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.

A Low-Cost AI Buoy System for Monitoring Water Quality at Offshore Aquaculture Cages.

The ocean resources have been rapidly depleted in the recent decade, and the complementary role of aquaculture to food security has become more critical than ever before. Water quality is one of the key factors in determining the success of aquaculture and real-time water quality monitoring is an important process for aquaculture. This paper proposes a low-cost and easy-to-build artificial intelligence (AI) buoy system that autonomously measures the related water quality data and instantly forwards them via wireless channels to the shore server. Furthermore, the data provide aquaculture staff with real-time water quality information and also assists server-side AI programs in implementing machine learning techniques to further provide short-term water quality predictions. In particular, we aim to provide a low-cost design by combining simple electronic devices and server-side AI programs for the proposed buoy system to measure water velocity. As a result, the cost for the practical implementation is approximately USD 2015 only to facilitate the proposed AI buoy system to measure the real-time data of dissolved oxygen, salinity, water temperature, and velocity. In addition, the AI buoy system also offers short-term estimations of water temperature and velocity, with mean square errors of 0.021 °C and 0.92 cm/s, respectively. Furthermore, we replaced the use of expensive current meters with a flow sensor tube of only USD 100 to measure water velocity.

Single-Cell Transcriptomic Census of Endothelial Changes Induced by Matrix Stiffness and the Association with Atherosclerosis.

Vascular endothelial cell (EC) plasticity plays a critical role in the progression of atherosclerosis by giving rise to mesenchymal phenotypes in the plaque lesion. Despite the evidence for arterial stiffening as a major contributor to atherosclerosis, the complex interplay among atherogenic stimuli in vivo has hindered attempts to determine the effects of extracellular matrix (ECM) stiffness on endothelial-mesenchymal transition (EndMT). To study the regulatory effects of ECM stiffness on EndMT, an in vitro model is developed in which human coronary artery ECs are cultured on physiological or pathological stiffness substrates. Leveraging single-cell RNA sequencing, cell clusters with mesenchymal transcriptional features are identified to be more prevalent on pathological substrates than physiological substrates. Trajectory inference analyses reveal a novel mesenchymal-to-endothelial reverse transition, which is blocked by pathological stiffness substrates, in addition to the expected EndMT trajectory. ECs pushed to a mesenchymal character by pathological stiffness substrates are enriched in transcriptional signatures of atherosclerotic ECs from human and murine plaques. This study characterizes at single-cell resolution the transcriptional programs that underpin EC plasticity in both physiological or pathological milieus, and thus serves as a valuable resource for more precisely defining EndMT and the transcriptional programs contributing to atherosclerosis.

Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-ß signaling.

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-ß signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-ß signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-ß signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

Transcriptomic alterations underline aging of osteogenic bone marrow stromal cells.

BACKGROUND: Multipotent bone marrow stromal cells (BMSCs) are adult stem cells that form functional osteoblasts and play a critical role in bone remodeling. During aging, an increase in bone loss and reduction in structural integrity lead to osteoporosis and result in an increased risk of fracture. We examined age-dependent histological changes in murine vertebrae and uncovered that bone loss begins as early as the age of 1 mo. AIM: To identify the functional alterations and transcriptomic dynamics of BMSCs during early bone loss. METHODS: We collected BMSCs from mice at early to middle ages and compared their self-renewal and differentiation potential. Subsequently, we obtained the transcriptomic profiles of BMSCs at 1 mo, 3 mo, and 7 mo. RESULTS: The colony-forming and osteogenic commitment capacity showed a comparable finding that decreased at the age of 1 mo. The transcriptomic analysis showed the enrichment of osteoblastic regulation genes at 1 mo and loss of osteogenic features at 3 mo. The BMSCs at 7 mo showed enrichment of adipogenic and DNA repair features. Moreover, we demonstrated that the WNT and MAPK signaling pathways were upregulated at 1 mo, followed by increased pro-inflammatory and apoptotic features. CONCLUSION: Our study uncovered the cellular and molecular dynamics of bone aging in mice and demonstrated the contribution of BMSCs to the early stage of age-related bone loss.

Novel Lineage-Tracing System to Identify Site-Specific Ectopic Bone Precursor Cells.

Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreERT2;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion.

A single cell transcriptional atlas of early synovial joint development.

Synovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization and in vitro characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.

Small molecules for mesenchymal stem cell fate determination.

Mesenchymal stem cells (MSCs) are adult stem cells harboring self-renewal and multilineage differentiation potential that are capable of differentiating into osteoblasts, adipocytes, or chondrocytes in vitro, and regulating the bone marrow microenvironment and adipose tissue remodeling in vivo. The process of fate determination is initiated by signaling molecules that drive MSCs into a specific lineage. Impairment of MSC fate determination leads to different bone and adipose tissue-related diseases, including aging, osteoporosis, and insulin resistance. Much progress has been made in recent years in discovering small molecules and their underlying mechanisms control the cell fate of MSCs both in vitro and in vivo. In this review, we summarize recent findings in applying small molecules to the trilineage commitment of MSCs, for instance, genistein, medicarpin, and icariin for the osteogenic cell fate commitment; isorhamnetin, risedronate, and arctigenin for pro-adipogenesis; and atractylenolides and dihydroartemisinin for chondrogenic fate determination. We highlight the underlying mechanisms, including direct regulation, epigenetic modification, and post-translational modification of signaling molecules in the AMPK, MAPK, Notch, PI3K/AKT, Hedgehog signaling pathways etc. and discuss the small molecules that are currently being studied in clinical trials. The target-based manipulation of lineage-specific commitment by small molecules offers substantial insights into bone marrow microenvironment regulation, adipose tissue homeostasis, and therapeutic strategies for MSC-related diseases.

