Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment.

A critical but molecularly uncharacterized step in heart formation and regeneration is the process that commits progenitor cells to differentiate into cardiomyocytes. Here, we show that the endoderm-derived dual Nodal/bone morphogenetic protein (BMP) antagonist Cerberus-1 (Cer1) in embryonic stem cell cultures orchestrates two signaling pathways that direct the SWI/SNF chromatin remodeling complex to cardiomyogenic loci in multipotent (KDR/Flk1+) progenitors, activating lineage-specific transcription. Transient inhibition of Nodal by Cer1 induces Brahma-associated factor 60c (Baf60c), one of three Baf60 variants (a, b, and c) that are mutually exclusively assembled into SWI/SNF. Blocking Nodal and BMP also induces lineage-specific transcription factors Gata4 and Tbx5, which interact with Baf60c. siRNA to Cer1, Baf60c, or the catalytic SWI/SNF subunit Brg1 prevented the developmental opening of chromatin surrounding the Nkx2.5 early cardiac enhancer and cardiomyocyte differentiation. Overexpression of Baf60c fully rescued these deficits, positioning Baf60c and SWI/SNF function downstream from Cer1. Thus, antagonism of Nodal and BMP coordinates induction of the myogenic Baf60c variant and interacting transcription factors to program the developmental opening of cardiomyocyte-specific loci in chromatin. This is the first demonstration that cues from the progenitor cell environment direct the subunit variant composition of SWI/SNF to remodel the transcriptional landscape for lineage-specific differentiation.

A Nodal-to-TGFß cascade exerts biphasic control over cardiopoiesis.

RATIONALE: The transforming growth factor-ß (TGFß) family member Nodal promotes cardiogenesis, but the mechanism is unclear despite the relevance of TGFß family proteins for myocardial remodeling and regeneration. OBJECTIVE: To determine the function(s) of TGFß family members during stem cell cardiogenesis. METHODS AND RESULTS: Murine embryonic stem cells were engineered with a constitutively active human type I Nodal receptor (caACVR1b) to mimic activation by Nodal and found to secrete a paracrine signal that promotes cardiogenesis. Transcriptome and gain- and loss-of-function studies identified the factor as TGFß2. Both Nodal and TGFß induced early cardiogenic progenitors in embryonic stem cell cultures at day 0 to 2 of differentiation. However, Nodal expression declines by day 4 due to feedback inhibition, whereas TGFß persists. At later stages (days 4-6), TGFß suppresses the formation of cardiomyocytes from multipotent Kdr(+) progenitors while promoting the differentiation of vascular smooth muscle and endothelial cells. CONCLUSIONS: Nodal induces TGFß, and both stimulate the formation of multipotent cardiovascular Kdr(+) progenitors. TGFß, however, becomes uniquely responsible for controlling subsequent lineage segregation by stimulating vascular smooth muscle and endothelial lineages and simultaneously blocking cardiomyocyte differentiation.

Efficient differentiation of human iPSC-derived mesenchymal stem cells to chondroprogenitor cells.

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell-based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC-derived mesenchymal-like progenitor population. We found the direct plating of undifferentiated iPSCs into high-density micromass cultures in the presence of BMP-2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP-2 treatment of iPSC-derived MSC-like (iPSC-MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage-specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell-based approaches to repair joint cartilage damage.

Differential chondrogenic differentiation between iPSC derived from healthy and OA cartilage is associated with changes in epigenetic regulation and metabolic transcriptomic signatures.

Induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine. The iPSCs exhibit a preference for lineage differentiation to the donor cell type indicating the existence of memory of origin. Although the intrinsic effect of the donor cell type on differentiation of iPSCs is well recognized, whether disease-specific factors of donor cells influence the differentiation capacity of iPSC remains unknown. Using viral based reprogramming, we demonstrated the generation of iPSCs from chondrocytes isolated from healthy (AC-iPSCs) and osteoarthritis cartilage (OA-iPSCs). These reprogrammed cells acquired markers of pluripotency and differentiated into uncommitted mesenchymal-like progenitors. Interestingly, AC-iPSCs exhibited enhanced chondrogenic potential as compared OA-iPSCs and showed increased expression of chondrogenic genes. Pan-transcriptome analysis showed that chondrocytes derived from AC-iPSCs were enriched in molecular pathways related to energy metabolism and epigenetic regulation, together with distinct expression signature that distinguishes them from OA-iPSCs. Our molecular tracing data demonstrated that dysregulation of epigenetic and metabolic factors seen in OA chondrocytes relative to healthy chondrocytes persisted following iPSC reprogramming and differentiation toward mesenchymal progenitors. Our results suggest that the epigenetic and metabolic memory of disease may predispose OA-iPSCs for their reduced chondrogenic differentiation and thus regulation at epigenetic and metabolic level may be an effective strategy for controlling the chondrogenic potential of iPSCs.

