Prevalence of and risk factors for chronic kidney disease in ten metropolitan areas of China: a cross-sectional study using three kidney damage markers.

INTRODUCTION: To estimate the up-to-date prevalence of chronic kidney disease among the health check-up population in economically developed areas of China using estimated glomerular filtration rate, urinary albumin creatinine ratio, and kidney ultrasound. METHODS: Healthcare data from 38,093 subjects in 10 megalopolises of China who had an annual health check-up in 2021 were used. The overall and stratified prevalence of chronic kidney disease by sex, age, region and comorbidity group was reported. The association between chronic kidney disease and covariates of demographics, and comorbidities were analyzed in the multivariable-adjusted logistic regression model. RESULTS: A total of 3837 CKD cases were detected meeting any of the three CKD diagnostic criteria, with a crude prevalence of 10.1% in the study population. Using one criterion of decreased glomerular filtration rate, albuminuria and kidney structural abnormalities alone detected 204 (5.3%), 3289 (85.7%) and 563 (14.7%) cases, respectively. The addition of kidney ultrasound detected 427 (11.1%) structural abnormality cases without decreased GFR and albuminuria. The most common abnormalities were renal masses, hydronephrosis due to obstruction and congenital anomalies of kidney and urinary tract. Female, older age, low city-tier, hypertension, diabetes, obesity, hypertriglyceridemia as well as early disease stages such as pre-hypertension, impaired fasting glucose and overweight were significantly associated with chronic kidney disease. CONCLUSION: Kidney ultrasound helps to amplify the detection of CKD patients, which is a supplement to kidney function and urine protein.

Microfluidic Isolation of CD34-Positive Skin Cells Enables Regeneration of Hair and Sebaceous Glands In Vivo.

Skin stem cells resident in the bulge area of hair follicles and at the basal layer of the epidermis are multipotent and able to self-renew when transplanted into full-thickness defects in nude mice. Based on cell surface markers such as CD34 and the α6-integrin, skin stem cells can be extracted from tissue-derived cell suspensions for engraftment using the gold standard cell separation technique of fluorescence-activated cell sorting (FACS). This paper describes an alternative separation method using microfluidic devices coated with degradable antibody-functionalized hydrogels. The microfluidic method allows direct injection of tissue digestate (no preprocessing tagging of cells is needed), is fast (45 minutes from injected sample to purified cells), and scalable. This method is used in this study to isolate CD34-positive (CD34+) cells from murine skin tissue digestate, and the functional capability of these cells is demonstrated by transplantation into nude mice using protocols developed by other groups for FACS-sorted cells. Specifically, the transplantation of microfluidic isolated CD34+ cells along with dermal and epidermal cells was observed to generate significant levels of hair follicles and sebaceous glands consistent with those observed previously with FACS-sorted cells.

Stem Cell Separation Technologies.

Stem cell therapy and translational stem cell research require large-scale supply of stem cells at high purity and viability, thus leading to the development of stem cell separation technologies. This review covers key technologies being applied to stem cell separation, and also highlights exciting new approaches in this field. First, we will cover conventional separation methods that are commercially available and have been widely adapted. These methods include Fluorescence-activated cell sorting (FACS), Magnet-activated cell sorting (MACS), pre-plating, conditioned expansion media, density gradient centrifugation, field flow fractionation (FFF), and dielectrophoresis (DEP). Next, we will introduce emerging novel methods that are currently under development. These methods include improved aqueous two-phase system, systematic evolution of ligands by exponential enrichment (SELEX), and various types of microfluidic platforms. Finally, we will discuss the challenges and directions towards future breakthroughs for stem cell isolation. Advancing stem cell separation techniques will be essential for clinical and research applications of stem cells.

Microfluidic enrichment of mouse epidermal stem cells and validation of stem cell proliferation in vitro.

