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Tumors were characterized as nonhealing wounds by Virchow in 1858 and Dvorak in 1986. Since then, researchers have analyzed tumors from a new perspective. The parallels between tumorigenesis and physiological wound healing can provide a new framework for developing antitumor therapeutics. One commonality between tumors and wounds is the involvement of the stromal environment, particularly adipose stromal/stem cells (ASCs). ASCs exhibit dual functions, in which they stimulate tumor progression and assist in tissue repair and regeneration. Numerous studies have focused on the role of ASCs in cancer and wound healing, but none to date has linked age, cancer, and wound healing. Furthermore, very few studies have focused on the role of donor-specific characteristics of ASCs, such as age and their role in facilitating ASC behavior in cancer and wound healing. This review article is designed to provide important insights into the impact of donor age on ASC tumor and wound response and their role in facilitating ASC behavior in cancer and wound healing.

Supplementary Tables 1-8 from Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells

Supplementary Figure 1 from Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells

Supplementary Figure 2C from Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) derived from bone marrow stem cells (BMSC) and adipose tissue stem cells (ASC) of humans and rhesus macaques were evaluated for their cell cycle properties during protracted culture in vitro. Human ASCs (hASC) and rhesus BMSCs (rBMSC) underwent significantly more total population doublings than human BMSCs (hBMSC) and rhesus ASCs (rASC). The cell cycle profile of all MSCs was altered as cultures aged. hMSCs underwent an increase in the frequency of cells in the S phase at P20 and P30. However, rhesus MSCs from both sources developed a distinct polyploid population of cells at P20, which progressed to aneuploidy by P30. Karyotype analysis of MSCs revealed the development of tetraploid or aneuploid karyotypes in the rhesus cells at P20 or P30. Analysis of the transcriptome of the MSCs from early and late passages revealed significant alterations in the patterns of gene expression (8.8% of the genes were differentially expressed in hBMSCs versus hASCs, and 5.5% in rBMSCs versus rASCs). Gene expression changes were much less evident within the same cell type as aging occurred (0.7% in hMSCs and 0.9% in rMSC). Gene ontology analysis showed that functions involved in protein catabolism and regulation of pol II transcription were overrepresented in rASCs, whereas the regulation of IκB/nuclear factor-κB cascade were overrepresented in hBMSCs. Functional analysis of genes that were differentially expressed in rASCs and hBMSCs revealed that pathways involved in cell cycle, cell cycle checkpoints, protein-ubiquitination, and apoptosis were altered. [Cancer Res 2008;68(11):4229–38]

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either “systemic administration” through all the medium streams or “intraarticular administration” by adding the drugs to only the shared “synovial fluid”-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

Dermal fibroblasts (DFs) share several qualities with mesenchymal stem cell/multipotent stromal cells (MSCs) derived from various tissues, including adipose-derived stromal/stem cells (ASCs). ASCs and DFs are morphologically comparable and both cell types can be culture expanded through the utilization of their plastic-adherence properties. Despite these similar characteristics, numerous studies indicate that ASC and DF display distinct therapeutic benefits in clinical applications. To more accurately distinguish between these cell types, human DFs and ASCs isolated from three individual donors were analyzed for multipotency and cell surface marker expressions. The detection of cell surface markers, CD29, CD34, CD44, CD73, CD90, and CD105, were used for phenotypic characterization of the DFs and ASCs. Furthermore, both cell types underwent lineage differentiation based on histochemical staining and the expression of adipogenic related genes, CCAAT/Enhancer-Binding Protein alpha (CEBPα), Peroxisome proliferator-activated receptor gamma (PPARγ), UCP1, Leptin (LEP), and Adiponectin (ADIPOQ); and osteogenic related genes, Runt related transcription factor 2 (Runx2), Alkaline phosphatase (ALPL), Osteocalcin (OCN), and Osteopontin (OPN). Evidence provided by this study demonstrates similarities between donor-matched ASC and DF with respect to morphology, surface marker expression, differentiation potential, and gene expression, although appearance of enhanced adipogenesis in the ASC based solely on spectrophotometric analyses with no significant difference in real-time polymerase chain reaction detection of adipogenic biomarkers. Thus, there is substantial overlap between the ASC and DF phenotypes based on biochemical and differentiation metrics.

Metaplastic breast carcinoma (MBC) is a rare breast cancer subtype with rapid growth, high rates of metastasis, recurrence and drug resistance, and diverse molecular and histological heterogeneity. Patient-derived xenografts (PDXs) provide a translational tool and physiologically relevant system to evaluate tumor biology of rare subtypes. Here, we provide an in-depth comprehensive characterization of a new PDX model for MBC, TU-BcX-4IC. TU-BcX-4IC is a clinically aggressive tumor exhibiting rapid growth in vivo, spontaneous metastases, and elevated levels of cell-free DNA and circulating tumor cell DNA. Relative chemosensitivity of primary cells derived from TU-BcX-4IC was performed using the National Cancer Institute (NCI) oncology drug set, crystal violet staining, and cytotoxic live/dead immunofluorescence stains in adherent and organoid culture conditions. We employed novel spheroid/organoid incubation methods (Pu·MA system) to demonstrate that TU-BcX-4IC is resistant to paclitaxel. An innovative physiologically relevant system using human adipose tissue was used to evaluate presence of cancer stem cell-like populations ex vivo. Tissue decellularization, cryogenic-scanning electron microscopy imaging and rheometry revealed consistent matrix architecture and stiffness were consistent despite serial transplantation. Matrix-associated gene pathways were essentially unchanged with serial passages, as determined by qPCR and RNA sequencing, suggesting utility of decellularized PDXs for in vitro screens. We determined type V collagen to be present throughout all serial passage of TU-BcX-4IC tumor, suggesting it is required for tumor maintenance and is a potential viable target for MBC. In this study we introduce an innovative and translational model system to study cell–matrix interactions in rare cancer types using higher passage PDX tissue.

