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The p53 transcription factor is a master regulator of cellular responses to stress that is commonly inactivated in diverse cancer types. Despite decades of research, the mechanisms by which p53 impedes tumorigenesis across vastly different cellular contexts requires further investigation. The bulk of research has been completed using in vitro studies of cancer cell lines or in vivo studies in mouse models, but much less is known about p53 action in diverse non-transformed human tissues. Here, we investigated how different cellular states modify the p53 transcriptional program in human cells through a combination of computational analyses of publicly available large-scale datasets and in vitro studies using an isogenic system consisting of induced pluripotent stem cells (iPSCs) and two derived lineages. Analysis of publicly available mRNA expression and genetic dependency data demonstrated wide variation in terms of expression and function of a core p53 transcriptional program across various tissues and lineages. To monitor the impact of cell differentiation on the p53 transcriptome within an isogenic cell culture system, we activated p53 by pharmacological inhibition of its negative regulator MDM2. Using cell phenotyping assays and genome wide transcriptome analyses, we demonstrated that cell differentiation confines and modifies the p53 transcriptional network in a lineage-specific fashion. Although hundreds of p53 target genes are transactivated in iPSCs, only a small fraction is transactivated in each of the differentiated lineages. Mechanistic studies using small molecule inhibitors and genetic knockdowns revealed the presence of two major regulatory mechanisms contributing to this massive heterogeneity across cellular states: gene silencing by epigenetic regulatory complexes and constitutive transactivation by lineage-specific transcription factors. Altogether, these results illuminate the impact of cell differentiation on the p53 program, thus advancing our understanding of how this tumor suppressor functions in different contexts.

SummaryBackgroundGene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies.MethodsWe developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar- free,COL7A1corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing.FindingsiPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT- treated mice.InterpretationDEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

Endothelial cells (ECs) are important components of the circulatory system. These cells can be used for in vitro modeling of cardiovascular diseases and in regenerative medicine to promote vascularization of engineered tissue constructs. However, low proliferative capacity and patient-to-patient variability limit the use of primary ECs in the clinic and disease modeling. ECs differentiated from human induced pluripotent stem cells (iPSCs) can serve as a viable alternative to primary ECs for these applications. This is because human iPSCs can proliferate indefinitely and have the potential to differentiate into a variety of somatic cell lines, providing a renewable source of patient-specific cells. Here, we present an optimized, highly reproducible method for the differentiation of human iPSCs toward vascular ECs. The protocol relies on the activation of the WNT signaling pathway and the use of growth factors and small molecules. The resulting iPSC-derived ECs can be cultured for multiple passages without losing their functionality and are suitable for both in vitro and in vivo studies.

Age‐related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen‐associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen‐associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely‐tuned mechanism regulating directional apical:basal sorting and secretion of drusen‐associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE‐derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.

Human induced pluripotent stem cells (iPSCs) have emerged as a promising alternative to bone-marrow derived mesenchymal stem/stromal cells for cartilage tissue engineering. However, the effect of biochemical and mechanical cues on iPSC chondrogenesis remains understudied. This study evaluated chondrogenesis of induced pluripotent mesenchymal progenitor cells (iPS-MPs) encapsulated in a cartilage-mimetic hydrogel under different culture conditions: free swelling versus dynamic compressive loading and different growth factors (TGFβ3 and/or BMP2). Human iPSCs were differentiated into iPS-MPs and chondrogenesis was evaluated by gene expression (qPCR) and protein expression (immunohistochemistry) after three weeks. In pellet culture, both TGFβ3 and BMP2 were required to promote chondrogenesis. However, the hydrogel in growth factor-free conditions promoted chondrogenesis, but rapidly progressed to hypertrophy. Dynamic loading in growth factor-free conditions supported chondrogenesis, but delayed the transition to hypertrophy. Findings were similar with TGFβ3, BMP2, and TGFβ3 + BMP2. Dynamic loading with TGFβ3, regardless of BMP2, was the only condition that promoted a stable chondrogenic phenotype (aggrecan + collagen II) accompanied by collagen X down-regulation. Positive TGFβRI expression with load-enhanced Smad2/3 signaling and low SMAD1/5/8 signaling was observed. In summary, this study reports a promising cartilage-mimetic hydrogel for iPS-MPs that when combined with appropriate biochemical and mechanical cues induces a stable chondrogenic phenotype.

