Approximately 10% of fractures will not heal without intervention. Current treatments can be marginally effective, costly, and some have adverse effects. A safe and manufacturable mimic of anabolic bone is the primary goal of bone engineering, but achieving this is challenging. Mesenchymal stem cells (MSCs), are excellent candidates for engineering bone, but lack reproducibility due to donor source and culture methodology. The need for a bioactive attachment substrate also hinders progress. Herein, we describe a highly osteogenic MSC line generated from induced pluripotent stem cells that generates high yields of an osteogenic cell-matrix (ihOCM) in vitro. In mice, the intrinsic osteogenic activity of ihOCM surpasses bone morphogenic protein 2 (BMP2) driving healing of calvarial defects in 4 weeks by a mechanism mediated in part by collagen VI and XII. We propose that ihOCM may represent an effective replacement for autograft and BMP products used commonly in bone tissue engineering.
Human mesenchymal stem cells (hMSCs) are effective in treating disorders resulting from an inflammatory or heightened immune response. The hMSCs derived from induced pluripotent stem cells (ihMSCs) share the characteristics of tissue derived hMSCs but lack challenges associated with limited tissue sources and donor variation. To meet the expected future demand for ihMSCs, there is a need to develop scalable methods for their production at clinical yields while retaining immunomodulatory efficacy. Herein, we describe a platform for the scalable expansion and rapid harvest of ihMSCs with robust immunomodulatory activity using degradable gelatin methacryloyl (GelMA) microcarriers. GelMA microcarriers were rapidly and reproducibly fabricated using a custom microfluidic step emulsification device at relatively low cost. Using vertical wheel bioreactors, 8.8 to 16.3-fold expansion of ihMSCs was achieved over 8 days.Complete recovery by 5-minute digestion of the microcarriers with standard cell dissociation reagents resulted in >95% viability. The ihMSCs matched or exceeded immunomodulatory potential in vitro when compared with ihMSCs expanded on monolayers. This is the first description of a robust, scalable, and cost-effective method for generation of immunomodulatory ihMSCs, representing a significant contribution to their translational potential.
Tetrahymena thermophila harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the ‘cell cycles’ of the diploid micronucleus and polyploid macronucleus are offset. Micronuclear S phase initiates just before cytokinesis and is completed in daughter cells prior to onset of macronuclear DNA replication. Mitotic micronuclear division occurs mid-cell cycle, while macronuclear amitosis is coupled to cell division. Here we report the first RNA-seq cell cycle analysis of a binucleated ciliated protozoan. RNA was isolated across 1.5 vegetative cell cycles, starting with a macronuclear G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the 26,000+ predicted genes were shown to be cell cycle regulated. Proteins present in both nuclei exhibit a single mRNA peak that always precedes their macronuclear function. Nucleus-limited genes, including nucleoporins and importins, are expressed prior to their respective nucleus-specific role. Cyclin D and A/B gene family members exhibit different expression patterns that suggest nucleus-restricted roles. Periodically expressed genes cluster into seven cyclic patterns. Four clusters have known PANTHER GO terms associated with G1/S and G2/M phase. We propose that these clusters encode known and novel factors that coordinate micro- and macronuclear-specific events such as mitosis, amitosis, DNA replication and cell division.