Osteoarthritis (OA) is a painful and disabling disease that affects millions of people worldwide. Symptom-alleviating treatments exist, although none with long-term efficacy. Furthermore, there are currently no disease-modifying OA drugs (DMOADs) with demonstrated efficacy in OA patients, which is, in part, attributed to a lack of full understanding of the pathogenesis of OA. The inability to translate findings from basic research to clinical applications also highlights the deficiencies in the available OA models at simulating the clinically relevant pathologies and responses to treatments in humans. In this review, the current status in the development of DMOADs will be first presented, with special attention to those in Phase II–IV clinical trials. Next, current in vitro, ex vivo, and in vivo OA models are summarized and the respective advantages and disadvantages of each are highlighted. Of note, the development and application of microphysiological or tissue-on-a-chip systems for modeling OA in humans are presented and the issues that need to be addressed in the future are discussed. Microphysiological systems should be given serious consideration for their inclusion in the DMOAD development pipeline, both for their ability to predict drug safety and efficacy in human clinical trials at present, as well as for their potential to serve as a test platform for personalized medicine. IMPACT STATEMENT: At present, no disease-modifying osteoarthritis (OA) drugs (DMOADs) have been approved for widespread clinical use by regulatory bodies. The failure of developing effective DMOADs is likely owing to multiple factors, not the least of which are the intrinsic differences between the intact human knee joint and the preclinical models. This work summarizes the current OA models for the development of DMOADs, discusses the advantages/disadvantages of each, and then proposes future model development to aid in the discovery of effective and personalized DMOADs. The review also highlights the microphysiological systems, which are emerging as a new platform for drug development.

Adipose tissue is widely recognized as an abundant and accessible human tissue that serves as a source of cells and extracellular matrix scaffolds for regenerative surgical applications. Increasingly, orthopedic surgeons are turning to adipose tissue as a resource in their treatment of osteoarthritis and related conditions. In the U.S., the regulatory landscape governing the orthopedic surgical utilization of autologous and allogeneic adipose tissue remains complex. This manuscript reviews the Food and Drug Administration's nomenclature and guidance regarding adipose tissue products. Additionally, it surveys recent pre-clinical and clinical trial literature relating to the application of adipose-derived cells and tissues in the treatment of osteoarthritis.

Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell-derived microphysiological system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell-derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial-like fibrous tissue, and adipose tissue are integrated into a microfluidics-enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin-1β mediated inflammation, which are similar to observations in joint diseases in humans. The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the "therapeutic effect" of the nonsteroidal anti-inflammatory drug, naproxen, as well as four other potential disease-modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions.

As musculoskeletal (MSK) disorders continue to increase globally, there is an increased need for novel, in vitro models to efficiently study human bone physiology in the context of both healthy and diseased conditions. For these models, the inclusion of innate immune cells is critical. Specifically, signaling factors generated from macrophages play key roles in the pathogenesis of many MSK processes and diseases, including fracture, osteoarthritis, infection etc. In this study, we aim to engineer three-dimensional (3D) and macrophage-encapsulated bone tissues in vitro, to model cell behavior, signaling, and other biological activities in vivo, in comparison to current two-dimensional models. We first investigated and optimized 3D culture conditions for macrophages, and then co-cultured macrophages with mesenchymal stem cells (MSCs), which were induced to undergo osteogenic differentiation to examine the effect of macrophage on new bone formation. Seeded within a 3D hydrogel scaffold fabricated from photocrosslinked methacrylated gelatin, macrophages maintained high viability and were polarized toward an M1 or M2 phenotype. In co-cultures of macrophages and human MSCs, MSCs displayed immunomodulatory activities by suppressing M1 and enhancing M2 macrophage phenotypes. Lastly, addition of macrophages, regardless of polarization state, increased MSC osteogenic differentiation, compared with MSCs alone, with proinflammatory M1 macrophages enhancing new bone formation most effectively. In summary, this study illustrates the important roles that macrophage signaling and inflammation play in bone tissue formation.

Breast cancer affects women globally; the majority of breast cancer-related mortalities are due to metastasis. Acquisition of a mesenchymal phenotype has been implicated in the progression of breast cancer cells to an invasive, metastatic state. Triple-negative breast cancer (TNBC) subtypes have high rates of metastases, recurrence, and have poorer prognoses compared to other breast cancer types, partially due to lack of commonly targeted receptors. Kinases have diverse and pivotal functions in metastasis in TNBC, and discovery of new kinase targets for TNBC is warranted. We previously used a screening approach to identify intermediate-synthesis nonpotent, nonselective small-molecule inhibitors from the Published Kinase Inhibitor Set that reversed the mesenchymal phenotype in TNBC cells. Two of these inhibitors (GSK346294A and GSK448459A) are structurally similar, but have unique kinase activity profiles and exhibited differential biologic effects on TNBC cells, specifically on epithelial-to-mesenchymal transition (EMT). Here, we further interrogate these effects and compare activity of these inhibitors on transwell migration, gene (qRT-PCR) and protein (western blot) expressions, and cancer stem cell-like behavior. We incorporated translational patient-derived xenograft models in these studies, and we focused on the lead inhibitor hit, GSK346294A, to demonstrate the utility of our comparative analysis as a screening modality to identify novel kinase targets and signaling pathways to pursue in TNBC. This study introduces a new method for discovering novel kinase targets that reverse the EMT phenotype; this screening approach can be applied to all cancer types and is not limited to breast cancer.

