Regenerative capacity of human pluripotent stem cell-derived articular chondrocytes in vitro.

The ideal cell source for articular cartilage repair remains elusive. Using developmentally inspired differentiation protocols, we induced human pluripotent stem cells (hPSCs) toward articular chondrocytes capable of joint cartilage repair in rodent models, which were distinct from growth plate chondrocytes, fated to be replaced by bone in vivo. Working toward clinical translation, we demonstrated controlled differentiation into chondrocytes by comprehensive gene expression analysis at each step of the differentiation. Articular chondrocytes derived from hPSCs could be expanded several passages in vitro without losing chondrogenic potential. Furthermore, chondrocytes isolated from these articular cartilage tissues had the potential to serially regenerate new articular and growth plate cartilage tissues. Finally, the ability to cryopreserve articular chondrocytes with the desired phenotype is critical for clinical translation and here we report no loss in cell viability or regenerative potential following cryopreservation. These results support the immense potential of hPSC-derived articular chondrocytes as a cell-based therapy for cartilage repair.

Multi-omic single cell sequencing: Overview and opportunities for kidney disease therapeutic development.

Single cell sequencing technologies have rapidly advanced in the last decade and are increasingly applied to gain unprecedented insights by deconstructing complex biology to its fundamental unit, the individual cell. First developed for measurement of gene expression, single cell sequencing approaches have evolved to allow simultaneous profiling of multiple additional features, including chromatin accessibility within the nucleus and protein expression at the cell surface. These multi-omic approaches can now further be applied to cells in situ, capturing the spatial context within which their biology occurs. To extract insights from these complex datasets, new computational tools have facilitated the integration of information across different data types and the use of machine learning approaches. Here, we summarize current experimental and computational methods for generation and integration of single cell multi-omic datasets. We focus on opportunities for multi-omic single cell sequencing to augment therapeutic development for kidney disease, including applications for biomarkers, disease stratification and target identification.

Lineage-specific differences and regulatory networks governing human chondrocyte development.

To address large gaps in our understanding of the molecular regulation of articular and growth plate cartilage development in humans, we used our directed differentiation approach to generate these distinct cartilage tissues from human embryonic stem cells. The resulting transcriptomic profiles of hESC-derived articular and growth plate chondrocytes were similar to fetal epiphyseal and growth plate chondrocytes, with respect to genes both known and previously unknown to cartilage biology. With the goal to characterize the regulatory landscapes accompanying these respective transcriptomes, we mapped chromatin accessibility in hESC-derived chondrocyte lineages, and mouse embryonic chondrocytes, using ATAC-sequencing. Integration of the expression dataset with the differentially accessible genomic regions revealed lineage-specific gene regulatory networks. We validated functional interactions of two transcription factors (TFs) (RUNX2 in growth plate chondrocytes and RELA in articular chondrocytes) with their predicted genomic targets. The maps we provide thus represent a framework for probing regulatory interactions governing chondrocyte differentiation. This work constitutes a substantial step towards comprehensive and comparative molecular characterizations of distinct chondrogenic lineages and sheds new light on human cartilage development and biology.

Reactivation of a developmental Bmp2 signaling center is required for therapeutic control of the murine periosteal niche.

Two decades after signals controlling bone length were discovered, the endogenous ligands determining bone width remain unknown. We show that postnatal establishment of normal bone width in mice, as mediated by bone-forming activity of the periosteum, requires BMP signaling at the innermost layer of the periosteal niche. This developmental signaling center becomes quiescent during adult life. Its reactivation however, is necessary for periosteal growth, enhanced bone strength, and accelerated fracture repair in response to bone-anabolic therapies used in clinical orthopedic settings. Although many BMPs are expressed in bone, periosteal BMP signaling and bone formation require only Bmp2 in the Prx1-Cre lineage. Mechanistically, BMP2 functions downstream of Lrp5/6 pathway to activate a conserved regulatory element upstream of Sp7 via recruitment of Smad1 and Grhl3. Consistent with our findings, human variants of BMP2 and GRHL3 are associated with increased risk of fractures.