Mesenchyme-specific loss of Dot1L histone methyltransferase leads to skeletal dysplasia phenotype in mice.

Chromatin modifying enzymes play essential roles in skeletal development and bone maintenance, and deregulation of epigenetic mechanisms can lead to skeletal growth and malformation disorders. Here, we report a novel skeletal dysplasia phenotype in mice with conditional loss of Disruptor of telomeric silencing 1-like (Dot1L) histone methyltransferase in limb mesenchymal progenitors and downstream descendants. Phenotypic characterizations of mice with Dot1L inactivation by Prrx1-Cre (Dot1L-cKOPrrx1) revealed limb shortening, abnormal bone morphologies, and forelimb dislocations. Our in vivo and in vitro data support a crucial role for Dot1L in regulating growth plate chondrocyte proliferation and differentiation, extracellular matrix production, and secondary ossification center formation. Micro-computed tomography analysis of femurs revealed that partial loss of Dot1L expression is sufficient to impair trabecular bone formation and microarchitecture in young mice. Moreover, RNAseq analysis of Dot1L deficient chondrocytes implicated Dot1L in the regulation of key genes and pathways necessary to promote cell cycle regulation and skeletal growth. Collectively, our data show that early expression of Dot1L in limb mesenchyme provides essential regulatory control of endochondral bone morphology, growth, and stability.

Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting.

BACKGROUND: Tail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown. RESULTS: TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1. CONCLUSION: Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

TIMP-1 Promotes Oligodendrocyte Differentiation Through Receptor-Mediated Signaling.

The extracellular protein tissue inhibitor of metalloproteinase (TIMP)-1 is both a matrix metalloproteinase (MMP) inhibitor and a trophic factor. Mice lacking TIMP-1 exhibit delayed central nervous system myelination during postnatal development and impaired remyelination following immune-mediated injury in adulthood. We have previously determined that the trophic action of TIMP-1 on oligodendrocyte progenitor cells (OPCs) to mature into oligodendrocytes is independent of its MMP inhibitory function. However, the mechanism by which TIMP-1 promotes OPC differentiation is not known. To address this gap in our understanding, herein, we report that TIMP-1 signals via a CD63/ß1-integrin receptor complex to activate Akt (protein kinase B) to promote ß-catenin signaling in OPCs. The regulation of ß-catenin by TIMP-1 to promote OPC differentiation was counteracted, but not abrogated, by canonical signaling evoked by Wnt7a. These data provide a previously uncharacterized trophic action of TIMP-1 to regulate oligodendrocyte maturation via a CD63/ß1-integrin/Akt pathway mechanism. These findings contribute to our emerging understanding on the role of TIMP-1 as a growth factor expressed to promote CNS myelination during development and induced in the adult to promote myelin repair.

Regeneration of Articular Cartilage by Human ESC-Derived Mesenchymal Progenitors Treated Sequentially with BMP-2 and Wnt5a.

The success of cell-based therapies to restore joint cartilage requires an optimal source of reparative progenitor cells and tight control of their differentiation into a permanent cartilage phenotype. Bone morphogenetic protein 2 (BMP-2) has been extensively shown to promote mesenchymal cell differentiation into chondrocytes in vitro and in vivo. Conversely, developmental studies have demonstrated decreased chondrocyte maturation by Wingless-Type MMTV Integration Site Family, Member 5A (Wnt5a). Thus, we hypothesized that treatment of human embryonic stem cell (hESC)-derived chondroprogenitors with BMP-2 followed by Wnt5a may control the maturational progression of these cells into a hyaline-like chondrocyte phenotype. We examined the effects of sustained exposure of hESC-derived mesenchymal-like progenitors to recombinant Wnt5a or BMP-2 in vitro. Our data indicate that BMP-2 promoted a strong chondrogenic response leading to terminal maturation, whereas recombinant Wnt5a induced a mild chondrogenic response without promoting hypertrophy. Moreover, Wnt5a suppressed BMP-2-mediated chondrocyte maturation, preventing the formation of fibrocartilaginous tissue in high-density cultures treated sequentially with BMP-2 and Wnt5a. Implantation of scaffoldless pellets of hESC-derived chondroprogenitors pretreated with BMP-2 followed by Wnt5a into rat chondral defects induced an articular-like phenotype in vivo. Together, the data establish a novel role for Wnt5a in controlling the progression from multipotency into an articular-like cartilage phenotype in vitro and in vivo. Stem Cells Translational Medicine 2017;6:40-50.