There are more than 3 million breast cancer survivors living in the United States of which a significant number have undergone mastectomy followed by breast and nipple-areolar complex (NAC) reconstruction. Current strategies for NAC reconstruction are dependent on nonliving or nonpermanent techniques, including tattooing, nipple prosthetics, or surgical nipple-like structures. Described herein is a tissue engineering approach demonstrating the feasibility of an allogeneic acellular graft for nipple reconstruction. Nonhuman primate (NHP)-derived NAC tissues were decellularized and their extracellular matrix components analyzed by both proteomic and histological analyses. Decellularized NHP nipple tissue showed the removal of intact cells and greatly diminished profiles for intracellular proteins, as compared with intact NHP nipple tissue. We further evaluated the biocompatibility of decellularized grafts and their potential to support host-mediated neovascularization against commercially available acellular dermal grafts by performing in vivo studies in a murine model. A follow-up NHP pilot study evaluated the host-mediated neovascularization and re-epithelialization of onlay engrafted decellularized NAC grafts. The murine model revealed greater neovascularization in the decellularized NAC than in the commercially available control grafts, with no observed biocompatibility issues. The in vivo NHP model confirmed that the decellularized NAC grafts encourage neovascularization as well as re-epithelialization. These results support the concept that a biologically derived acellular nipple graft is a feasible approach for nipple reconstruction, supporting neovascularization in the absence of adverse systemic responses. Impact statement Currently, women in the United States most often undergo a mastectomy, followed by reconstruction, after being diagnosed with breast cancer. These breast cancer survivors are often left with nipple-areolar complex (NAC) reconstructions that are subsatisfactory, nonliving, and/or nonpermanent. Utilizing an acellular biologically derived whole NAC graft would allow these patients a living and permanent tissue engineering solution to nipple reconstruction.

Despite a decline in overall incidence rates for cancer in the past decade, due in part to impressive advancements in both diagnosis and treatment, breast cancer (BC) remains the leading cause of cancer-related deaths in women. BC alone accounts for ∼30% of all new cancer diagnoses in women worldwide. Triple-negative BC (TNBC), defined as having no expression of the estrogen or progesterone receptors and no amplification of the HER2 receptor, is a subtype of BC that does not benefit from the use of estrogen receptor-targeting or HER2-targeting therapies. Differences in socioeconomic factors and cell intrinsic and extrinsic characteristics have been demonstrated in Black and White TNBC patient tumors. The emergence of patient-derived xenograft (PDX) models as a surrogate, translational, and functional representation of the patient with TNBC has led to the advances in drug discovery and testing of novel targeted approaches and combination therapies. However, current established TNBC PDX models fail to represent the diverse patient population and, most importantly, the specific ethnic patient populations that have higher rates of incidence and mortality. The primary aim of this review is to emphasize the importance of using clinically relevant translatable tumor models that reflect TNBC human tumor biology and heterogeneity in high-risk patient populations. The focus is to highlight the complexity of BC as it specifically relates to the management of TNBC in Black women. We discuss the importance of utilizing PDX models to study the extracellular matrix (ECM), and the distinct differences in ECM composition and biophysical properties in Black and White women. Finally, we demonstrate the crucial importance of PDX models toward novel drug discovery in this patient population.

In vitro models of angiogenesis are valuable tools for understanding the underlying mechanisms of pathological conditions and for the preclinical evaluation of therapies. Our laboratory developed the rat mesentery culture model as a new tool for investigating mechanistic cell–cell interactions at specific locations across intact blood and lymphatic microvascular networks ex vivo. The objective of this study was to report a method for evaluating the effect of aging on human stem cell differentiation into pericytes during angiogenesis in cultured microvascular networks. DiI labeled exogenous stem cells were seeded onto harvested adult Wistar rat mesenteric tissues and cultured in alpha-MEM + 1% serum for up to 5 days according to four experimental groups: (1) adult human adipose–derived stem cells (hASCs), (2) aged hASCs, (3) adult human bone marrow-derived stem cells (hBMSCs), and (4) aged hBMSCs. Angiogenesis per experimental group was supported by observation of increased vessel density and capillary sprouting. For each tissue per experimental group, a subset of cells was observed in typical pericyte location wrapped along blood vessels. Stem cell differentiation into pericytes was supported by the adoption of elongated pericyte morphology along endothelial cells and positive NG2 labeling. The percentage of cells in pericyte locations was not significantly different across the experimental groups, suggesting that aged mesenchymal stem cells are able to retain their differentiation capacity. Our results showcase an application of the rat mesentery culture model for aging research and the evaluation of stem cell fate within intact microvascular networks.

There are over 3M breast cancer survivors in the US, many of whom have undergone breast reconstructive surgery. With one-third of early stage and two-thirds of late-stage breast cancer patients opting for mastectomies without a standard, viable method to reliably reconstruct a permanent and natural nipple-areolar complex (NAC). NAC reconstruction is of key psychological importance for these patients, decreasing feelings of distress and increasing body image and self-esteem. Current NAC reconstruction options include rubber prostheses, 3D tattoos, and surgical techniques to create NAC-like structures from a patient’s own skin tissue. These approaches produce NACs that either lack physical depth or fail to maintain a protrusion. The objectives of the non-human primate 6 week engraftment studies with our decellularized human nipple areolar complex (hNAC) grafts were 1) to determine the biocompatibility of the hNAC graft and 2) to measure host-mediated recellularization of the hNAC graft on a nearest-to-human model species. If the hNAC graft maintains cell-derived insoluble matrix components yet is devoid of host cells, intracellular material, and pathogenic organisms, then the hNAC graft will not elicit an adverse humoral response and will show both good biocompatibility and recellularization. The methods employed for our studies involved surgical engraftment of the hNAC graft along the dorsal midline of three adult male non-human primates (rhesus macaque). The number of hNAC grafts engrafted for each NHP was 12 for one NHP and 18 for the other two. Gross animal weight, complete blood count data, and metabolite data were collected weekly from periods before, during (span of 6-weeks), and after hNAC engraftment for each animal. In addition, hNACs were surgically removed at defined weekly time points during the study for histological analysis of recellularization. All data was compiled for each study and statistically analyzed separately and combined. Statistical analyses included unpaired, parametric comparisons of systemic indicators (i.e. weight, complete blood count, metabolites) at each period of the study and of re-epithelialization and neovascular content for individual hNAC grafts during host-mediated recellularization over time. The results from this NHP study demonstrate good evidence of biocompatibility of the hNAC on non-human primates. Gross body weight remained unchanged for all NHPs and complete blood counts (e.g. red blood cells, white blood cells, platelets) and metabolites remained mostly within normal ranges for all NHPs. The hNAC grafts did not effect liver, kidney, or pancreas function. The hNAC grafts showed good recellularization during the study with significant increases measured for re-epithelialization and neovascularization. Histological analysis further revealed functioning microvasculature within grafts as early as 3-weeks post-engraftment. These data suggest that the hNAC graft is biocompatible and capable of recellularization. The presence of de novo epithelia and vasculature within the hNAC grafts collectively support recellularization and regeneration of a hNAC. Citation Format: David Graham, William Heim, Vincent Caronna, Abigail Chaffin, Brooke Grasperge, Scott Sullivan, Bruce A Bunnell, Nicholas C Pashos. New approach to nipple reconstruction: In vivo evaluation of acellular nipple-areolar complex grafts [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-14-13.

Osteoarthritis (OA) is a degenerative joint disease resulting in limited mobility and severe disability. Type II diabetes mellitus (T2D) is a weight-independent risk factor for OA, but a link between the two diseases has not been elucidated. Adipose stem cells (ASCs) isolated from the infrapatellar fat pad (IPFP) may be a viable regenerative cell for OA treatment. This study analyzed the expression profiles of inflammatory and adipokine-related genes in IPFP-ASCs of non-diabetic (Non-T2D), pre-diabetic (Pre-T2D), and T2D donors. Pre-T2D ASCs exhibited a substantial decrease in levels of mesenchymal markers CD90 and CD105 with no change in adipogenic differentiation compared to Non-T2D and T2D IPFP-ASCs. In addition, Cyclooxygenase-2 (

Liver kinase B1 (LKB1) is a potent tumor suppressor that regulates cellular energy balance and metabolism as an upstream kinase of the AMP-activated protein kinase (AMPK) pathway. LKB1 regulates cancer cell invasion and metastasis in multiple cancer types, including breast cancer. In this study, we evaluated LKB1’s role as a regulator of the tumor microenvironment (TME). This was achieved by seeding the MDA-MB-231-LKB1 overexpressing cell line onto adipose and tumor scaffolds, followed by the evaluation of tumor matrix-induced tumorigenesis and metastasis. Results demonstrated that the presence of tumor matrix enhanced tumorigenesis in both MDA-MB-231 and MDA-MB-231-LKB1 cell lines. Metastasis was increased in both MDA-MB-231 and -LKB1 cells seeded on the tumor scaffold. Endpoint analysis of tumor and adipose scaffolds revealed LKB1-mediated tumor microenvironment remodeling as evident through altered matrix protein production. The proteomic analysis determined that LKB1 overexpression preferentially decreased all major and minor fibril collagens (collagens I, III, V, and XI). In addition, proteins observed to be absent in tumor scaffolds in the LKB1 overexpressing cell line included those associated with the adipose matrix (COL6A2) and regulators of adipogenesis (IL17RB and IGFBP4), suggesting a role for LKB1 in tumor-mediated adipogenesis. Histological analysis of MDA-MB-231-LKB1-seeded tumors demonstrated decreased total fibril collagen and indicated decreased stromal cell presence. In accordance with this, in vitro condition medium studies demonstrated that the MDA-MB-231-LKB1 secretome inhibited adipogenesis of adipose-derived stem cells. Taken together, these data demonstrate a role for LKB1 in regulating the tumor microenvironment through fibril matrix remodeling and suppression of adipogenesis.

Due to their significant therapeutic potential, adipose stem cells (ASCs) have become the focus of intense of preclinical and clinical research. Characterization of ASCs that is both comprehensive and well standardized is essential for the successful progression of the regenerative medicine field. This chapter provides a detailed outline of the in vitro assays commonly employed for the characterization of ASCs, including flow cytometric assessments of surface markers, self-renewal, and multipotent differentiation. An in-depth examination of adipogenic, osteogenic, and chondrogenic differentiation is covered, including signaling pathways, methodology, and confirmation assays. Furthermore, ASCs possess significant immunomodulatory abilities that make them ideal therapeutic tools. This chapter defines the influence of ASCs on immune cell populations in vitro, including paracrine and cell contact-dependent mechanisms, and summarizes the assays used to quantify this immunoregulation. Research on ASCs is a rapidly evolving field. Rigorous characterization standards, as recommended by the International Federation of Adipose Therapeutics and Sciences (IFATS) and others, must be adopted and maintained by researchers to promote high-impact research, reproducibility, effective collaborations, and acceleration of scientific discoveries in regenerative medicine.

Pain is the predominant symptom of osteoarthritis (OA) that drives patients to seek medical care. Currently, there are no pharmacological treatments that can reverse or halt the progression of OA. Safe and efficacious medications for long-term management of OA pain are also unavailable. Understanding the mechanisms behind OA pain generation at onset and over time is critical for developing effective treatments. In this narrative review, we first summarize our current knowledge on the innervation of the knee joint, and then discuss the molecular mechanism(s) currently thought to underlie OA pain. In particular, we focus on the contribution of each joint component to the generation of pain. Next, the current experimental models for studying OA pain are summarized, and the methods to assess pain in rodents are presented. The potential application of emerging microphysiological systems in OA pain research is especially highlighted. Lastly, we discuss the current challenge in standardizing models and the selection of appropriate systems to address specific questions.

Obesity rates are climbing, representing a confounding and contributing factor to many disease states, including cancer. With respect to breast cancer, obesity plays a prominent role in the etiology of this disease, with certain subtypes such as triple-negative breast cancer having a strong correlation between obesity and poor outcomes. Therefore, it is critical to examine the obesity-related alterations to the normal stroma and the tumor microenvironment (TME). Adipocytes and adipose stem cells (ASCs) are major components of breast tissue stroma that have essential functions in both physiological and pathological states, including energy storage and metabolic homeostasis, physical support of breast epithelial cells, and directing inflammatory and wound healing responses through secreted factors. However, these processes can become dysregulated in both metabolic disorders, such as obesity and also in the context of breast cancer. Given the well-established obesity-neoplasia axis, it is critical to understand how interactions between different cell types in the tumor microenvironment, including adipocytes and ASCs, govern carcinogenesis, tumorigenesis, and ultimately metastasis. ASCs and adipocytes have multifactorial roles in cancer progression; however, due to the plastic nature of these cells, they also have a role in regenerative medicine, making them promising tools for tissue engineering. At the physiological level, the interactions between obesity and breast cancer have been examined; here, we will delineate the mechanisms that regulate ASCs and adipocytes in these different contexts through interactions between cancer cells, immune cells, and other cell types present in the tumor microenvironment. We will define the current state of understanding of how adipocytes and ASCs contribute to tumor progression through their role in the tumor microenvironment and how this is altered in the context of obesity. We will also introduce recent developments in utilizing adipocytes and ASCs in novel approaches to breast reconstruction and regenerative medicine.

Fibroblasts in the synovial membrane secrete molecules essential to forming the extracellular matrix (ECM) and supporting joint homeostasis. While evidence suggests that fibroblasts contribute to the response to joint injury, the outcomes appear to be patient-specific and dependent on interactions between resident immune cells, particularly macrophages (Mφs). On the other hand, the response of Mφs to injury depends on their functional phenotype. The goal of these studies was to further explore these issues in an in vitro 3D microtissue model that simulates a pathophysiological disease-specific microenvironment. Two sources of fibroblasts were used to assess patient-specific influences: mesenchymal stem cell (MSC)- and induced pluripotent stem cell (iPSC)-derived fibroblasts. These were co-cultured with either M1 or M2 Mφs, and the cultures were challenged with polyethylene particles coated with lipopolysaccharide (cPE) to model wear debris generated from total joint arthroplasties. Our results indicated that the fibroblast response to cPE was dependent on the source of the fibroblasts and the presence of M1 or M2 Mφs: the fibroblast response as measured by gene expression changes was amplified by the presence of M2 Mφs. These results demonstrate that the immune system modulates the function of fibroblasts; furthermore, different sources of differentiated fibroblasts may lead to divergent results. Overall, our research suggests that M2 Mφs may be a critical target for the clinical treatment of cPE induced fibrosis.

Triple-negative breast cancer (TNBC) is an aggressive, molecularly heterogeneous subtype of breast cancer. Obesity is associated with increased incidence and worse prognosis in TNBC through various potential mechanisms. Recent evidence suggests that the gut microbiome plays a central role in the progression of cancer, and that imbalances or dysbiosis in the population of commensal microbiota can lead to inflammation and contribute to tumor progression. Obesity is characterized by low-grade inflammation, and gut dysbiosis is associated with obesity, chronic inflammation, and failure of cancer immunotherapy. However, the debate on what constitutes a “healthy” gut microbiome is ongoing, and the connection among the gut microbiome, obesity, and TNBC has not yet been addressed. This study aims to characterize the role of obesity in modulating the gut microbiome in a syngeneic mouse model of TNBC. 16S rRNA sequencing and metagenomic analyses were performed to analyze and annotate genus and taxonomic profiles. Our results suggest that obesity decreases alpha diversity in the gut microbiome. Metagenomic analysis revealed that obesity was the only significant factor explaining the similarity of the bacterial communities according to their taxonomic profiles. In contrast to the analysis of taxonomic profiles, the analysis of variation of functional profiles suggested that obesity status, tumor presence, and the obesity–tumor interaction were significant in explaining the variation of profiles, with obesity having the strongest correlation. The presence of tumor modified the profiles to a greater extent in obese than in lean animals. Further research is warranted to understand the impact of the gut microbiome on TNBC progression and immunotherapy.

Purpose Diffusion MRI offers insight into ischemic stroke progression in both human and rodent models. However, diffusion MRI to evaluate therapeutic application of mesenchymal stem cells is limited. Robust analytical techniques are required to identify potential physiological changes as a function of cell therapy in stroke. Here, we seek to establish Neurite Orientation Dispersion and Density Imaging (NODDI) as a feasible method in evaluating stroke evolution in response to cell-based therapeutics. Methods Diffusion MRI data at 21.1T were acquired from 16 male rats. Rats were grouped randomly: naive (baseline, N = 5), stroke with injections of phosphate buffered saline (N = 6), stroke with injection of 2D human mesenchymal stem cells (hMSC, N = 5). Data were acquired on days 1, 3, 7, and 21 post-surgery. DTI and NODDI maps were generated, with regions of interest placed in the ischemic hemisphere external capsule and striatum. Diffusion parameters were compared between groups each day, and within groups across hemispheres and longitudinally. Behavioral characterizations were on days 0 (pre-surgery), 3, 7, 14, and 21. Results The 2D hMSC preserved diffusional restriction in the external capsule compared to saline (day 1: MD, P = .4060; AD, P = .0220). NODDI indicates that hMSC may have preserved intracellular volume fractions (ICVF: day 1, P = .0086; day 3, P = .0021; day 21, P = .0383). Diffusion metrics of hMSC treated animals were comparable to naive for the external capsule. Conclusions NODDI compliments DTI metrics, enhances interpretation of tissue outcome in ischemic stroke following hMSC application, and may be useful in evaluating or predicting therapeutic response.

In vivo mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to nonengraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the survival of MSC implants is unknown. Here, we employ a biomarker of cellular aging, the decoy TRAIL receptor CD264, to compare the survival kinetics of two cell populations in human bone marrow MSC (hBM-MSC) cultures. Sorted CD264+ hBM-MSCs from two age-matched donors have elevated β-galactosidase activity, decreased differentiation potential and form in vitro colonies inefficiently relative to CD264- hBM-MSCs. Counterintuitive to their aging phenotype, CD264+ hBM-MSCs exhibited comparable survival to matched CD264- hBM-MSCs from the same culture during in vitro colony formation and in vivo when implanted ectopically in immunodeficient NIH III mice. In vitro and in vivo survival of these two cell populations were independent of colony-forming efficiency. These findings have ramifications for the preparation of hBM-MSC therapies given the prevalence of aging CD264+ cells in hBM-MSC cultures and the popularity of colony-forming efficiency as a quality control metric in preclinical and clinical studies with MSCs.

Chronic wounds are characterized by an increased bacterial presence, alkaline pH, and excessive wound drainage. Hydrogel biomaterials composed of the carbohydrate polymer chitosan are advantageous for wound healing applications because of their innate antimicrobial and hemostatic properties. Here, genipin-cross-linked-chitosan hydrogels were synthesized and characterized, and their

The development of resistance to therapy is a significant obstacle to effective therapeutic regimens. Evaluating the effects of oncology drugs in the laboratory setting is limited by the lack of translational models that accurately recapitulate cell–microenvironment interactions present in tumors. Acquisition of resistance to therapy is facilitated, in part, by the composition of the tumor extracellular matrix (ECM), with the primary current in vitro model using collagen I (COL I). Here we seek to identify the prevalence of COL I-enhanced expression in the triple-negative breast cancer (TNBC) subtype. Furthermore, we identify if methods of response to therapy are altered depending on matrix composition. We demonstrated that collagen content varies in patient tumor samples across subtypes, with COL I expression dramatically increased in typically less aggressive estrogen receptor (ER)-positive(ER(+))/progesterone receptor (PGR)-positive (PGR(+)) cancers irrespective of patient age or race. These findings are of significance considering how frequently COL I is implicated in tumor progression. In vitro analyses of ER(+) and ER-negative (ER(–)) cell lines were used to determine the effects of ECM content (collagen I, collagen IV, fibronectin, and laminin) on proliferation, cellular phenotype, and survival. Neither ER(+) nor ER(–) cells demonstrated significant increases in proliferation when cultured on these ECM substrates. ER(–) cells cultured on these substrates were sensitized to both chemotherapy and targeted therapy. In addition, MDA-MB-231 cells expressed different morphologies, binding affinities, and stiffness across these substrates. We also demonstrated that ECM composition significantly alters transcription of senescence-associated pathways across ER(+) and ER(–) cell lines. Together, these results suggest that complex matrix composites should be incorporated into in vitro tumor models, especially for the drug-resistant TNBC subtype. IMPACT STATEMENT: The importance of tumor extracellular matrix (ECM) in disease progression is often inadequately represented in models of breast cancer that rely heavily on collagen I and Matrigel. Through immunohistochemistry analysis of patient breast tumors, we show a wide variation in collagen content based on subtype, specifically a repression of fibril collagens in the receptor negative subtype, irrespective of age and race. We also demonstrated that tumor ECM composition alters cellular elasticity and oncogenic pathway activation demonstrating that physiologically relevant three-dimensional models of breast cancer should include an ECM that is subtype specific.