The Role of Bmp2 in the Maturation and Maintenance of the Murine Knee Joint.

Bone morphogenetic proteins (BMPs) are key regulators of skeletal development, growth, and repair. Although BMP signaling is required for synovial joint formation and is also involved in preserving joint function after birth, the role of specific BMP ligands in adult joint homeostasis remains unclear. The purpose of this study was to define the role of Bmp2 in the morphogenesis and maintenance of the knee joint. To do this, we first created Bmp2-LacZ and Gdf5-LacZ knock-in mice and compared their expression patterns in the developing and postnatal murine knee joint. We then generated a knockout mouse model using the Gdf5-cre transgene to specifically delete Bmp2 within synovial joint-forming cells. Joint formation, maturation, and homeostasis were analyzed using histology, immunohistochemistry, qRT-PCR, and atomic force microscopy (AFM)-based nanoindentation to assess the cellular, molecular, and biomechanical changes in meniscus and articular cartilage. Bmp2 is expressed in the articular cartilage and meniscus of the embryonic and adult mouse knee in a pattern distinct from Gdf5. The knee joints of the Bmp2 knockout mice form normally but fail to mature properly. In the absence of Bmp2, the extracellular matrix and shape of the meniscus are altered, resulting in functional deficits in the meniscus and articular cartilage that lead to a progressive osteoarthritis (OA) like knee pathology as the animals age. These findings demonstrate that BMP activity provided by Bmp2 is required for the maturation and maintenance of the murine knee joint and reveal a unique role for Bmp2 that is distinct from Gdf5 in knee joint biology. © 2018 American Society for Bone and Mineral Research.

Impact of broad regulatory regions on Gdf5 expression and function in knee development and susceptibility to osteoarthritis.

OBJECTIVES: Given the role of growth and differentiation factor 5 (GDF5) in knee development and osteoarthritis risk, we sought to characterise knee defects resulting from Gdf5 loss of function and how its regulatory regions control knee formation and morphology. METHODS: The brachypodism (bp) mouse line, which harbours an inactivating mutation in Gdf5, was used to survey how Gdf5 loss of function impacts knee morphology, while two transgenic Gdf5 reporter bacterial artificial chromosome mouse lines were used to assess the spatiotemporal activity and function of Gdf5 regulatory sequences in the context of clinically relevant knee anatomical features. RESULTS: Knees from homozygous bp mice (bp/bp) exhibit underdeveloped femoral condyles and tibial plateaus, no cruciate ligaments, and poorly developed menisci. Secondary ossification is also delayed in the distal femur and proximal tibia. bp/bp mice have significantly narrower femoral condyles, femoral notches and tibial plateaus, and curvier medial femoral condyles, shallower trochlea, steeper lateral tibial slopes and smaller tibial spines. Regulatory sequences upstream from Gdf5 were weakly active in the prenatal knee, while downstream regulatory sequences were active throughout life. Importantly, downstream but not upstream Gdf5 regulatory sequences fully restored all the key morphological features disrupted in the bp/bp mice. CONCLUSIONS: Knee morphology is profoundly affected by Gdf5 absence, and downstream regulatory sequences mediate its effects by controlling Gdf5 expression in knee tissues. This downstream region contains numerous enhancers harbouring human variants that span the osteoarthritis association interval. We posit that subtle alterations to morphology driven by changes in downstream regulatory sequence underlie this locus' role in osteoarthritis risk.

A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal.

Growth and differentiation factors (GDFs) are secreted signaling molecules within the BMP family that have critical roles in joint morphogenesis during skeletal development in mice and humans. Using genetic data obtained from a six-generation Chinese family, we identified a missense variant in GDF6 (NP_001001557.1; p.Y444N) that fully segregates with a novel autosomal dominant synostoses (SYNS) phenotype, which we designate as SYNS4. Affected individuals display bilateral wrist and ankle deformities at birth and progressive conductive deafness after age 40 years. We find that the Y444N variant affects a highly conserved residue of GDF6 in a region critical for binding of GDF6 to its receptor(s) and to the BMP antagonist NOG, and show that this mutant GDF6 is a more potent stimulator of the canonical BMP signaling pathway compared with wild-type GDF6. Further, we determine that the enhanced BMP activity exhibited by mutant GDF6 is attributable to resistance to NOG-mediated antagonism. Collectively, our findings indicate that increased BMP signaling owing to a GDF6 gain-of-function mutation is responsible for loss of joint formation and profound functional impairment in patients with SYNS4. More broadly, our study highlights the delicate balance of BMP signaling required for proper joint morphogenesis and reinforces the critical role of BMP signaling in skeletal development.