Differentiating human induced pluripotent stem cells (iPSCs) into multipotent mesenchymal stem/stromal cells (MSCs) offers a renewable source of therapeutically invaluable cells. However, the process of MSC derivation from iPSCs suffers from an undesirably low efficiency. In this chapter, we present an optimized procedure to produce MSCs from human iPSCs with a high efficiency. The protocol depends on the generation of embryoid bodies (EBs) and requires the treatment of EBs with transforming growth factor beta 1 (TGF-β1). The resulting MSCs can be purified based on the expression of CD73, CD105, and CD90 markers and expanded for multiple passages without losing their characteristics.

Differentiating human induced pluripotent stem cells (iPSCs) into multipotent mesenchymal stem/stromal cells (MSCs) offers a renewable source of therapeutically invaluable cells. However, the process of MSC derivation from iPSCs suffers from an undesirably low efficiency. In this chapter, we present an optimized procedure to produce MSCs from human iPSCs with a high efficiency. The protocol depends on the generation of embryoid bodies (EBs) and requires the treatment of EBs with transforming growth factor beta 1 (TGF-β1). The resulting MSCs can be purified based on the expression of CD73, CD105, and CD90 markers and expanded for multiple passages without losing their characteristics.

Solute carrier family 6 member 1 (SLC6A1) is abundantly expressed in the developing brain even before the central nervous system is formed. Its encoded GABA transporter 1 is responsible for the reuptake of GABA into presynaptic neurons and glia, thereby modulating neurotransmission. GABA transporter 1 is expressed globally in the brain, in both astrocytes and neurons. The GABA uptake function of GABA transporter 1 in neurons cannot be compensated for by other GABA transporters, while the function in glia can be partially replaced by GABA transporter 3. Recently, many variants in SLC6A1 have been associated with a spectrum of epilepsy syndromes and neurodevelopmental disorders, including myoclonic atonic epilepsy, childhood absence epilepsy, autism, and intellectual disability, but the patho-mechanisms associated with these phenotypes remain unclear. The presence of GABA transporter 1 in both neurons and astrocytes further obscures the role of abnormal GABA transporter 1 in the heterogenous disease phenotype manifestations. Here we examine the impact on transporter trafficking and function of twenty-two SLC6A1 variants identified in patients with a broad spectrum of phenotypes. We also evaluate changes in protein expression and subcellular localization of the variant GABA transporter 1 in various cell types, including neurons and astrocytes derived from human patient induced pluripotent stem cells. We found that a partial or complete loss of function represents a common disease mechanism, although the extent of GABA uptake reduction is variable. The reduced GABA uptake appears to be due to reduced cell surface expression of the variant transporter caused by variant protein misfolding, endoplasmic reticulum retention, and subsequent degradation. Although the extent of reduction of the total protein, surface protein, and the GABA uptake level of the variant transporters is variable, the loss of GABA uptake function and endoplasmic reticulum retention is consistent across induced pluripotent stem cell-derived cell types, including astrocytes and neurons, for the surveyed variants. Interestingly, we did not find a clear correlation of GABA uptake function and the disease phenotypes, such as myoclonic atonic epilepsy vs developmental delay, in this study. Together, our study suggests that impaired transporter protein trafficking and surface expression are the major disease-associated mechanisms associated with pathogenic SLC6A1 variants. Our results resemble findings from pathogenic variants in other genes affecting the GABA pathway, such as GABAA receptors. This study provides critical insight into therapeutic developments for SLC6A1 variant-mediated disorders and implicates that boosting transporter function by either genetic or pharmacologic approaches would be beneficial.