Malignant bone disease (MBD) occurs when tumors establish in bone, causing catastrophic tissue damage as a result of accelerated bone destruction and inhibition of repair. The resultant so-called osteolytic lesions (OL) take the form of tumor-filled cavities in bone that cause pain, fractures, and associated morbidity. Furthermore, the OL microenvironment can support survival of tumor cells and resistance to chemotherapy. Therefore, a deeper understanding of OL formation and MBD progression is imperative for the development of future therapeutic strategies. Herein, we describe a novel in vitro platform to study bone–tumor interactions based on three-dimensional co-culture of osteogenically enhanced human mesenchymal stem cells (OEhMSCs) in a rotating wall vessel bioreactor (RWV) while attached to micro-carrier beads coated with extracellular matrix (ECM) composed of factors found in anabolic bone tissue. Osteoinhibition was recapitulated in this model by co-culturing the OEhMSCs with a bone–tumor cell line (MOSJ-Dkk1) that secretes the canonical Wnt (cWnt) inhibitor Dkk-1, a tumor-borne osteoinhibitory factor widely associated with several forms of MBD, or intact tumor fragments from Dkk-1 positive patient-derived xenografts (PDX). Using the model, we observed that depending on the conditions of growth, tumor cells can biochemically inhibit osteogenesis by disrupting cWnt activity in OEhMSCs, while simultaneously co-engrafting with OEhMSCs, displacing them from the niche, perturbing their activity, and promoting cell death. In the absence of detectable co-engraftment with OEhMSCs, Dkk-1 positive PDX fragments had the capacity to enhance OEhMSC proliferation while inhibiting their osteogenic differentiation. The model described has the capacity to provide new and quantifiable insights into the multiple pathological mechanisms of MBD that are not readily measured using monolayer culture or animal models.
Radiotherapy of head and neck cancers frequently leads to irreversible hypofunction of salivary glands, which severely compromises the quality of life and is extremely difficult to treat. We found recently that salivary gland resident macrophages are sensitive to radiation and interact with epithelial progenitors and endothelial cells through homeostatic paracrine factors. Heterogeneous subpopulations of resident macrophages are present in other organs with distinct functions, whereas subpopulations of salivary gland resident macrophages with distinct functions or transcriptional profiles have not been reported yet. Using single-cell RNA sequencing, we found that mouse submandibular glands (SMGs) contain 2 distinct self-renewing resident macrophage subsets, an MHC-IIhi subset present in many other organs and an uncommon Csf2r+ subset. The main source of Csf2 in SMGs are innate lymphoid cells (ILCs) that rely on IL15 for maintenance, while the main source of IL15 protein is Csf2r+ resident macrophages, indicating a homeostatic paracrine interaction between these cells. Csf2r+ resident macrophages are the major source of hepatocyte growth factor (Hgf) that regulates homeostasis of SMG epithelial progenitors. Meanwhile, Csf2r+ resident macrophages are responsive to Hedgehog signaling that can rescue salivary function impaired by radiation. Consistently, irradiation persistently decreased numbers of ILCs and levels of IL15 and Csf2 in SMGs, which were all recovered by transient activation of Hedgehog signaling after radiation. Csf2r+ resident macrophages and MHC-IIhi resident macrophages share transcriptome profiles of perivascular macrophages and macrophages associated with nerves and/or epithelial cells in other organs, respectively, and such niche preferences were supported by lineage tracing and immunofluorescent staining. These findings reveal an uncommon resident macrophage subset that regulates the homeostasis of the salivary gland and is promising as the target to restore salivary gland function impaired by radiation.
As a prototypic ciliated protozoan, Tetrahymena thermophila harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the 'cell cycles' of the diploid germline micronucleus and polyploid somatic macronucleus are offset. Micronuclear S phase initiates just before cell division and is completed in daughter cells prior to the onset of macronuclear S phase. Whereas mitotic micronuclear division occurs mid-cell cycle, amitotic macronuclear division immediately precedes cytokinesis. Here we report the first RNA-seq analysis across the cell cycle of a binucleated organism. RNA was isolated at 30 min intervals across 1.5 vegetative cell cycles, starting with a macronuclear G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the predicted 26,000+ T. thermophila genes were shown to be cell cycle regulated. Proteins that are required in micro- and macronuclei exhibit a single mRNA peak that correlates with their macronuclear function, while the expression of nucleus-limited protein-coding genes, including nucleoporins and importins, peak prior to their respective nucleus-specific role. Cyclin D and cyclin A/B genes showed distinct expression patterns that predict nucleus-specific functions. Clustering of periodically expressed genes revealed seven gene expression patterns. Four clusters have known PANTHER GO biological processes that are overrepresented for G1/S and G2/M phase functions. We propose that these clusters encode known and novel factors that coordinate micro- and macronuclear-specific events such as mitosis, amitosis, DNA replication and cell division.
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