Bulge stem cells reside in the lowest permanent portion of hair follicles and are responsible for the renewal of these follicles along with the repair of the epidermis during wound healing. These cells are identified by surface expression of CD34 and the α6-integrin. When CD34 and α6 double-positive cells are isolated and implanted into murine skin, they give rise to epidermis and hair follicle structures. The current gold standard for isolation of these stem cells is fluorescence-activated cell sorting (FACS) based on cell surface markers. Here, we describe an alternative method for CD34 bulge stem cell isolation: a microfluidic platform that captures stem cells based on cell surface markers. This method is relatively fast, requiring 30 min of time from direct introduction of murine skin tissue digestate into a two-stage microfluidic device to one-pass elution of CD34(+) enriched cells with a purity of 55.8% ± 5.1%. The recovered cells remain viable and formed colonies with characteristic morphologies. When grown in culture, enriched cells contain a larger α6(+) population than un-enriched cells.

Development of a total atherosclerotic occlusion with cell-mediated calcium deposits in a rabbit femoral artery using tissue-engineering scaffolds.

This study sought to establish a chronic total occlusion (CTO) model with cell-mediated calcium deposits in rabbit femoral arteries. CTO is the most severe case in atherosclerosis and contains calcium deposits. Previous animal models of CTO do not mimic the gradual occlusion of arteries or have calcium in physiological form. In the present study we tested the strategy of placing tissue-engineering scaffolds preloaded with cells in arteries to develop a novel CTO model. Primary human osteoblasts (HOBs) were first cultured in vitro on polycaprolactone (PCL) scaffolds with 5 ng TGFß1 loading for 28 days for precalcification. The HOB-PCL construct was then implanted into a rabbit femoral artery for an additional 3, 10 or 28 days. At the time of sacrifice, angiograms and gross histology of arteries were captured to examine the occlusion of arteries. Fluorescent staining of calcium and EDS detection of calcium were used to evaluate the presence and distribution of calcium inside arteries. Rabbit femoral arteries were totally occluded over 28 days. Calcium was presented at CTO sites at 3, 10 and 28 days, with the day 10 specimens showing the maximum calcium. Chronic inflammatory response and recanalization were observed in day 28 CTO sites. A novel CTO model with cell-mediated calcium has been successfully established in a rabbit femoral artery. This model can be used to develop new devices and therapies to treat severe atherosclerotic occlusion.

Calcification of primary human osteoblast cultures under flow conditions using polycaprolactone scaffolds for intravascular applications.

Total atherosclerotic occlusion is a leading cause of death. Recent animal models of this disease are devoid of cell-mediated calcification and arteries are often not occluded gradually. This study is part of a project with the objective of developing a new model featuring the above two characteristics, using a tissue-engineering scaffold. The amount and distribution of calcium deposits in primary human osteoblast (HOB) cultures on polycaprolactone (PCL) scaffolds under flow conditions were investigated. HOBs were cultured on PCL scaffolds with TGF-ß1 loadings of 0 (control), 5 and 50 ng. HOB-PCL constructs were cultured in spinner flasks. Under flow conditions, cell numbers present in HOB cultures on PCL scaffolds increased from day 7 to day 14, and most calcification was induced at day 21. TGF-ß1 loadings of 5 and 50 ng did not show a significant difference in ALP activity, cell numbers and amount of calcium deposited in HOB cultures, but calcium staining showed that 50 ng TGF-ß1 had higher calcium deposited on both days 21 and 28 under flow conditions compared with 5 ng of loading. Amount of calcium deposited by HOBs on day 28 showed a decrease from their levels on day 21. PCL degradation may be a factor contributing to this loss. The results indicate that cell-induced calcification can be achieved on PCL scaffolds under flow conditions. In conclusion, TGFß1-HOB loaded PCL can be applied to create a model for total atherosclerotic occlusion with cell-deposited calcium in animal arteries.

Tumor antigen-pulsed CD8α(+) dendritic cells induce T cell-mediated graft-versus-tumor effect in vitro.

The graft-versus-tumor (GVT) effect of T cells induced by tumor antigen-pulsed CD8α(+) dendritic cells (DCs) in vitro was investigated in this study. