Antibody-activation of connexin hemichannels in bone osteocytes with ATP release suppresses breast cancer and osteosarcoma malignancy.

Bone tissue represents the most frequent site of cancer metastasis. We developed a hemichannel-activating antibody, Cx43-M2. Cx43-M2, directly targeting osteocytes in situ, activates osteocytic hemichannels and elevates extracellular ATP, thereby inhibiting the growth and migration of cultured breast and osteosarcoma cancer cells. Cx43-M2 significantly decreases breast cancer metastasis, osteosarcoma growth, and osteolytic activity, while improving survival rates in mice. The antibody's inhibition of breast cancer and osteosarcoma is dose dependent in both mouse and human cancer metastatic models. Furthermore, Cx43-M2 enhances anti-tumor immunity by increasing the population and activation of tumor-infiltrating immune-promoting effector T lymphocytes, while reducing immune-suppressive regulatory T cells. Our results suggest that the Cx43-M2 antibody, by activating Cx43 hemichannels and facilitating ATP release and purinergic signaling, transforms the cancer microenvironment from a supportive to a suppressive state. Collectively, our study underscores the potential of Cx43-M2 as a therapeutic for treating breast cancer bone metastasis and osteosarcoma.

Evaluation of Connexin Hemichannel Activity In Vivo.

Connexin hemichannels (Cx HCs) are hexameric structures at the cell plasma membrane, whose function as membrane transport proteins allows for the passive flow of small hydrophilic molecules and ions (≤1 kDa) between the cytosol and the extracellular environment. Activation of Cx HCs is highly dependent on pathological conditions. HC activity provokes changes in the microenvironment, inducing the dissemination of signaling molecules in both an autocrine and paracrine manner. Given the elicitation of a variety of signaling pathways, and assortment of Cx species and dispersion throughout the body, Cx HCs have been implicated in a range of processes such as cell proliferation, differentiation, cell death, and tissue modeling and remodeling. While studying the expression and localization of Cx HCs can be done using traditional laboratory techniques, such as immunoblot analysis, measuring the functionality/activity of the HCs requires a more explicit methodology and is essential for determining Cx-mediated physiological changes. The study of Cx HC function/activity has focused mainly on in vitro measurements through electrophysiological characterization or, more commonly, using HC-permeable dye uptake studies. Here, we describe the use of dye uptake to measure Cx HC activity in vivo using mechanically stimulated osteocytic Cx43 HCs with Evans blue dye as our model.

Gap Junctions or Hemichannel-Dependent and Independent Roles of Connexins in Fibrosis, Epithelial-Mesenchymal Transitions, and Wound Healing.

Fibrosis initially appears as a normal response to damage, where activated fibroblasts produce large amounts of the extracellular matrix (ECM) during the wound healing process to assist in the repair of injured tissue. However, the excessive accumulation of the ECM, unresolved by remodeling mechanisms, leads to organ dysfunction. Connexins, a family of transmembrane channel proteins, are widely recognized for their major roles in fibrosis, the epithelial-mesenchymal transition (EMT), and wound healing. Efforts have been made in recent years to identify novel mediators and targets for this regulation. Connexins form gap junctions and hemichannels, mediating communications between neighboring cells and inside and outside of cells, respectively. Recent evidence suggests that connexins, beyond forming channels, possess channel-independent functions in fibrosis, the EMT, and wound healing. One crucial channel-independent function is their role as the primary functional component for cell adhesion. Other channel-independent functions of connexins involve their roles in mitochondria and exosomes. This review summarizes the latest advances in the channel-dependent and independent roles of connexins in fibrosis, the EMT, and wound healing, with a particular focus on eye diseases, emphasizing their potential as novel, promising therapeutic targets.

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens.

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection.

Connexins (Cxs) play a crucial role in maintaining lens transparency. Here, we present a protocol for altering Cx hemichannel (HC) function in primary chicken lens fiber cells using high-titer retroviral replication competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor (A) infection. We describe steps for incubating eggs, isolating lenses, culturing cells, preparing reagents, and infecting cells. We then detail cell treatment and detection of apoptosis and death. This protocol can assess protein kinase A, HC activity, and increased glutathione transport for protecting lens fiber cells against oxidative stress. For complete details on the use and execution of this protocol, please refer to Liu et al.,1 Riquelme et al.,2 Shi et al.,3 Jiang,4 and Rath et al.5.

