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Urinary bladder dysfunction can be caused by environmental, genetic, and developmental insults. Depending upon insult severity, the bladder may lose its ability to maintain volumetric capacity and intravesical pressure resulting in renal deterioration. Bladder augmentation enterocystoplasty (BAE) is utilized to increase bladder capacity to preserve renal function using autologous bowel tissue as a “patch.” To avoid the clinical complications associated with this procedure, we have engineered composite grafts comprised of autologous bone marrow mesenchymal stem cells (MSCs) co-seeded with CD34+ hematopoietic stem/progenitor cells (HSPCs) onto a pliable synthetic scaffold [poly(1,8-octamethylene-citrate-co-octanol)(POCO)] or a biological scaffold (SIS; small intestinal submucosa) to regenerate bladder tissue in our baboon bladder augmentation model. We set out to determine the global protein expression profile of bladder tissue that has undergone regeneration with the aforementioned stem cell seeded scaffolds along with baboons that underwent BAE. Data demonstrate that POCO and SIS grafted animals share high protein homogeneity between native and regenerated tissues while BAE animals displayed heterogeneous protein expression between the tissues following long-term engraftment. We posit that stem cell-seeded scaffolds can recapitulate tissue that is nearly indistinguishable from native tissue at the protein level and may be used in lieu of procedures such as BAE.

Chronic bladder dysfunction due to bladder disease or trauma is detrimental to affected patients as it can lead to increased risk of upper urinary tract dysfunction. Current treatment options include surgical interventions that enlarge the bladder with autologous bowel tissue to alleviate pressure on the upper urinary tract. This highly invasive procedure, termed bladder augmentation enterocystoplasty (BAE), significantly increases the risk of patient morbidity and mortality due to the incompatibility between bowel and bladder tissue. Therefore, patients would significantly benefit from an alternative treatment strategy that can regenerate healthy tissue and restore overall bladder function. Previous research has demonstrated the potential of citrate-based scaffolds co-seeded with bone marrow-derived stem/progenitor cells as an alternative graft for bladder augmentation. Recognizing that contact guidance can potentially influence tissue regeneration, we hypothesized that microtopographically patterned scaffolds would modulate cell responses and improve overall quality of the regenerated bladder tissue. We fabricated microgrooved (MG) scaffolds using the citrate-based biomaterial poly (1,8-octamethylene-citrate-co-octanol) (POCO) and co-seeded them with human bone marrow-derived mesenchymal stromal cells (MSCs) and CD34+ hematopoietic stem/progenitor cells (HSPCs). MG POCO scaffolds supported MSC and HSPC attachment, and MSC alignment within the microgrooves. All scaffolds were characterized and assessed for bladder tissue regeneration in an established nude rat bladder augmentation model. In all cases, normal physiological function was maintained post-augmentation, even without the presence of stem/progenitor cells. Urodynamic testing at 4-weeks post-augmentation for all experimental groups demonstrated that bladder capacity increased and bladder compliance was normal. Histological evaluation of the regenerated tissue revealed that cell-seeded scaffolds restored normal bladder smooth muscle content and resulted in increased revascularization and peripheral nerve regeneration. The presence of microgrooves on the cell-seeded scaffolds increased microvasculature formation by 20 % and urothelial layer thickness by 25 % in the regenerating tissue. Thus, this work demonstrates that microtopography engineering can influence bladder tissue regeneration to improve overall anatomical structure and re-establish bladder physiology. [Display omitted]

To date, there are no efficacious translational solutions for end-stage urinary bladder dysfunction. Current surgical strategies, including urinary diversion and bladder augmentation enterocystoplasty (BAE), utilize autologous intestinal segments (e.g. ileum) to increase bladder capacity to protect renal function. Considered the standard of care, BAE is fraught with numerous short- and long-term clinical complications. Previous clinical trials employing tissue engineering approaches for bladder tissue regeneration have also been unable to translate bench-top findings into clinical practice. Major obstacles still persist that need to be overcome in order to advance tissue-engineered products into the clinical arena. These include scaffold/bladder incongruencies, the acquisition and utility of appropriate cells for anatomic and physiologic tissue recapitulation, and the choice of an appropriate animal model for testing. In this study, we demonstrate that the elastomeric, bladder biomechanocompatible poly(1,8-octamethylene-citrate-co-octanol) (PRS; synthetic) scaffold coseeded with autologous bone marrow-derived mesenchymal stem cells and CD34+ hematopoietic stem/progenitor cells support robust long-term, functional bladder tissue regeneration within the context of a clinically relevant baboon bladder augmentation model simulating bladder trauma. Partially cystectomized baboons were independently augmented with either autologous ileum or stem-cell-seeded small-intestinal submucosa (SIS; a commercially available biological scaffold) or PRS grafts. Stem-cell synergism promoted functional trilayer bladder tissue regeneration, including whole-graft neurovascularization, in both cell-seeded grafts. However, PRS-augmented animals demonstrated fewer clinical complications and more advantageous tissue characterization metrics compared to ileum and SIS-augmented animals. Two-year study data demonstrate that PRS/stem-cell-seeded grafts drive bladder tissue regeneration and are a suitable alternative to BAE.

Urinary bladder insult can be caused by environmental, genetic, and developmental factors. Depending upon insult severity, the bladder may lose its ability to maintain capacity and intravesical pressures resulting in renal deterioration. Bladder augmentation enterocystoplasty (BAE) is employed to increase bladder capacity to preserve renal function using autologous bowel tissue as a \"patch.\" To avoid the clinical complications associated with this procedure, we have engineered composite grafts comprised of autologous bone marrow mesenchymal stem cells (MSCs) with CD34+ hematopoietic stem/progenitor cells (HSPCs) co-seeded onto a pliable synthetic scaffold [POCO; poly(1,8-octamethylene-citrate-co-octanol)] or a biological scaffold (SIS; small intestinal submucosa) to regenerate bladder tissue in a baboon bladder augmentation model. We set out to determine the protein expression profile of bladder tissue that has undergone regeneration with the aforementioned stem cell seeded scaffolds along with baboons that underwent BAE. Data demonstrate that POCO and SIS grafted animals share high protein homogeneity between native and regenerated tissues while BAE animals displayed heterogenous protein expression between the tissues following long-term engraftment. We posit that stem cell seeded scaffolds can recapitulate tissue that is almost indistinguishable from native tissue at the protein level and may be used in lieu of procedures such as BAE.

Chronic bladder dysfunction due to bladder disease or trauma is detrimental to affected patients as it can lead to increased risk of upper urinary tract dysfunction. Current treatment options include surgical intervention that enlarge the bladder with autologous bowel tissue to alleviate pressure on the upper urinary tract. This highly invasive procedure, termed bladder augmentation enterocystoplasty (BAE), significantly increases risk of patient morbidity and mortality due to the incompatibility between the bowel and bladder tissue. Therefore, patients would significantly benefit from an alternative treatment strategy that can regenerate healthy tissue and restore overall bladder function. Previous research has demonstrated the potential of citrate-based scaffolds co-seeded with bone marrow-derived stem/progenitor cells as an alternative graft for bladder augmentation. Recognizing that contact guidance is known to influence tissue regeneration, we hypothesized that patterned scaffolds would modulate cell responses and improve overall quality of the regenerated bladder tissue. We fabricated microgrooved (MG) scaffolds using citrate-based biomaterial poly(1,8-octamethylene-citrate-co-octanol) (POCO) and co-seeded them with human bone marrow derived mesenchymal stem cells (MSCs) and CD34+ hematopoietic stem/progenitor cells (HSPCs). Microgrooved POCO scaffolds supported MSC and HSPC attachment, and MSC alignment within the microgrooves. All scaffolds were characterized and assessed for bladder tissue regeneration in an established nude rat bladder augmentation model. In all cases, normal physiological function was maintained post-augmentation, even without the presence of stem/progenitor cells. Urodynamic testing at 4-weeks post-augmentation for all experimental groups demonstrated that capacity increased and compliance was normal. Histological evaluation of the regenerated tissue revealed that cell-seeded scaffolds restored normal bladder smooth muscle content and resulted in increased revascularization and peripheral nerve regeneration. The presence of microgrooves on the cell-seeded scaffolds increased microvasculature formation by 20% and urothelium layer thickness by 25% in the regenerating tissue. Thus, this work demonstrates that micropatterning affects bladder regeneration to improve overall anatomical structure and re-establish bladder physiology.Competing Interest StatementThe authors have declared no competing interest.Footnotes* The statistical analysis of the in vivo experiments was redone to better reflect the observed differences between experimental groups.

Transcriptional regulation is an important part within the cell. Transcription factors bind specific DNA sequences, regulate expression of their target genes, and can be identified by the presence of their DNA binding domains. Transcription factors are pivotal in lineage commitment and differentiation in hematopoietic tissue. Hematopioesis is a highly regulated system giving rise to all mature blood cell types through a series of cell proliferations, commitment, and differentiation. Thus the objective of this study was to identify novel transcription factors and their associated proteins that participate in the transcriptional regulation of myeloid differentiation. By utilizing microarray technology and the genomic databases, the identification of transcription factors was done in a cell model of monocytic differentiation, and the identified proteins (1) contained motifs that were consistent with transcription factors, (2) were expressed in undifferentiated hematopoietic progenitor cells, and (3) had increased expression over time during differentiation. Using the U937 cells for our monocytic differentiation cell model, a total of 347 cDNAs was found that had increased expression during induced monocytic differentiation using GeneFilter filter array system. Of these, a total of eleven genes contained motifs consistent with transcription factors and other DNA binding domains. Of these selected genes, two were known transcription factors in myeloid differentiation-MAFB and PU.1, three novel zinc finger proteins, and 6 nuclear-DNA binding proteins. Next, real-time RT-PCR was used in an attempt to validate the expression patterns in our U937 cell model as well as another myeloid model system, HL60. For both known genes, their expression pattern was as predicted, and even MAFB was shown for the first lineage specificity toward monocytic differentiation in this system. For the three novel genes, the zinc fingers, their expression pattern could not be validated. Thus a possible explanation for this difference might be due the possible non-specific cross-hybridizations of similar zinc finger proteins when the GeneFilters were used. However, since the known proteins were identified and validated using our study model system, we concluded that our methodology was successful in identifying in identifying transcription factors that were highly differentially expressed during myeloid differentiation such as MAFB.

Coronavirus Disease-19 (COVID-19), caused by the coronavirus SARS-CoV-2, was initially recognized in Wuhan, China and subsequently spread to all continents. The disease primarily affects the lower respiratory system, but may involve other organs and systems. Histopathologic evaluation of tissue from affected patients is crucial for diagnostic purposes, but also for advancing our understanding of the disease. For that reason, we developed immunohistochemical (IHC) and in situ hybridization (ISH) assays for detection of the. virus. A total of eight autopsy lungs, one placenta, and ten kidney biopsies from COVID-19 patients were stained with a panel of commercially available antibodies for IHC and commercially available RNA probes for ISH. Similarly, autopsy lungs, placentas and renal biopsies from non-COVID-19 patients were stained with the same antibodies and probes. All eight lungs and the placenta from COVID-19 patients stained positive by IHC and ISH, while the kidney biopsies stained negative by both methodologies. As expected, all specimens from non-COVID-19 patients were IHC and ISH negative. These two assays represent a sensitive and specific method for detecting the virus in tissue samples. We provide the protocols and the list of commercially available antibodies and probes for these assays, so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes. Detailed protocols for immunohistochemical and in situ hybridization assays for the detection of SARS-CoV-2 are provide so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes. These assays were found to represent a sensitive and specific method for detecting the virus in tissue samples.

ObjectivesEssential healthcare workers (HCW) uniquely serve as both COVID-19 healers and, potentially, as carriers of SARS-CoV-2. We assessed COVID-19-related stigma and bullying against HCW controlling for social, psychological, medical and community variables.DesignWe nested an analytical cross-sectional study of COVID-19-related stigma and bullying among HCW within a larger mixed-methods effort assessing COVID-19-related lived experience and impact. Adjusted OR (aOR) and 95% CIs evaluated the association between working in healthcare settings and experience of COVID-19-related bullying and stigma, controlling for confounders. Thematic qualitative analysis provided insight into lived experience of COVID-19-related bullying.SettingWe recruited potential participants in four languages (English, Spanish, French, Italian) through Amazon Mechanical Turk’s online workforce and Facebook.ParticipantsOur sample included 7411 people from 173 countries who were aged 18 years or over.FindingsHCW significantly experienced more COVID-19-related bullying after controlling for the confounding effects of job-related, personal, geographic and sociocultural variables (aOR: 1.5; 95% CI 1.2 to 2.0). HCW more frequently believed that people gossip about others with COVID-19 (OR: 2.2; 95% CI 1.9 to 2.6) and that people with COVID-19 lose respect in the community (OR: 2.3; 95% CI 2.0 to 2.7), both which elevate bullying risk (OR: 2.7; 95% CI 2.3 to 3.2, and OR: 3.5; 95% CI 2.9 to 4.2, respectively). The lived experience of COVID-19-related bullying relates frequently to public identities as HCW traverse through the community, intersecting with other domains (eg, police, racism, violence).InterpretationAfter controlling for a range of confounding factors, HCW are significantly more likely to experience COVID-19-related stigma and bullying, often in the intersectional context of racism, violence and police involvement in community settings.

Single-cell sequencing technologies have revealed an unexpectedly broad repertoire of cells required to mediate complex functions in multicellular organisms. Despite the multiple roles of adipose tissue in maintaining systemic metabolic homeostasis, adipocytes are thought to be largely homogenous with only 2 major subtypes recognized in humans so far. Here we report the existence and characteristics of 4 distinct human adipocyte subtypes, and of their respective mesenchymal progenitors. The phenotypes of these distinct adipocyte subtypes are differentially associated with key adipose tissue functions, including thermogenesis, lipid storage, and adipokine secretion. The transcriptomic signature of “brite/beige” thermogenic adipocytes reveals mechanisms for iron accumulation and protection from oxidative stress, necessary for mitochondrial biogenesis and respiration upon activation. Importantly, this signature is enriched in human supraclavicular adipose tissue, confirming that these cells comprise thermogenic depots in vivo, and explain previous findings of a rate-limiting role of iron in adipose tissue browning. The mesenchymal progenitors that give rise to beige/brite adipocytes express a unique set of cytokines and transcriptional regulators involved in immune cell modulation of adipose tissue browning. Unexpectedly, we also find adipocyte subtypes specialized for high-level expression of the adipokines adiponectin or leptin, associated with distinct transcription factors previously implicated in adipocyte differentiation. The finding of a broad adipocyte repertoire derived from a distinct set of mesenchymal progenitors, and of the transcriptional regulators that can control their development, provides a framework for understanding human adipose tissue function and role in metabolic disease.

BackgroundCancer-associated fibroblasts (CAFs) are recruited to the tumor microenvironment (TME) and are critical drivers of breast cancer (BC) malignancy. Circulating tumor cells (CTCs) travel through hematogenous routes to establish metastases. CTCs circulate both individually and, more rarely, in clusters with other cell types. Clusters of CTCs have higher metastatic potential than single CTCs. Previously, we identified circulating CAFs (cCAFs) in patients with BC and found that while healthy donors had no CTCs or cCAFs, both were present in most Stage IV patients. cCAFs circulate individually, as cCAF–cCAF homotypic clusters, and in heterotypic clusters with CTCs.MethodsIn this study, we evaluate CTCs, cCAFs, and heterotypic cCAF-CTC clusters in patients with stage I–IV BC. We evaluate the association of heterotypic clusters with BC disease progression and metastasis in a spontaneous mouse model. Using previously established primary BC and CAF cell lines, we examine the metastatic propensity of heterotypic cCAF-CTC clusters in orthotopic and tail vein xenograft mouse models of BC. Using an in vitro clustering assay, we determine factors that may be involved in clustering between CAF and BC cells.ResultsWe report that the dissemination of CTCs, cCAFs, and clusters is an early event in BC progression, and we find these clusters in all clinical stages of BC. Furthermore, cCAFs-CTC heterotypic clusters have a higher metastatic potential than homotypic CTC clusters in vivo. We also demonstrate that the adhesion and stemness marker CD44, found on a subset of CTCs and CAF cells, is involved in heterotypic clustering of these cells.ConclusionWe identify a novel subset of circulating tumor cell clusters that are enriched with stromal CAF cells in BC patient blood and preclinical mouse models of BC metastasis. Our data suggest that clustering of CTCs with cCAFs augments their metastatic potential and that CD44 might be an important mediator of heterotypic clustering of cCAFs and BC cells.