Gene expression profiling in prognosis of distant recurrence in HR-positive and HER2-negative breast cancer patients.

There had been several studies using gene-expression profiling in predicting distant recurrence in breast cancer. In this study, we developed an 18-gene classifier (18-GC) to predict distant recurrence of breast cancer and compared it with the 21-gene panel (Oncotype DX®, ODx) in performance. Included were 224 breast cancer patients with positive hormonal receptor (HR+) and negative human epidermal growth factor receptor 2 (HER2-). We compared the demographic, clinical, and survival information of the patients, and further compared the prediction of recurrence risk obtained by using the 18-GC with that by ODx. To have the best combined sensitivity and specificity, receiver operating characteristics (ROC) curve analysis was performed to determine the cutoff values for several breakpoint scores. For the new 18-GC, a breakpoint score of 21 was adopted to produce a combined highest sensitivity (95%) and specificity (39%) in detecting distant recurrence. At this breakpoint score, 164 of the 224 patients were classified by the 18-GC in the same risk level as by ODx, giving a concordance rate of 73%. Along with patient age and tumor stage, this 18-GC was found to be an independent significant prognostic factor of distant metastasis of breast cancer. We have thus created a new gene panel assay for prediction of distant recurrence in HR+ and HER2- breast cancer patients. With a high concordance rate with ODx, this new assay may serve as a good tool for individual breast cancer patients to make an informed decision on whether adjuvant chemotherapy should be performed post-surgery.

Validation of the 18-gene classifier as a prognostic biomarker of distant metastasis in breast cancer.

We validated an 18-gene classifier (GC) initially developed to predict local/regional recurrence after mastectomy in estimating distant metastasis risk. The 18-gene scoring algorithm defines scores as: <21, low risk; ≥21, high risk. Six hundred eighty-three patients with primary operable breast cancer and fresh frozen tumor tissues available were included. The primary outcome was the 5-year probability of freedom from distant metastasis (DMFP). Two external datasets were used to test the predictive accuracy of 18-GC. The 5-year rates of DMFP for patients classified as low-risk (n = 146, 21.7%) and high-risk (n = 537, 78.6%) were 96.2% (95% CI, 91.1%-98.8%) and 80.9% (74.6%-81.9%), respectively (median follow-up interval, 71.8 months). The 5-year rates of DMFP of the low-risk group in stage I (n = 62, 35.6%), stage II (n = 66, 20.1%), and stage III (n = 18, 10.3%) were 100%, 94.2% (78.5%-98.5%), and 90.9% (50.8%-98.7%), respectively. Multivariate analysis revealed that 18-GC is an independent prognostic factor of distant metastasis (adjusted hazard ratio, 5.1; 95% CI, 1.8-14.1; p = 0.0017) for scores of ≥21. External validation showed that the 5-year rate of DMFP in the low- and high-risk patients was 94.1% (82.9%-100%) and 80.3% (70.7%-89.9%, p = 0.06) in a Singapore dataset, and 89.5% (81.9%-94.1%) and 73.6% (67.2%-79.0%, p = 0.0039) in the GEO-GSE20685 dataset, respectively. In conclusion, 18-GC is a viable prognostic biomarker for breast cancer to estimate distant metastasis risk.

Nanoparticle-mediated conversion of primary human astrocytes into neurons and oligodendrocytes.

Central nervous system (CNS) diseases and injuries are accompanied by reactive gliosis and scarring involving the activation and proliferation of astrocytes to form hypertrophic and dense structures, which present a significant barrier to neural regeneration. Engineering astrocytes to functional neurons or oligodendrocytes may constitute a novel therapeutic strategy for CNS diseases and injuries. Such direct cellular programming has been successfully demonstrated using viral vectors via the transduction of transcriptional factors, such as Sox2, which could program resident astrocytes into neurons in the adult brain and spinal cord, albeit the efficiency was low. Here we report a non-viral nanoparticle-based transfection method to deliver Sox2 or Olig2 into primary human astrocytes and demonstrate the effective conversion of the astrocytes into neurons and oligodendrocyte progenitors following the transgene expression of Sox2 and Olig2, respectively. This approach is highly translatable for engineering astrocytes to repair injured CNS tissues.

Nanoparticle-mediated transcriptional modification enhances neuronal differentiation of human neural stem cells following transplantation in rat brain.

Strategies to enhance survival and direct the differentiation of stem cells in vivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy.

Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering.

Liver transplantation is the ultimate treatment for severe hepatic failure to date. However, the limited supply of donor organs has severely hampered this treatment. So far, great potentials of using mesenchymal stem cells (MSCs) to replenish the hepatic cell population have been shown; nevertheless, there still is a lack of an optimal three-dimensional scaffold for generation of well-transplantable hepatic tissues. In this study, we utilized a cryo-chemical decellularization method which combines physical and chemical approach to generate acellular liver scaffolds (ALS) from the whole liver. The produced ALS provides a biomimetic three-dimensional environment to support hepatic differentiation of MSCs, evidenced by expression of hepatic-associated genes and marker protein, glycogen storage, albumin secretion, and urea production. It is also found that hepatic differentiation of MSCs within the ALS is much more efficient than two-dimensional culture in vitro. Importantly, the hepatic-like tissues (HLT) generated by repopulating ALS with MSCs are able to act as functional grafts and rescue lethal hepatic failure after transplantation in vivo. In summary, the cryo-chemical method used in this study is suitable for decellularization of liver and create acellular scaffolds that can support hepatic differentiation of MSCs and be used to fabricate functional tissue-engineered liver constructs.