The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. Here, we describe a protocol that provides a straightforward method for introducing a stable, highly expressed fluorescent protein into iPSCs using the CRISPR/Cas9 system and a standardized donor vector. The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.

Reprogramming a patient’s somatic cells into induced pluripotent stem cells (iPSCs) holds great promise for disease modeling and the development of autologous cellular therapeutics. However, it remains challenging to consistently reprogram primary human cells, as they are frequently aged, diseased, or in low abundance. Here we present a modified highly efficient and clinically relevant RNA-based method for reprogramming disease-associated and other difficult-to-reprogram human primary fibroblast lines into iPSCs. We also describe optimizations that can be employed for consistent reprogramming of these difficult-to-reprogram cells. With the provided protocol, integration-free iPSC lines can be successfully generated from a small number of primary human fibroblasts in approximately 5–7 weeks.

Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide-binding mode, however, remains unclear. In this work, we addressed this key question using a "clogged" Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide-binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide-binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide-binding mode in other vesicle-fusion pathways.

SUMMARYHuman trisomy 21 (Down syndrome) is the most common genetic cause of intellectual disability, and is associated with complex perturbations in protein expression during development. Brain region-specific alterations in neuronal density and composition originate prenatally in trisomy 21 individuals, and are presumed to underlie the intellectual disability and early onset neurodegeneration that characterizes Down syndrome. However, the mechanisms by which chromosome 21 aneuploidy drives alterations in the central nervous system are not well understood, particularly in brain regions that are uniquely human and thus inaccessible to established animal models. Cerebral organoids are pluripotent stem cell derived models of prenatal brain development that have been used to deepen our understanding of the atypical processes associated with human neurobiological disorders, and thus provide a promising avenue to explore the molecular basis for neurodevelopmental alterations in trisomy 21. Here, we employ high-resolution label-free mass spectrometry to map proteomic changes over the course of trisomy 21 cerebral organoid development, and evaluate the proteomic alterations in response to treatment with harmine, a small molecule inhibitor of the chromosome 21 encoded protein kinase DYRK1A. Our results reveal trisomy 21 specific dysregulation of networks associated with neurogenesis, axon guidance and extracellular matrix remodeling. We find significant overlap of these networks show significant overlap with previously identified dysregulated gene expression modules identified in trisomy 21 fetal brain tissue. We show that harmine leads to partial normalization of key regulators of cortical development, including WNT7A and the transcription factors TBR1, BCL11A, and POU3F2, pointing to a causative role for DYRK1A over-expression in neurodevelopmental effects of human trisomy 21.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts. As a result, we can now generate up to 4,019 iPSC colonies from only 500 starting human primary neonatal fibroblasts and reprogram up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant, integration-free iPSCs from a variety of human patient’s fibroblasts under feeder-free conditions and can be applicable for the clinical translation of iPSCs and studying the biology of reprogramming., Induced pluripotent stem cells (iPSCs) have potential for regenerative medicine applications, but are generated with very low efficiency. Here, the authors show highly efficient reprogramming of human primary fibroblasts to iPSCs via the synergistic activity of synthetic modified mRNAs, mature miRNA mimics, and optimized culture methods.

Environment plays a crucial role in the differentiation of cells during embryogenesis and defines the identity of stem cells and progenitor populations in the developing organism. Here, we will discuss our current understanding of external and internal factors affecting the development of the skin, epidermal progenitors and epidermal stem cell niches, including cell–cell interactions, signaling pathways, extracellular matrix and mechanophysical properties of the microenvironment.

Cell competition in the skin promotes tissue youthfulness, but the same process later leads to skin ageing. This finding helps to explain how skin is maintained, and uncovers potential anti-ageing therapeutic targets. Cell competition drives skin youthfulness but also skin ageing.