Dynamics and cellular localization of Bmp2, Bmp4, and Noggin transcription in the postnatal mouse skeleton.

Transcription of BMPs and their antagonists in precise spatiotemporal patterns is essential for proper skeletal development, maturation, maintenance, and repair. Nevertheless, transcriptional activity of these molecules in skeletal tissues beyond embryogenesis has not been well characterized. In this study, we used several transgenic reporter mouse lines to define the transcriptional activity of two potent BMP ligands, Bmp2 and Bmp4, and their antagonist, Noggin, in the postnatal skeleton. At 3 to 4 weeks of age, Bmp4 and Noggin reporter activity was readily apparent in most cells of the osteogenic or chondrogenic lineages, respectively, whereas Bmp2 reporter activity was strongest in terminally differentiated cells of both lineages. By 5 to 6 months, activity of the reporters had generally abated; however, the Noggin and Bmp2 reporters remained remarkably active in articular chondrocytes and persisted there indefinitely. We further found that endogenous Bmp2, Bmp4, and Noggin transcript levels in postnatal bone and cartilage mirrored the activity of their respective reporters in these tissues. Finally, we found that the activity of the Bmp2, Bmp4, and Noggin reporters in bone and cartilage at 3 to 4 weeks could be recapitulated in both osteogenic and chondrogenic culture models. These results reveal that Bmp2, Bmp4, and Noggin transcription persists to varying degrees in skeletal tissues postnatally, with each gene exhibiting its own cell type-specific pattern of activity. Illuminating these patterns and their dynamics will guide future studies aimed at elucidating both the causes and consequences of aberrant BMP signaling in the postnatal skeleton.

Identifying functional annotation for noncoding genomic sequences.

The recent success of genome-wide association studies has generated a trove of biologically significant variants implicated in human disease. However, many, if not most, of these variants fall in noncoding regions that have traditionally lacked much functional annotation. New data sets and tools allow for a more detailed assessment of potential importance of noncoding genetic variants. An overview of types of regulatory annotation that are currently available, and approaches to analyzing this data are provided with emphasis on usage of the UCSC genome browser.

An autonomous BMP2 regulatory element in mesenchymal cells.

BMP2 is a morphogen that controls mesenchymal cell differentiation and behavior. For example, BMP2 concentration controls the differentiation of mesenchymal precursors into myocytes, adipocytes, chondrocytes, and osteoblasts. Sequences within the 3'untranslated region (UTR) of the Bmp2 mRNA mediate a post-transcriptional block of protein synthesis. Interaction of cell and developmental stage-specific trans-regulatory factors with the 3'UTR is a nimble and versatile mechanism for modulating this potent morphogen in different cell types. We show here, that an ultra-conserved sequence in the 3'UTR functions independently of promoter, coding region, and 3'UTR context in primary and immortalized tissue culture cells and in transgenic mice. Our findings indicate that the ultra-conserved sequence is an autonomously functioning post-transcriptional element that may be used to modulate the level of BMP2 and other proteins while retaining tissue specific regulatory elements.

Control of BMP gene expression by long-range regulatory elements.

Much evidence suggests that "developmental regulator" genes, like those encoding transcription factors and signaling molecules, are typically controlled by many modular, tissue-specific cis-regulatory elements that function during embryogenesis. These elements are often far from gene coding regions and promoters. Bone morphogenetic proteins (BMPs) drive many processes in development relating to organogenesis and differentiation. Four BMP family members, Bmp2, Bmp4, Bmp5, and Gdf6, are now known to be under the control of distant cis-regulatory elements. BMPs are thus firmly placed in the category of genes prone to this phenomenon. The analysis of distant BMP regulatory elements has provided insight into the many pleiotropic effects of BMP genes, and underscores the biological importance of non-coding genomic DNA elements.