Human mesenchymal stem or stromal cells (hMSCs) are known for their potential in regenerative medicine due to their differentiation abilities, secretion of trophic factors, and regulation of immune responses in damaged tissues. Due to the limited quantity of hMSCs typically isolated from bone marrow, other tissue sources, such as adipose tissue-derived mesenchymal stem cells (hASCs), are considered a promising alternative. However, differences have been observed for hASCs in the context of metabolic characteristics and response to in vitro culture stress compared to bone marrow derived hMSCs (BM-hMSCs). In particular, the relationship between metabolic homeostasis and stem cell functions, especially the immune phenotype and immunomodulation of hASCs, remains unknown. This study thoroughly assessed the changes in metabolism, redox cycles, and immune phenotype of hASCs during in vitro expansion. In contrast to BM-hMSCs, hASCs did not respond to culture stress significantly during expansion as limited cellular senescence was observed. Notably, hASCs exhibited the increased secretion of pro-inflammatory cytokines and the decreased secretion of anti-inflammatory cytokines after extended culture expansion. The NAD+/NADH redox cycle and other metabolic characteristics associated with aging were relatively stable, indicating that hASC functional decline may be regulated through an alternative mechanism rather than NAD+/Sirtuin aging pathways as observed in BM-hMSCs. Furthermore, transcriptome analysis by mRNA-sequencing revealed the upregulation of genes for pro-inflammatory cytokines/chemokines and the downregulation of genes for anti-inflammatory cytokines for hASCs at high passage. Proteomics analysis indicated key pathways (e.g., tRNA charging, EIF2 signaling, protein ubiquitination pathway) that may be associated with the immune phenotype shift of hASCs. Together, this study advances our understanding of the metabolism and senescence of hASCs and may offer vital insights for the biomanufacturing of hASCs for clinical use.

Lipedema is an underdiagnosed painful adipose tissue disorder that occurs almost exclusively in women, with onset manifesting at puberty or at times of hormonal change. Unlike many fat disorders, diet and exercise have little to no impact on the prevention or progression of this disease. Estrogens control the distribution of body fat and food intake, regulate leptin expression, increase insulin sensitivity, and reduce inflammation through signaling pathways mediated by its receptors, estrogen receptor alpha (ERα) and ERβ. This review will focus on understanding the role of estrogen in the pathogenesis of the disease and envisage potential hormonal therapy for lipedema patients.

Human adipose-derived stem cells (hASCs) are potent modulators of inflammation and promising candidates for the treatment of inflammatory and autoimmune diseases. Strategies to improve hASC survival and immunoregulation are active areas of investigation. Autophagy, a homeostatic and stress-induced degradative pathway, plays a crucial role in hASC paracrine signaling—a primary mechanism of therapeutic action. Therefore, induction of autophagy with rapamycin (Rapa), or inhibition with 3-methyladenine (3-MA), was examined as a preconditioning strategy to enhance therapeutic efficacy. Following preconditioning, both Rapa and 3-MA-treated hASCs demonstrated preservation of stemness, as well as upregulated transcription of cyclooxygenase-2 (COX2) and interleukin-6 (IL-6). Rapa-ASCs further upregulated TNFα-stimulated gene-6 (TSG-6) and interleukin-1 beta (IL-1β), indicating additional enhancement of immunomodulatory potential. Preconditioned cells were then stimulated with the inflammatory cytokine interferon-gamma (IFNγ) and assessed for immunomodulatory factor production. Rapa-pretreated cells, but not 3-MA-pretreated cells, further amplified COX2 and IL-6 transcripts following IFNγ exposure, and both groups upregulated secretion of prostaglandin-E2 (PGE2), the enzymatic product of COX2. These findings suggest that a 4-h Rapa preconditioning strategy may bestow the greatest improvement to hASC expression of cytokines known to promote tissue repair and regeneration and may hold promise for augmenting the therapeutic potential of hASCs for inflammation-driven pathological conditions.

Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic., Removal of integrated HIV DNA remains a roadblock for HIV cure. Here, Mancuso et al. show that intravenous administration of an adeno-associated virus-based CRISPR/Cas9 gene editing construct to SIV-infected macaques results in excision of integrated proviral DNA from infected blood cells and tissues known to be viral reservoirs.

Human adipose-derived stem cells (ASCs) show immense promise for treating inflammatory diseases, attributed primarily to their potent paracrine signaling. Previous investigations demonstrated that short-term Rapamycin preconditioning of bone marrow-derived stem cells (BMSCs) elevated secretion of prostaglandin E2, a pleiotropic molecule with therapeutic effects in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), and enhanced immunosuppressive capacity in vitro. However, this has yet to be examined in ASCs. The present study examined the therapeutic potential of short-term Rapamycin-preconditioned ASCs in the EAE model. Animals were treated at peak disease with control ASCs (EAE-ASCs), Rapa-preconditioned ASCs (EAE-Rapa-ASCs), or vehicle control (EAE). Results show that EAE-ASCs improved clinical disease scores and elevated intact myelin compared to both EAE and EAE-Rapa-ASC animals. These results correlated with augmented CD4+ T helper (Th) and T regulatory (Treg) cell populations in the spinal cord, and increased gene expression of interleukin-10 (IL-10), an anti-inflammatory cytokine. Conversely, EAE-Rapa-ASC mice showed no improvement in clinical disease scores, reduced myelin levels, and significantly less Th and Treg cells in the spinal cord. These findings suggest that short-term Rapamycin preconditioning reduces the therapeutic efficacy of ASCs when applied to late-stage EAE.

Osteoarthritis (OA) is a common joint disorder with a significant economic and healthcare impact. The knee joint is composed of cartilage and the adjoining bone, a synovial capsule, the infrapatellar fat pad (IPFP), and other connective tissues such as tendons and ligaments. Adipose tissue has recently been highlighted as a major contributor to OA through strong inflammation mediating effects. In this study, methacrylated gelatin (GelMA) constructs seeded with adipose tissue-derived mesenchymal stem cells (ASCs) and cultured in a 3D printed bioreactor were investigated for use in microphysiological systems to model adipose tissue in the knee joint. Four patient-derived ASC populations were seeded at a density of 20 million cells/mL in GelMA. Live/Dead and boron-dipyrromethene/4&prime, 6-diamidino-2-phenylindole (BODIPY/DAPI) staining of cells within the constructs demonstrated robust cell viability after 28 days in a growth (control) medium, and robust cell viability and lipid accumulation in adipogenic differentiation medium. qPCR gene expression analysis and protein analysis demonstrated an upregulated expression of key adipogenesis-associated genes. Overall, these data indicate that ASCs retain their adipogenic potential when seeded within GelMA hydrogels and cultured within perfusion bioreactors, and thus can be used in a 3D organ-on-a-chip system to study the role of the IPFP in the pathobiology of the knee OA.

Preclinical animal studies are essential to the development of safe and effective stem cell therapies. Bioluminescence imaging (BLI) is a powerful tool in animal studies that enables the real-time longitudinal monitoring of stem cells in vivo to elucidate their regenerative properties. This review describes the application of BLI in preclinical stem cell research to address critical challenges in producing successful stem cell therapeutics. These challenges include stem cell survival, proliferation, homing, stress response, and differentiation. The applications presented here utilize bioluminescence to investigate a variety of stem and progenitor cells in several different in vivo models of disease and implantation. An overview of luciferase reporters is provided, along with the advantages and disadvantages of BLI. Additionally, BLI is compared to other preclinical imaging modalities and potential future applications of this technology are discussed in emerging areas of stem cell research.

While decellularized adipose tissue (DAT) has potential as an "off-the-shelf" biomaterial product for regenerative medicine, it remains to be determined if donor-source body mass index (BMI) impacts the functionality of DAT. This study set out to comparatively characterize lean versus overweight/obese-donor derived DAT hydrogel based on proteome and to analyze their respective effects on adipose stromal/stem cell (ASC) viability, and differentiation in vitro. Decellularized adipose tissue from lean (lDAT) and overweight/obese (oDAT) donors is produced and characterized. Variability in the fibril microstructures is found, with dense fibrotic fiber clusters and large pore area uniquely present in the oDAT samples. Proteomic analysis reveals that lDAT contains a greater proportion of enriched extracellular proteins and a smaller proportion of enriched intracellular proteins relative to oDAT. Biocompatibility studies show that ASCs cultured in lDAT and oDAT hydrogels remain viable. The adipogenic and osteogenic differentiation capability of ASCs seeded in lDAT and oDAT hydrogels is confirmed by an upregulation in marker gene expression and phenotypic analysis. In conclusion, this study establishes that DAT hydrogels derived from lean and overweight/obese adipose donors present similar physicochemical profiles with some distinctive features while comparably supporting the viability and adipogenic differentiation of ASCs in vitro.

In late 2019, a novel coronavirus (SARS-CoV-2) emerged in Wuhan, capital city of Hubei province in China. Cases of SARS-CoV-2 infection quickly grew by several thousand per day. Less than 100 days later, the World Health Organization declared that the rapidly spreading viral outbreak had become a global pandemic. Coronavirus disease 2019 (COVID-19) is typically associated with fever and respiratory symptoms. It often progresses to severe respiratory distress and multi-organ failure which carry a high mortality rate. Older patients or those with medical comorbidities are at greater risk for severe disease. Inflammation, pulmonary edema and an over-reactive immune response can lead to hypoxia, respiratory distress and lung damage. Mesenchymal stromal/stem cells (MSCs) possess potent and broad-ranging immunomodulatory activities. Multiple in vivo studies in animal models and ex vivo human lung models have demonstrated the MSC’s impressive capacity to inhibit lung damage, reduce inflammation, dampen immune responses and aid with alveolar fluid clearance. Additionally, MSCs produce molecules that are antimicrobial and reduce pain. Upon administration by the intravenous route, the cells travel directly to the lungs where the majority are sequestered, a great benefit for the treatment of pulmonary disease. The in vivo safety of local and intravenous administration of MSCs has been demonstrated in multiple human clinical trials, including studies of acute respiratory distress syndrome (ARDS). Recently, the application of MSCs in the context of ongoing COVID-19 disease and other viral respiratory illnesses has demonstrated reduced patient mortality and, in some cases, improved long-term pulmonary function. Adipose-derived stem cells (ASC), an abundant type of MSC, are proposed as a therapeutic option for the treatment of COVID-19 in order to reduce morbidity and mortality. Additionally, when proven to be safe and effective, ASC treatments may reduce the demand on critical hospital resources. The ongoing COVID-19 outbreak has resulted in significant healthcare and socioeconomic burdens across the globe. There is a desperate need for safe and effective treatments. Cellular based therapies hold great promise for the treatment of COVID-19. This literature summary reviews the scientific rationale and need for clinical studies of adipose-derived stem cells and other types of mesenchymal stem cells in the treatment of patients who suffer with COVID-19.