The hedgehog (Hh) signaling pathway plays fundamental roles during embryonic development and tumorigenesis. Previously, we have shown that ablation of the tumor suppressor and negative regulator, Suppressor of fused (Sufu), within this pathway causes embryonic lethality around E9.5 in the mouse. In this study, we examine how lack of Sufu influences early cell fate determination processes. We established embryonic stem cell (ESC) lines from preimplantation Sufu(-/-) and wild-type mouse embryos and show that these ESCs express the typical pluripotency markers, alkaline phosphatase, SSEA-1, Oct4, Sox2, and Nanog. We demonstrate that these ESCs express all core Hh pathway components and that glioma-associated protein (Gli)1 mRNA levels are increased in Sufu(-/-) ESCs. Upon spontaneous differentiation of Sufu(-/-) ESCs into embryoid bodies (EBs) in vitro, the Hh pathway is strongly upregulated as indicated by an increase in both Gli1 and patched1 (Ptch1) gene expression. Interestingly, developing Sufu(-/-) EBs were smaller than their wild-type counterparts and showed decreased expression of the ectodermal markers, Fgf5 and Sox1. In vivo teratoma formation revealed that Sufu(-/-) ESCs have a limited capacity for differentiation as the resulting tumors lacked the mesodermal derivatives, cartilage and bone. However, Sufu(-/-) ESCs were able to develop into chondrocytes and osteocytes in vitro, which suggests a differential response of ESCs compared with in vivo conditions. Our findings suggest a regulatory function of the Hh signaling pathway in early mesodermal cell fate determination and emphasize the role of Sufu as a key molecule in this process.

The extracellular matrix (ECM) controls keratinocyte proliferation, migration, and differentiation through β-integrin signaling. Wound-healing research requires expanding cells in vitro while maintaining replicative capacity; however, early terminal differentiation under traditional culture conditions limits expansion. Here, a design of experiments approach identifies poly(ethylene glycol)-based hydrogel formulations with mechanical properties (elastic modulus, E = 20.9 ± 0.56 kPa) and bioactive peptide sequences that mimic the epidermal ECM. These hydrogels enable systematic investigation of the influence of cell-binding domains from fibronectin (RGDS), laminin (YIGSR), and collagen IV (HepIII) on keratinocyte stemness and β1 integrin expression. Quantification of 14-day keratin protein expression shows four hydrogels improve stemness compared to standard techniques. Three hydrogels increase β1 integrin expression, demonstrating a positive linear relationship between stemness and β1 integrin expression. Multifactorial statistical analysis predicts an optimal peptide combination ([RGDS] = 0.67 mm, [YIGSR] = 0.13 mm, and [HepIII] = 0.02 mm) for maintaining stemness in vitro. Best-performing hydrogels exhibit no decrease in Ki-67-positive cells compared to standards (15% decrease, day 7 to 14; p < 0.05, Tukey Test). These data demonstrate that precisely designed hydrogel biomaterials direct integrin expression and promote proliferation, improving the regenerative capability of cultured keratinocytes for basic science and translational work.

Human lung research has made remarkable progress over the last century largely through the use of animal models of disease. The challenge for the future is to translate these findings into human disease and bring about meaningful disease modification or even cure. The ability to generate transformative therapies in the future will require human tissue, currently scarce under the best of circumstances. Unfortunately, patient-derived somatic cells are often poorly characterized and have a limited life span in culture. Moreover, these cells are frequently obtained from patients with end-stage disease exposed to multiple drug therapies, leaving researchers with questions about whether their findings recapitulate disease-initiating processes or are simply the result of pharmacological intervention or subsequent host responses. The goal of studying early disease in multiple cell and tissue types has driven interest in the use of induced pluripotent stem cells (iPSCs) to model lung disease. These cells provide an alternative model for relevant lung research and hold promise in particular for studying the initiation of disease processes in genetic conditions such as heritable pulmonary arterial hypertension as well as other lung diseases. In this Perspective, we focus on potential iPSC use in pulmonary vascular disease research as a model for iPSC use in many types of advanced lung disease.