Diabetes diminishes muscle precursor cell-mediated microvascular angiogenesis.

The skeletal muscles of Type II diabetic (T2D) patients can be characterized by a reduced vessel density, corresponding to deficiencies in microvascular angiogenesis. Interestingly, T2D also inhibits the function of many myogenic cells resident within skeletal muscle, including satellite cells, which are well-known for the role they play in maintaining homeostasis. The current study was undertaken to gain a better understanding of the mechanisms whereby satellite cell progeny, muscle precursor cells (MPCs), influence microvascular angiogenesis. Network growth and the expression of genes associated with angiogenesis were reduced when microvessels were treated with conditioned media generated by proliferating MPCs isolated from diabetic, as compared to control rat skeletal muscle, a phenomenon that was also observed when myoblasts from control or diabetic human skeletal muscle were used. When only exosomes derived from diabetic or control MPCs were used to treat microvessels, no differences in microvascular growth were observed. An evaluation of the angiogenesis factors in control and diabetic MPCs revealed differences in Leptin, vascular endothelial growth factor (VEGF), IL1-ß, interleukin 10, and IP-10, and an evaluation of the MPC secretome revealed differences in interleukin 6, MCP-1, VEGF, and interleukin 4 exist. Angiogenesis was also reduced in tissue-engineered skeletal muscles (TE-SkM) containing microvessels when they were generated from MPCs isolated from diabetic as compared to control skeletal muscle. Lastly, the secretome of injured control, but not diabetic, TE-SkM was able to increase VEGF and increase microvascular angiogenesis. This comprehensive analysis of the interaction between MPCs and microvessels in the context of diabetes points to an area for alleviating the deleterious effects of diabetes on skeletal muscle.

Connexin 43 hemichannels and prostaglandin E2 release in anabolic function of the skeletal tissue to mechanical stimulation.

Bone adapts to changes in the physical environment by modulating remodeling through bone resorption and formation to maintain optimal bone mass. As the most abundant connexin subtype in bone tissue, connexin 43 (Cx43)-forming hemichannels are highly responsive to mechanical stimulation by permitting the exchange of small molecules (<1.2 kDa) between bone cells and the extracellular environment. Upon mechanical stimulation, Cx43 hemichannels facilitate the release of prostaglandins E2 (PGE2), a vital bone anabolic factor from osteocytes. Although most bone cells are involved in mechanosensing, osteocytes are the principal mechanosensitive cells, and PGE2 biosynthesis is greatly enhanced by mechanical stimulation. Mechanical stimulation-induced PGE2 released from osteocytic Cx43 hemichannels acts as autocrine effects that promote ß-catenin nuclear accumulation, Cx43 expression, gap junction function, and protects osteocytes against glucocorticoid-induced osteoporosis in cultured osteocytes. In vivo, Cx43 hemichannels with PGE2 release promote bone formation and anabolism in response to mechanical loading. This review summarizes current in vitro and in vivo understanding of Cx43 hemichannels and extracellular PGE2 release, and their roles in bone function and mechanical responses. Cx43 hemichannels could be a significant potential new therapeutic target for treating bone loss and osteoporosis.

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Mechanosensitive piezo1 calcium channel activates connexin 43 hemichannels through PI3K signaling pathway in bone.

BACKGROUND: Mechanical loading promotes bone formation and osteocytes are a major mechanosensory cell in the bone. Both Piezo1 channels and connexin 43 hemichannels (Cx43 HCs) in osteocytes are important players in mechanotransduction and anabolic function by mechanical loading. However, the mechanism underlying mechanotransduction involving Piezo1 channels and Cx43 HCs in osteocytes and bone remains unknown. RESULTS: We showed that, like mechanical loading, Piezo1 specific agonist Yoda1 was able to increase intracellular Ca2+ signaling and activate Cx43 HCs, while Yoda1 antagonist Dooku1 inhibited Ca2+ and Cx43 HC activation induced by both mechanical loading and Yoda1. Moreover, the intracellular Ca2+ signal activated by Yoda1 was reduced by the inhibition of Cx43 HCs and pannexin1 (Panx1) channels, as well as ATP-P2X receptor signaling. Piezo1 and Cx43 HCs were co-localized on the osteocyte cell surface, and Yoda1-activated PI3K-Akt signaling regulated the opening of Cx43 HCs. Furthermore, Cx43 HCs opening by mechanical loading on tibias was ablated by inhibition of Piezo1 activation in vivo. CONCLUSION: We demonstrated that upon mechanical stress, increased intracellular Ca2+ activated by Piezo1 regulates the opening of HCs through PI3K-Akt and opened Cx43 HCs, along with Panx1 channels, and ATP-P2X signaling sustain the intracellular Ca2+ signal, leading to bone anabolic function.

Connexin 43 hemichannels regulate mitochondrial ATP generation, mobilization, and mitochondrial homeostasis against oxidative stress.

Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. Here, we reported a new role of mitochondrial Cx43 (mtCx43) and hemichannels (HCs) in modulating mitochondria homeostasis and function in bone osteocytes under oxidative stress. In murine long bone osteocyte-Y4 cells, the translocation of Cx43 to mitochondria was increased under H2O2-induced oxidative stress. H2O2 increased the mtCx43 level accompanied by elevated mtCx43 HC activity, determined by dye uptake assay. Cx43 knockdown (KD) by the CRISPR-Cas9 lentivirus system resulted in impairment of mitochondrial function, primarily manifested as decreased ATP production. Cx43 KD had reduced intracellular reactive oxidative species levels and mitochondrial membrane potential. Additionally, live-cell imaging results demonstrated that the proton flux was dependent on mtCx43 HCs because its activity was specifically inhibited by an antibody targeting Cx43 C-terminus. The co-localization and interaction of mtCx43 and ATP synthase subunit F (ATP5J2) were confirmed by Förster resonance energy transfer and a protein pull-down assay. Together, our study suggests that mtCx43 HCs regulate mitochondrial ATP generation by mediating K+, H+, and ATP transfer across the mitochondrial inner membrane and the interaction with mitochondrial ATP synthase, contributing to the maintenance of mitochondrial redox levels in response to oxidative stress.

Association of adenosine signaling gene signature with estrogen receptor-positive breast and prostate cancer bone metastasis.

Bone metastasis is a common and devastating consequence of several major cancer types, including breast and prostate. Osteocytes are the predominant bone cell, and through connexin (Cx) 43 hemichannels release ATP to the bone microenvironment that can be hydrolyzed to adenosine. Here, we investigated how genes related to ATP paracrine signaling are involved in two common bone-metastasizing malignancies, estrogen receptor positive (ER+) breast and prostate cancers. Compared to other sites, bone metastases of both cancer types expressed higher levels of ENTPD1 and NT5E, which encode CD39 and CD73, respectively, and hydrolyze ATP to adenosine. ADORA3, encoding the adenosine A3 receptor, had a similar expression pattern. In primary ER+ breast cancer, high levels of the triplet ENTPD1/NT5E/ADORA3 expression signature was correlated with lower overall, distant metastasis-free, and progression-free survival. In ER+ bone metastasis biopsies, this expression signature is associated with lower survival. This expression signature was also higher in bone-metastasizing primary prostate cancers than in those that caused other tumor events or did not lead to progressive disease. In 3D culture, a non-hydrolyzable ATP analog inhibited the growth of breast and prostate cancer cell lines more than ATP did. A3 inhibition also reduced spheroid growth. Large-scale screens by the Drug Repurposing Hub found ER+ breast cancer cell lines were uniquely sensitive to adenosine receptor antagonists. Together, these data suggest a vital role for extracellular ATP degradation and adenosine receptor signaling in cancer bone metastasis, and this study provides potential diagnostic means for bone metastasis and specific targets for treatment and prevention.

Engineering Functional Vascularized Beige Adipose Tissue from Microvascular Fragments of Models of Healthy and Type II Diabetes Conditions.

Engineered beige adipose tissues could be used for screening therapeutic strategies or as a direct treatment for obesity and metabolic disease. Microvascular fragments are vessel structures that can be directly isolated from adipose tissue and may contain cells capable of differentiation into thermogenic, or beige, adipocytes. In this study, culture conditions were investigated to engineer three-dimensional, vascularized functional beige adipose tissue using microvascular fragments isolated from both healthy animals and a model of type II diabetes (T2D). Vascularized beige adipose tissues were engineered and exhibited increased expression of beige adipose markers, enhanced function, and improved cellular respiration. While microvascular fragments isolated from both lean and diabetic models were able to generate functional tissues, differences were observed in regard to vessel assembly and tissue function. This study introduces an approach that could be employed to engineer vascularized beige adipose tissues from a single, potentially autologous source of cells.

Engineering Human Beige Adipose Tissue.

In this study, we described a method for generating functional, beige (thermogenic) adipose microtissues from human microvascular fragments (MVFs). The MVFs were isolated from adipose tissue acquired from adults over 50 years of age. The tissues express thermogenic gene markers and reproduce functions essential for the potential therapeutic impact of beige adipose tissues such as enhanced lipid metabolism and increased mitochondrial respiration. MVFs serve as a potential single, autologous source of cells that can be isolated from adult patients, induced to recreate functional aspects of beige adipose tissue and enable rapid vascularization post-transplantation. This approach has the potential to be used as an autologous therapy for metabolic diseases or as a model for the development of a personalized approach to high-throughput drug development/screening for adipose tissue.

Studying macrophage activation in immune-privileged lens through CSF-1 protein intravitreal injection in mouse model.

Macrophage (MΦ) activation and promotion of fibrosis are critical processes in lens capsule healing after injury. Here, we detail a protocol that induces MΦ2 formation within the vitreous body of the eye. Our procedure combines the use of an intravitreal injection of a growth factor (CSF-1) and immunofluorescence to confirm the presence of MΦ2 and fibrotic tissue formation. This protocol allows assessment of the distribution of macrophages and quantification of fibrotic tissue formation/sealing within the vitreous body of mouse eyes. For complete details on the use and execution of this profile, please refer to Li et al. (2021), Gerhardt et al. (2003), Kubota et al. (2009).

Adipogenic Differentiation Alters Properties of Vascularized Tissue-Engineered Skeletal Muscle.

Advances in the engineering of comprehensive skeletal muscle models in vitro will improve drug screening platforms and can lead to better therapeutic approaches for the treatment of skeletal muscle injuries. To this end, a vascularized tissue-engineered skeletal muscle (TE-SkM) model that includes adipocytes was developed to better emulate the intramuscular adipose tissue that is observed in skeletal muscles of patients with diseases such as diabetes. Muscle precursor cells cultured with and without microvessels derived from adipose tissue (microvascular fragments) were used to generate TE-SkM constructs, with and without a microvasculature, respectively. TE-SkM constructs were treated with adipogenic induction media to induce varying levels of adipogenesis. With a delayed addition of induction media to allow for angiogenesis, a robust microvasculature in conjunction with an increased content of adipocytes was achieved. The augmentation of vascularized TE-SkM constructs with adipocytes caused a reduction in maturation (compaction), mechanical integrity (Young's modulus), and myotube and vessel alignment. An increase in basal glucose uptake was observed in both levels of adipogenic induction, and a diminished insulin-stimulated glucose uptake was associated with the higher level of adipogenic differentiation and the greater number of adipocytes.

ATP Inhibits Breast Cancer Migration and Bone Metastasis through Down-Regulation of CXCR4 and Purinergic Receptor P2Y11.

ATP released by bone osteocytes is shown to activate purinergic signaling and inhibit the metastasis of breast cancer cells into the bone. However, the underlying molecular mechanism is not well understood. Here, we demonstrate the important roles of the CXCR4 and P2Y11 purinergic receptors in mediating the inhibitory effect of ATP on breast cancer cell migration and bone metastasis. Wound-healing and transwell migration assays showed that non-hydrolysable ATP analogue, ATPγS, inhibited migration of bone-tropic human breast cancer cells in a dose-dependent manner. BzATP, an agonist for P2X7 and an inducer for P2Y11 internalization, had a similar dose-dependent inhibition on cell migration. Both ATPγS and BzATP suppressed the expression of CXCR4, a chemokine receptor known to promote breast cancer bone metastasis, and knocking down CXCR4 expression by siRNA attenuated the inhibitory effect of ATPγS on cancer cell migration. While a P2X7 antagonist A804598 had no effect on the impact of ATPγS on cell migration, antagonizing P2Y11 by NF157 ablated the effect of ATPγS. Moreover, the reduction in P2Y11 expression by siRNA decreased cancer cell migration and abolished the impact of ATPγS on cell migration and CXCR4 expression. Similar to the effect of ATPγS on cell migration, antagonizing P2Y11 inhibited bone-tropic breast cancer cell migration in a dose-dependent manner. An in vivo study using an intratibial bone metastatic model showed that ATPγS inhibited breast cancer growth in the bone. Taken together, these results suggest that ATP inhibits bone-tropic breast cancer cells by down-regulating the P2Y11 purinergic receptor and the down-regulation of CXCR4 expression.

Osteocytic Connexin43 Channels Regulate Bone-Muscle Crosstalk.

Bone-muscle crosstalk plays an important role in skeletal biomechanical function, the progression of numerous pathological conditions, and the modulation of local and distant cellular environments. Previous work has revealed that the deletion of connexin (Cx) 43 in osteoblasts, and consequently, osteocytes, indirectly compromises skeletal muscle formation and function. However, the respective roles of Cx43-formed gap junction channels (GJs) and hemichannels (HCs) in the bone-muscle crosstalk are poorly understood. To this end, we used two Cx43 osteocyte-specific transgenic mouse models expressing dominant negative mutants, Δ130-136 (GJs and HCs functions are inhibited), and R76W (only GJs function is blocked), to determine the effect of these two types of Cx43 channels on neighboring skeletal muscle. Blockage of osteocyte Cx43 GJs and HCs in Δ130-136 mice decreased fast-twitch muscle mass with reduced muscle protein synthesis and increased muscle protein degradation. Both R76W and Δ130-136 mice exhibited decreased muscle contractile force accompanied by a fast-to-slow fiber transition in typically fast-twitch muscles. In vitro results further showed that myotube formation of C2C12 myoblasts was inhibited after treatment with the primary osteocyte conditioned media (PO CM) from R76W and Δ130-136 mice. Additionally, prostaglandin E2 (PGE2) level was significantly reduced in both the circulation and PO CM of the transgenic mice. Interestingly, the injection of PGE2 to the transgenic mice rescued fast-twitch muscle mass and function; however, this had little effect on protein synthesis and degradation. These findings indicate a channel-specific response: inhibition of osteocytic Cx43 HCs decreases fast-twitch skeletal muscle mass alongside reduced protein synthesis and increased protein degradation. In contrast, blockage of Cx43 GJs results in decreased fast-twitch skeletal muscle contractile force and myogenesis, with PGE2 partially accounting for the measured differences.

Diabetic Conditions Confer Metabolic and Structural Modifications to Tissue-Engineered Skeletal Muscle.

Skeletal muscle is a tissue that is directly involved in the progression and persistence of type 2 diabetes (T2D), a disease that is becoming increasingly common. Gaining better insight into the mechanisms that are affecting skeletal muscle dysfunction in the context of T2D has the potential to lead to novel treatments for a large number of patients. Through its ability to emulate skeletal muscle architecture while also incorporating aspects of disease, tissue-engineered skeletal muscle (TE-SkM) has the potential to provide a means for rapid high-throughput discovery of therapies to treat skeletal muscle dysfunction, to include that which occurs with T2D. Muscle precursor cells isolated from lean or obese male Zucker diabetic fatty rats were used to generate TE-SkM constructs. Some constructs were treated with adipogenic induction media to accentuate the presence of adipocytes that is a characteristic feature of T2D skeletal muscle. The maturity (compaction and creatine kinase activity), mechanical integrity (Young's modulus), organization (myotube orientation), and metabolic capacity (insulin-stimulated glucose uptake) were all reduced by diabetes. Treating constructs with adipogenic induction media increased the quantity of lipid within the diabetic TE-SkM constructs, and caused changes in construct compaction, cell orientation, and insulin-stimulated glucose uptake in both lean and diabetic samples. Collectively, the findings herein suggest that the recapitulation of structural and metabolic aspects of T2D can be accomplished by engineering skeletal muscle in vitro.