iPS-derived neural progenitor cells from PPMS patients reveal defect in myelin injury response.

Primary progressive multiple sclerosis (PPMS) is a chronic demyelinating disease of the central nervous system (CNS) currently lacking any effective treatment. Promoting endogenous brain repair offers a potential strategy to halt and possibly restore neurologic function in PPMS. To understand how the microenvironment within white matter lesions plays a role in repair we have focused on neural progenitor cells (NPCs) since these are found in lesions in PPMS and have been found to influence oligodendrocyte progenitor cell maturation (OPCs). To better understand the cellular nature of NPCs in PPMS we developed iPS cells from blood samples of PPMS patients and age matched non-disease spouse or blood relative controls. Using these iPS cell lines we determined that the NPCs from PPMS cases provided no neuroprotection against active CNS demyelination compared to NPCs from control iPS lines which were capable of completely preventing injury. Conditioned media (CM) from PPMS NPCs provides no protection to OPCs and prevents maturation of OPCs into oligodendrocytes in vitro. We also found that CM from PPMS iPS NPCs elicited patient-specific differences in the response to compounds that should foster oligodendrocyte (OL) maturation. Together, these data establish a new model for understanding the nature of myelination defects in PPMS which may lead to novel targeted approaches for preventing demyelination in these patients.

Embryonic heart induction.

We have characterized two signaling pathways that induce heart tissue during embryonic development. The first is initiated by the Wnt antagonist Dickkopf1 (Dkk1) and involves the homeodomain transcription factor Hex. Other Wnt antagonists are less effective and the potency of Dkk1 might be due to synergy between Wnt antagonizing and another, novel activity emanating from its amino terminal cysteine-rich domain. The second signal is initiated by Nodal and its co-receptor Cripto. Importantly, both the Dkk1/Wnt antagonism and Nodal pathways act on the endoderm that underlies the future heart to control secretion of diffusible factors that induce cardiogenesis in adjacent mesoderm. In this article, we summarize data that Dkk1 induces cardiogenic differentiation cell non-autonomously through the action of the homeodomain transcription factor Hex. We also discuss recent data showing that Nodal also acts indirectly through stimulation of the secreted protein Cerberus, which is a member of the differential-screening selected aberrant in neuroblastoma (DAN) family of secreted proteins. Finally, we present the model that signaling from Dkk1 regulates novel activities, in addition to Wnt antagonism, which are essential for progression beyond initiation of cardiogenesis to control later stages of cardiomyocyte differentiation and myocardial tissue organization.

Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity.

The bHLH transcription factor Twist1 has emerged as a negative regulator of chondrogenesis in skeletal progenitor cells and as an inhibitor of maturation in growth plate chondrocytes. However, its role in articular cartilage remains obscure. Here we examine Twist1 expression during re-differentiation of expanded human articular chondrocytes, the distribution of Twist1 proteins in normal versus OA human articular cartilage, and its role in modulating OA development in mice. High levels of Twist1 transcripts were detected by qPCR analyses of expanded de-differentiated human articular chondrocytes that had acquired mesenchymal-like features. The induction of hallmark cartilage genes by Bmp-2 mediated chondrogenic differentiation was paralleled by the dramatic suppression of Twist1 in vitro. In normal human articular cartilage, Twist1-expressing chondrocytes were most abundant in the superficial zone with little to no expression in the middle and deep zones. However, our analyses revealed a higher proportion of deep zone articular chondrocytes expressing Twist1 in human OA cartilage as compared to normal articular cartilage. Moreover, Twist1 expression was prominent within proliferative cell clusters near fissure sites in more severely affected OA samples. To assess the role of Twist1 in OA pathophysiology, we subjected wild type mice and transgenic mice with gain of Twist1 function in cartilage to surgical destabilization of the medial meniscus. At 12 weeks post-surgery, micro-CT and histological analyses revealed attenuation of the OA phenotype in Twist1 transgenic mice compared to wild type mice. Collectively, the data reveal a role for Twist in articular cartilage maintenance and the attenuation of cartilage degeneration.