Understanding differences in gene expression that increase risk for pulmonary arterial hypertension (PAH) is essential to understanding the molecular basis for disease. Previous studies on patient samples were limited by end-stage disease effects or by use of nonadherent cells, which are not ideal to model vascular cells in vivo. These studies addressed the hypothesis that pathological processes associated with PAH may be identified via a genetic signature common across multiple cell types. Expression array experiments were initially conducted to analyze cell types at different stages of vascular differentiation (mesenchymal stromal and endothelial) derived from PAH patient-specific induced pluripotent stem (iPS) cells. Molecular pathways that were altered in the PAH cell lines were then compared with those in fibroblasts from 21 patients, including those with idiopathic and heritable PAH. Wnt was identified as a target pathway and was validated in vitro using primary patient mesenchymal and endothelial cells. Taken together, our data suggest that the molecular lesions that cause PAH are present in all cell types evaluated, regardless of origin, and that stimulation of the Wnt signaling pathway was a common molecular defect in both heritable and idiopathic PAH.

Recent advances in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) offer the possibility of developing new therapeutic approaches for the treatment of a variety of diseases, including inherited skin disorders. While the ultimate goal is the use of iPSCs in the treatment of human diseases, extensive research is still required with preclinical mouse models before iPSC technology can be introduced into the clinic. Therefore, the methodology for the derivation of multipotent keratinocytes from mouse iPSCs is of particular importance since it may allow for the assessment of the feasibility of using iPSCs in the treatment of inherited skin disorders using mouse models which mimic these diseases. Here, we describe two alternative protocols for the efficient differentiation of mouse iPSCs into functional keratinocytes capable of reconstituting a normal stratified epidermis, hair follicles, and sebaceous glands when grafted onto mice. Each protocol results in a different yield and efficiency of keratinocyte derivation depending on the mouse genetic background used in the study. Both protocols employ applications of retinoic acid and bone-morphogenetic protein-4 and growth on collagen type IV-coated dishes to induce iPSC differentiation toward a keratinocyte lineage.

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides an opportunity to develop novel personalized treatment options for numerous diseases and to advance current approaches for cell-based drug discoveries and disease modeling. The ability to differentiate iPSCs into relevant cell types is an important prerequisite for the successful development of iPSC-based treatment and modeling strategies. Here, we describe a protocol for the efficient differentiation of human iPSCs into functional keratinocytes. The protocol employs treating iPSCs with retinoic acid and bone-morphogenetic protein-4 to induce differentiation toward a keratinocyte lineage, which is then followed by the growth of differentiated iPSCs on collagen type I- and collagen type IV-coated dishes to enrich for iPSC-derived keratinocytes.

Recent advances in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) offer the possibility of developing new therapeutic approaches for the treatment of a variety of diseases, including inherited skin disorders. While the ultimate goal is the use of iPSCs in the treatment of human diseases, extensive research is still required with preclinical mouse models before iPSC technology can be introduced into the clinic. Therefore, the methodology for the derivation of multipotent keratinocytes from mouse iPSCs is of particular importance since it may allow for the assessment of the feasibility of using iPSCs in the treatment of inherited skin disorders using mouse models which mimic these diseases. Here, we describe two alternative protocols for the efficient differentiation of mouse iPSCs into functional keratinocytes capable of reconstituting a normal stratified epidermis, hair follicles, and sebaceous glands when grafted onto mice. Each protocol results in a different yield and efficiency of keratinocyte derivation depending on the mouse genetic background used in the study. Both protocols employ applications of retinoic acid and bone-morphogenetic protein-4 and growth on collagen type IV-coated dishes to induce iPSC differentiation toward a keratinocyte lineage.

Reprogramming somatic cells into an ESC-like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8–12 weeks. Differentiation was assessed by lineage-specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS-derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage-specific genes. Isolated implants of iPS cell-derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease.

In a recent study in Nature, Bonfanti et al. (2010) report that the skin microenvironment can convert thymic epithelial cells into skin stem cells. This finding suggests that somatic stem cells may have a broader potential for lineage switching than previously thought.