Biology subjects

Robey, P. G.

Publications and source records attributed to Robey, P. G..

7 recordsLinked to original sources

bioRxiv2024

Hyaluronic Acid and Emergent Tissue Mechanics Orchestrate Digit Tip Regeneration

Tissue regeneration is a dynamic process requiring coordinated cell fate decisions to restore structure and function. For instance, the tips of human and rodent digits fully regrow after amputation, although more proximal injuries fail to regenerate. While biochemical cues have been widely studied in wound healing, the role of the physical microenvironment remains less understood. Here, we discovered that tissue mechanics and extracellular matrix (ECM) composition differ markedly between non-regenerating and regenerating wounds. These differences are due to specific fibroblast sub-types which predominate in the non-regenerating wound and form fibrotic collagen networks. Conversely, bone-forming progenitors in regenerating digits synthesize abundant hyaluronic acid, which mediates collagen fibrillogenesis. By modeling the tissue mechanics of non-regenerative and regenerative wounds using hydrogels, we showed that substrate stiffness regulates the synthesis of fibrotic and regenerative ECM. We further revealed that mimicking the regenerative wound mechanics upregulated bone morphogenic protein signaling and osteogenic differentiation. We ultimately demonstrated that the link protein HAPLN1 promotes hyaluronic acid deposition in vivo, reduces scar formation, and induces bone repair after non-regenerative amputations. These findings emphasize the interplay among the ECM, tissue mechanics, and cell behavior in digit regeneration and point towards ECM modulation as a strategy to improve wound healing. HighlightsO_LINon-regenerating and regenerating digit tips diverge in their cellular and ECM composition C_LIO_LIRegeneration requires hyaluronic acid matrix, which mediates collagen assembly and tissue mechanics C_LIO_LISoft substrates enhance BMP signaling and propagate the synthesis of regenerative ECM C_LIO_LIHAPLN1 overexpression initiates rescue of non-regenerative digit tip amputations C_LI

developmental biology↗

bioRxiv2024

The Hippo-YAP/TAZ-TEAD axis promotes multipotency in bone marrow stromal cells

The mechanisms by which bone marrow stromal cells (BMSCs) maintain multilineage potency in vitro remain elusive. To identify the transcriptional regulatory circuits that contribute to BMSC multipotency, we performed paired single-nucleus multiomics of the expansion of freshly isolated BMSCs and of BMSCs undergoing tri-lineage differentiation. By computationally reconstructing the regulatory programs associated with initial stages of differentiation and early expansion, we identified the TEAD family of transcription factors, which is inhibited by Hippo signaling, as highly active in the BMSC in vitro multipotent state. Pharmacological inhibition of TEAD enhanced BMSC osteogenic and adipogenic differentiation, whereas its activation maintained BMSCs in an undifferentiated state, supporting a model whereby isolation of BMSCs coincides with a TEAD-controlled transcriptional state linked to multipotency. Our study highlights the Hippo pathway as a pivotal regulator of BMSC multipotency, and our regulatory network inferences are a reservoir of testable hypotheses that link transcription factors and their regulons to specific aspects of BMSC behavior.

genomics↗

bioRxiv2024

Metabolic Quadrivalency in RSeT Human Embryonic Stem Cells

One of the most important properties of human embryonic stem cells (hESCs) is related to their pluripotent states. In our recent study, we identified a previously unrecognized pluripotent state induced by RSeT medium. This state makes primed hESCs resistant to conversion to naive pluripotent state. In this study, we have further characterized the metabolic features in these RSeT hESCs, including metabolic gene expression, metabolomic analysis, and various functional assays. The commonly reported metabolic modes include glycolysis or both glycolysis and oxidative phosphorylation (i.e., metabolic bivalency) in pluripotent stem cells. However, besides the presence of metabolic bivalency, RSeT hESCs exhibited a unique metabolome with additional fatty acid oxidation and imbalanced nucleotide metabolism. This metabolic quadrivalency is linked to hESC growth independent of oxygen tension and restricted capacity for naive reprogramming in these cells. Thus, this study provides new insights into pluripotent state transitions and metabolic stress-associated hPSC growth in vitro.

biochemistry↗

bioRxiv2024

Resistance to Naive and Formative Pluripotency Conversion in RSeT Human Embryonic Stem Cells

One of the most important properties of human embryonic stem cells (hESCs) is related to their primed and naive pluripotent states. Our previous meta-analysis indicates the existence of heterogeneous pluripotent states derived from diverse naive protocols. In this study, we have characterized a commercial medium (RSeT)-based pluripotent state under various growth conditions. Notably, RSeT hESCs can circumvent hypoxic growth conditions as required by naive hESCs, in which some RSeT cells (e.g., H1 cells) exhibit much lower single cell plating efficiency, having altered or much retarded cell growth under both normoxia and hypoxia. Evidently, hPSCs lack many transcriptomic hallmarks of naive and formative pluripotency (a phase between naive and primed states). Integrative transcriptome analysis suggests our primed and RSeT hESCs are close to the early stage of post-implantation embryos, similar to the previously reported primary hESCs and early hESC cultures. Moreover, RSeT hESCs did not express naive surface markers such as CD75, SUSD2, and CD130 at a significant level. Biochemically, RSeT hESCs exhibit a differential dependency of FGF2 and co-independency of both Janus kinase (JAK) and TGF{beta} signaling in a cell-line-specific manner. Thus, RSeT hESCs represent a previously unrecognized pluripotent state downstream of formative pluripotency. Our data suggest that human naive pluripotent potentials may be restricted in RSeT medium. Hence, this study provides new insights into pluripotent state transitions in vitro.