Progressive recruitment of Runx2 to genomic targets despite decreasing expression during osteoblast differentiation.

The mRNAs encoding Runx2, a master osteoblast transcription factor, and its target gene Osteocalcin (OC), are commonly used as markers of osteoblast differentiation. We found that while OC mRNA levels do indeed increase during development of the osteoblast phenotype in MC3T3-E1 cultures, Runx2 mRNA levels surprisingly decrease. Neither translational control of Runx2 (based on Western analysis) nor regulation of its DNA-binding ability (assessed by electrophoretic mobility shift assay) could explain the unexpected opposite patterns of Runx2 and OC expression. Instead, a series of chromatin immunoprecipitation (ChIP) assays during osteoblast differentiation revealed that early on, when Runx2 protein amount and DNA-binding activity are maximal, it is practically absent from the OC promoter. At later stages, Runx2 is recruited to the OC promoter while Runx2 mRNA, protein, and in vitro DNA binding progressively decrease. We also followed Runx2 occupancy at a novel genomic target discovered by ChIP-Chip analysis of cells in which the OC promoter is maximally occupied. The results revealed that Runx2 is recruited to this locus and to the OC promoter with a remarkably similar temporal pattern. These observations highlight a mechanism that restrains Runx2-mediated transcriptional control by confining its access to genomic targets to a narrow window of time. The need for such stringent control is consistent with the severe consequences of Runx2 over-expression in vivo.

Identification of transcription factor target genes by ChIP display.

Transcription factors play pivotal roles in the control of cell growth and differentiation in health and disease. In the post-genomic era, it has become possible to locate the regions occupied by transcription factors throughout the genome, leading to better understanding of their mechanism of action and the genes that they regulate. All methods for transcription factor location analysis utilize chromatin immunoprecipitation (ChIP). Although ChIP was initially used to test whether a protein binds to a candidate promoter in living cells, newly developed methods allow the unbiased identification of novel targets of transcription factors. This chapter describes ChIP Display, an affordable method for transcription factor location analysis. Despite being relatively low throughput compared with alternative methods such as ChIP-chip and ChIP-SAGE, ChIP Display provides even small molecular biology laboratories with the opportunity to discover novel targets of any transcription factor, for which high-quality antibodies are available.

Identification of novel Runx2 targets in osteoblasts: cell type-specific BMP-dependent regulation of Tram2.

Runx2 is an osteoblast master transcription factor and a target for bone morphogenetic protein (BMP) signaling, but our knowledge of events downstream of Runx2 is limited. In this study, we used ChIP Display to discover seven novel genomic regions occupied by Runx2 in living MC3T3-E1 osteoblastic cells. Six of these regions are found within or up to 1-kb away from annotated genes, but only two are found within 5'-gene flanking sequences. One of the newly identified Runx2 target genes is Tram2, whose product facilitates proper folding of type I collagen. We demonstrate that Tram2 mRNA is suppressed in non-osteoblasts when Runx2 is over-expressed, and that this suppression is alleviated upon treatment with BMP-2. Moreover, we show that BMP-induced Runx2 expression in the C3H10T1/2, ST2, C2C12, and MC3T3-E1 cell lines coincides with an increase in Tram2 mRNA levels. Thus, Runx2 may regulate Tram2 expression in a BMP-dependent manner, and Tram2 may participate in the overall osteogenic function of Runx2. Among the other Runx2 target genes discovered in this study are Lnx2, an intracellular scaffolding protein that may play a role in Notch signaling, and Tnfrsf12a, a Tumor Necrosis Factor receptor family member that influences both osteoblast and osteoclast differentiation. Expanding our knowledge of Runx2 target genes, and manipulation of these genes, are warranted to better understand the regulation of osteoblast function and to provide opportunities for the development of new bone anabolics.