Scaffold Architecture and Matrix Strain Modulate Mesenchymal Cell and Microvascular Growth and Development in a Time Dependent Manner.

BACKGROUND: Volumetric tissue-engineered constructs are limited in development due to the dependence on well-formed vascular networks. Scaffold pore size and the mechanical properties of the matrix dictates cell attachment, proliferation and successive tissue morphogenesis. We hypothesize scaffold pore architecture also controls stromal-vessel interactions during morphogenesis. METHODS: The interaction between mesenchymal stem cells (MSCs) seeded on hydroxyapatite scaffolds of 450, 340, and 250 µm pores and microvascular fragments (MVFs) seeded within 20 mg/mL fibrin hydrogels that were cast into the cell-seeded scaffolds, was assessed in vitro over 21 days and compared to the fibrin hydrogels without scaffold but containing both MSCs and MVFs. mRNA sequencing was performed across all groups and a computational mechanics model was developed to validate architecture effects on predicting vascularization driven by stiffer matrix behavior at scaffold surfaces compared to the pore interior. RESULTS: Lectin staining of decalcified scaffolds showed continued vessel growth, branching and network formation at 14 days. The fibrin gel provides no resistance to spread-out capillary networks formation, with greater vessel loops within the 450 µm pores and vessels bridging across 250 µm pores. Vessel growth in the scaffolds was observed to be stimulated by hypoxia and successive angiogenic signaling. Fibrin gels showed linear fold increase in VEGF expression and no change in BMP2. Within scaffolds, there was multiple fold increase in VEGF between days 7 and 14 and early multiple fold increases in BMP2 between days 3 and 7, relative to fibrin. There was evidence of yap/taz based hippo signaling and mechanotransduction in the scaffold groups. The vessel growth models determined by computational modeling matched the trends observed experimentally. CONCLUSION: The differing nature of hypoxia signaling between scaffold systems and mechano-transduction sensing matrix mechanics were primarily responsible for differences in osteogenic cell and microvessel growth. The computational model implicated scaffold architecture in dictating branching morphology and strain in the hydrogel within pores in dictating vessel lengths.

Divergent effects of myogenic differentiation and diabetes on the capacity for muscle precursor cell adipogenic differentiation in a fibrin matrix.

The development of ectopic adipose tissue in skeletal muscle is associated with several skeletal muscle and metabolic pathologies, including Type II Diabetes Mellitus. The adipogenic differentiation of muscle precursor cells (MPCs) has been postulated to occur in skeletal muscle in vivo in a three-dimensional (3-D) configuration; therefore, it is appropriate to investigate this phenomenon using 3-D matrices in vitro. The capacity for MPC adipogenic differentiation in a 3-D environment was investigated in fibrin hydrogels by treating MPCs derived from healthy or diabetic animals with adipogenic induction medias that differed in their ability to increase lipid accumulation and activate the expression of genes associated with adipogenic differentiation (peroxisome proliferator-activated receptor gamma (PPARG), adiponectin (ADIPOQ), and fatty acid synthase (FAS)). The capacity for adipogenic differentiation was diminished, but not prevented, if myogenic differentiation preceded MPC exposure to adipogenic induction conditions. Conversely, adipogenic differentiation was greater in hydrogels containing MPCs from diabetic rats as compared to those derived from lean rats, as evidenced by an increase in lipid accumulation and adipogenic gene expression. Collectively, the data herein support a role for the MPCs in adipogenesis in a 3-D environment and that they may contribute to the ectopic accumulation of adipose tissue. The observation that the potential for adipogenic differentiation is maintained even after a period of myogenic differentiation alludes to the possibility that adipogenesis may occur during different phases of muscle development. Finally, the increase in adipogenic differentiation in hydrogels containing MPCs derived from diabetic animals provides strong evidence that a pathological environment in vivo increases their capacity for adipogenesis.