Regulated expression and temporal induction of the tail-anchored sarcolemmal-membrane-associated protein is critical for myoblast fusion.

Sarcolemmal-membrane-associated proteins (SLMAPs) define a new class of coiled-coil tail-anchored membrane proteins generated by alternative splicing mechanisms. An in vivo expression analysis indicated that SLMAPs are present in somites (11 days post-coitum) as well as in fusing myotubes and reside at the level of the sarcoplasmic reticulum and transverse tubules in adult skeletal muscles. Skeletal-muscle myoblasts were found to express a single 5.9 kb transcript, which encodes the full-length approximately 91 kDa SLMAP3 isoform. Myoblast differentiation was accompanied by the stable expression of the approximately 91 kDa SLMAP protein as well as the appearance of an approximately 80 kDa isoform. Deregulation of SLMAPs by ectopic expression in myoblasts resulted in a potent inhibition of fusion without affecting the expression of muscle-specific genes. Membrane targeting of the de-regulated SLMAPs was not critical for the inhibition of myotube development. Protein-protein interaction assays indicated that SLMAPs are capable of self-assembling, and the de-regulated expression of mutants that were not capable of forming SLMAP homodimers also inhibited myotube formation. These results imply that regulated levels and the temporal induction of SLMAP isoforms are important for normal muscle development.

Differentiation of Human Induced Pluripotent Stem Cells to Chondrocytes.

Human induced pluripotent stem (iPS) cells are relevant tools for modeling human skeletal development and disease, and represent a promising source of patient-specific cells for the regeneration of skeletal tissue, such as articular cartilage. Devising efficient and reproducible strategies, which closely mimic the physiological chondrogenic differentiation process, will be necessary to generate functional chondrocytes from human iPS cells. Our previous study demonstrated the generation of chondrogenically committed human iPS cells via the enrichment of a mesenchymal-like progenitor population, application of appropriate high-density culture conditions, and stimulation with bone morphogenetic protein-2 (Bmp-2). The differentiated iPS cells showed temporal expression of cartilage genes and the accumulation of a cartilaginous extracellular matrix in vitro. In this chapter, we provide detailed methodologies for the differentiation of human iPS cells to the chondrogenic lineage and describe protocols for the analysis of chondrogenic differentiation.

Derivation and Chondrogenic Commitment of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors.

The induction of human embryonic stem cells to a mesenchymal-like progenitor population constitutes a developmentally relevant approach for efficient directed differentiation of human embryonic stem (hES) cells to the chondrogenic lineage. The initial enrichment of a hemangioblast intermediate has been shown to yield a replenishable population of highly purified progenitor cells that exhibit the typical mesenchymal stem cell (MSC) surface markers as well as the capacity for multilineage differentiation to bone, fat, and cartilage. Herein, we provide detailed methodologies for the derivation and characterization of potent mesenchymal-like progenitors from hES cells and describe in vitro assays for bone morphogenetic protein (BMP)-2-mediated differentiation to the chondrogenic lineage.

Establishment of human cell type-specific iPS cells with enhanced chondrogenic potential.

The propensity of induced pluripotent stem (iPS) cells to differentiate into specific lineages may be influenced by a number of factors, including the selection of the somatic cell type used for reprogramming. Herein we report the generation of new iPS cells, which we derived from human articular chondrocytes and from cord blood mononucleocytes via lentiviral-mediated delivery of Oct4, Klf4, Sox2, and cMyc. Molecular, cytochemical, and cytogenic analyses confirmed the acquisition of hallmark features of pluripotency, as well as the retention of normal karyotypes following reprogramming of both the human articular chondrocytes (AC) and the cord blood (CB) cells. In vitro and in vivo functional analyses formally established the pluripotent differentiation capacity of all cell lines. Chondrogenic differentiation assays comparing iPS cells derived from AC, CB, and a well established dermal fibroblast cell line (HDFa-Yk26) identified enhanced proteoglycan-rich matrix formation and cartilage-associated gene expression from AC-derived iPS cells. These findings suggest that the tissue of origin may impact the fate potential of iPS cells for differentiating into specialized cell types, such as chondrocytes. Thus, we generated new cellular tools for the identification of inherent features driving high chondrogenic potential of reprogrammed cells.