cell biology↗

bioRxiv2021

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from human induced pluripotent stem cells

The human definitive yolk sac is an important organ supporting the early developing embryo through nutrient supply and by facilitating the establishment of the embryonic circulatory system. However, the molecular and cellular biology of the human yolk sac remains largely obscure due to the lack of suitable in vitro models. Here, we show that human induced pluripotent stem cells (hiPSCs) co-cultured with various types of stromal cells as spheroids self-organize into yolk sac-like organoids without the addition of exogenous factors. Yolk sac-like organoids recapitulated a yolk sac specific cellular complement and structures as well as the functional ability to generate definitive hematopoietic progenitor cells (HPCs). Furthermore, sequential hemato-vascular ontogenesis could be observed during organoid formation. Notably, our organoid system can be performed in a scalable, autologous, and xeno-free condition, thereby providing an important model of human definitive yolk sac development and allows for efficient bulk generation of hiPSC-derived HPCs.

cell biology↗

bioRxiv2020

Characterisation of ovine bone marrow-derived stromal cells (oBMSC) and evaluation of chondrogenically induced micro-pellets for cartilage tissue repair in vivo

BackgroundBone marrow stromal cells (BMSC) show promise in cartilage repair, and sheep are the most common large animal pre-clinical model. The objective of this study was to characterize ovine BMSC (oBMSC) in vitro, and to evaluate the capacity of chondrogenic micro-pellets manufactured from oBMSC or ovine articular chondrocytes (oACh) to repair osteochondral defects in sheep. MethodsoBMSC were characterised for surface marker expression using flow cytometry and evaluated for tri-lineage differentiation. oBMSC micro-pellets were manufactured in a microwell platform, and chondrogenesis was compared at 2%, 5%, and 20% O2. The capacity of cartilage micro-pellets manufactured from oBMSC or oACh to repair osteochondral defects in adult sheep was evaluated in an 8-week pilot study. Expanded oBMSC were positive for CD44 and CD146 and negative for CD45. ResultsThe common adipogenic induction medium ingredient, 3-Isobutyl-1-methylxanthine (IBMX) was toxic to oBMSC, but adipogenesis could be restored by excluding IBMX from the medium. BMSC chondrogenesis was optimal in a 2% O2 atmosphere. Micro-pellets formed from oBMSC or oACh appeared morphologically similar, but hypertrophic genes were elevated in oBMSC micro-pellets. While oACh micro-pellets formed cartilage-like repair tissue in sheep, oBMSC micro-pellets did not. ConclusionThe sensitivity of oBMSC to IBMX highlights species-species differences between oBMSC and hBMSC. Micro-pellets manufactured from oBMSC were not effective in repairing osteochondral defects, while oACh micro-pellets enabled modest repair. While oBMSC can be driven to form cartilage-like tissue in vitro, their effective use in cartilage repair will require mitigation of hypertrophy.

bioengineering↗

bioRxiv2019

Micro-pellet culture reveals that bone marrow mesenchymal stromal cell (BMSC) chondrogenic induction is triggered by a single day of TGF-β1 exposure

Despite immense promise, engineering of stable cartilage tissue from bone marrow-derived stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") remains elusive. Relative cartilage-like matrix deposition is commonly used to guide BMSC chondrogenic optimisation efforts. However, matrix deposition is heterogeneous in most models, and notably, it lags behind cell fate decisions. We reason that the lag time between cell fate decision and matrix accumulation, coupled with matrix heterogeneity, has obscured basic BMSC biological characteristics, such as differentiation kinetics. Here, we utilize a customized microwell platform to assemble hundreds of small-diameter BMSC micro-pellets and characterized chondrogenic differentiation kinetics in response to the canonical signaling molecule, transforming growth factor-{beta}1 (TGF-{beta}1). Micro-pellets provide a homogeneous readout, and our experimental design accounts for the significant time delay between growth factor signal and deposition of cartilage-like matrix. While 14-to-21-day induction protocols are routine, BMSC micro-pellet cultures reveal that a single day of TGF-{beta}1 exposure was sufficient to trigger chondrogenic differentiation cascades resulting in outcomes similar to micro-pellets exposed to TGF-{beta}1 for 21 days. RNA-sequencing analysis demonstrated that one day of TGF-{beta}1 exposure was also sufficient to induce hypertrophic cascades in BMSC, not observed in articular chondrocytes. Refocusing chondrogenic induction optimisation efforts from weeks to the first hours or days of culture, using homogeneous model systems, may benefit efforts to build stable cartilage formed by BMSCs. SignificanceThe macro-pellet model, and assumptions generated using it, have permeated BMSC-based cartilage tissue engineering strategies since the 1990s. Using a micro-pellet model, we show that BMSC chondrogenic kinetics are significantly more rapid than historical macro-pellets data suggests, and that BMSC chondrogenic and hypertrophic commitment is instructed by a single day of TGF-{beta}1 exposure. This highly relevant study demonstrates that: (1) macro-pellets, which are large heterogeneous tissue models confound the differentiation kinetics visible in micro-pellet models; (2) induction strategies should focus on the first hours or days of culture; (3) even a single day of TGF-{beta}1 exposure drives BMSC to form hypertrophic tissue in vivo, requiring early intervention to prevent hypertrophy; and (4) articular chondrocytes and BMSCs respond distinctly to TGF-{beta}1.

cell biology↗