. Using specific chemical inhibitors, we uncovered that the downstream regulatory pathways AKT/PI3K, MEK/ERK, and JNK/cJun may be critical in Sox10 and FoxD3 regulation in KC-NC. The TGF-ß1 pathway was also implicated in suppressing Sox10 expression and NC proliferation. In summary, our study shed light into the role of FGF2, IGF1, and TGF-ß1 on the induction of NC from KC cultures and the pathways that regulate Sox10 and FoxD3. We also established culture conditions for sustaining KC-NC multipotency and, therefore, the potential of these cells for regenerative medicine and cellular therapies.

Efficient and high yield isolation of myoblasts from skeletal muscle.

Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10-20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish.

NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings.

Adult skeletal muscle regeneration relies on the activity of satellite cells residing in the skeletal muscle niche. However, systemic and intrinsic factors decrease the myogenic differentiation potential of satellite cells thereby impairing muscle regeneration. Here we present data showing that late passage C2C12 myoblasts exhibited significantly impaired myogenic differentiation potential that was accompanied by impaired expression of myogenic regulatory factors (Myf5, MyoD, Myogenin, and MRF4) and members of myocyte enhancer factor 2 family. Notably, ectopic expression of NANOG preserved the morphology and restored the myogenic differentiation capacity of late passage myoblasts, possibly by restoring the expression level of these myogenic factors. Muscle regeneration was effective in 2D cultures and in 3D skeletal microtissues mimicking the skeletal muscle niche. The presence of NANOG was required for at least 15days to reverse the impaired differentiation potential of myoblasts. However, it was critical to remove NANOG during the process of maturation, as it inhibited myotube formation. Finally, myoblasts that were primed by NANOG maintained their differentiation capacity for 20days after NANOG withdrawal, suggesting potential epigenetic changes. In conclusion, these results shed light on the potential of NANOG to restore the myogenic differentiation potential of myoblasts, which is impaired after multiple rounds of cellular division, and to reverse the loss of muscle regeneration.

Laminin-111-derived peptide conjugated fibrin hydrogel restores salivary gland function.

Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as compared to animal-derived proteins. Therefore, the goal of this study was to demonstrate whether a 20 day treatment with laminin-111-derived peptide conjugated fibrin hydrogel promotes tissue regeneration in submandibular glands of a wound healing mouse model. In this study, laminin-111-derived peptide conjugated fibrin hydrogel significantly accelerated formation of salivary gland tissue. The regenerated gland tissues displayed not only structural but also functional restoration.

Reprogramming Postnatal Human Epidermal Keratinocytes Toward Functional Neural Crest Fates.

During development, neural crest (NC) cells are induced by signaling events at the neural plate border of all vertebrate embryos. Initially arising within the central nervous system, NC cells subsequently undergo an epithelial to mesenchymal transition to migrate into the periphery, where they differentiate into diverse cell types. Here we provide evidence that postnatal human epidermal keratinocytes (KC), in response to fibroblast growth factor 2 and insulin like growth factor 1 signals, can be reprogrammed toward a NC fate. Genome-wide transcriptome analyses show that keratinocyte-derived NC cells are similar to those derived from human embryonic stem cells. Moreover, they give rise in vitro and in vivo to NC derivatives such as peripheral neurons, melanocytes, Schwann cells and mesenchymal cells (osteocytes, chondrocytes, adipocytes, and smooth muscle cells). By demonstrating that human keratin-14+ KC can form NC cells, even from clones of single cells, our results have important implications in stem cell biology and regenerative medicine. Stem Cells 2017;35:1402-1415.

NANOG Reverses the Myogenic Differentiation Potential of Senescent Stem Cells by Restoring ACTIN Filamentous Organization and SRF-Dependent Gene Expression.

Cellular senescence as a result of organismal aging or progeroid diseases leads to stem cell pool exhaustion hindering tissue regeneration and contributing to the progression of age related disorders. Here we discovered that ectopic expression of the pluripotent factor NANOG in senescent or progeroid myogenic progenitors reversed cellular aging and restored completely the ability to generate contractile force. To elicit its effects, NANOG enabled reactivation of the ROCK and Transforming Growth Factor (TGF)-ß pathways-both of which were impaired in senescent cells-leading to ACTIN polymerization, MRTF-A translocation into the nucleus and serum response factor (SRF)-dependent myogenic gene expression. Collectively our data reveal that cellular senescence can be reversed and provide a novel strategy to regain the lost function of aged stem cells without reprogramming to the pluripotent state. Stem Cells 2017;35:207-221.