Persistent expression of Twist1 in chondrocytes causes growth plate abnormalities and dwarfism in mice.

Evidence from various in vitro gain and loss of function studies indicate that the bHLH transcription factor Twist1 negatively regulates chondrocyte differentiation; however limited information regarding Twist1 function in postnatal cartilage development and maintenance is available. Twist1 expression within the postnatal growth plate is restricted to immature, proliferating chondrocytes, and is significantly decreased or absent in hypertrophic chondrocytes. In order to examine the effect of maintaining the expression of Twist1 at later stages of chondocyte differentiation, we used type II collagen Cre (Col2-Cre) mice to activate a Cre-inducible Twist1 transgene specifically in chondrocytes (Col2-Twist1). At two weeks, postnatal growth was inhibited in Col2-Twist1 mice, as evidenced by limb shortening. Histological examination revealed abnormal growth plate structure, characterized by poor columnar organization of proliferating cartilaginous cells, decreased cellularity, and expansion of the hypertrophic zone. Moreover, structural defects within the growth plates of Col2-Twist1 transgenic mice included abnormal vascular invasion and focal regions of bony formation. Quantitative analysis of endochondral bone formation via micro-computed topography revealed impaired trabecular bone formation in the hindlimbs of Col2-Twist1 transgenic mice at various timepoints of postnatal development. Taken together, these findings indicate that regulated Twist1 expression contributes to growth plate organization and endochondral ossification to modulate postnatal longitudinal bone growth.

Molecular properties of cardiac tail-anchored membrane protein SLMAP are consistent with structural role in arrangement of excitation-contraction coupling apparatus.

The spatial arrangement of the cell-surface membranes (sarcolemma and transverse tubules) and internal membranes of the sarcoplasmic reticulum relative to the myofibril is critical for effective excitation-contraction (E-C) coupling in cardiac myocytes; however, the molecular determinants of this order remain to be defined. Here, we ascribe molecular and cellular properties to the coiled-coil, tail-anchored sarcolemmal membrane-associated protein (SLMAP) that are consistent with a potential role in organizing the E-C coupling apparatus of the cardiomyocyte. The expression of SLMAP was developmentally regulated and its localization was distinctly apparent at the level of the membranes involved in regulating the E-C coupling mechanism. Several SLMAP isoforms were expressed in the cardiac myocyte with unique COOH-terminal membrane anchors that could target this molecule to distinct subcellular membranes. Protein interaction analysis indicated that SLMAPs could self assemble and bind myosin in cardiac muscle. The cardiac-specific expression of SLMAP isoforms that can be targeted to distinct subcellular membranes, self assemble, and interact with the myofibril suggests a potential role for this molecule in the structural arrangement of the E-C coupling apparatus.

A novel isoform of sarcolemmal membrane-associated protein (SLMAP) is a component of the microtubule organizing centre.

The microtubule organizing centre (MTOC) or the centrosome serves a crucial role in the establishment of cellular polarity, organization of interphase microtubules and the formation of the bipolar mitotic spindle. We have elucidated the genomic structure of a gene encoding the sarcolemmal membrane-associated protein (SLMAP), which encodes a 91 kDa polypeptide with a previously uncharacterized N-terminal sequence encompassing a forkhead-associated (FHA) domain that resides at the centrosome. Anti-peptide antibodies directed against SLMAP N-terminal sequences showed colocalization with gamma-tubulin at the centrosomes at all phases of the cell cycle. Agents that specifically disrupt microtubules did not affect SLMAP association with centrosomes. Furthermore, SLMAP sequences directed a reporter green fluorescent protein (GFP) to the centrosome, and deletions of the newly identified N-terminal sequence from SLMAP prevented the centrosomal targeting. Deletion-mutant analysis concluded that overall, structural determinants in SLMAP were responsible for centrosomal targeting. Elevated levels of centrosomal SLMAP were found to be lethal, whereas mutants that lacked centrosomal targeting inhibited cell growth accompanied by an accumulation of cells at the G2/M phase of the cell cycle.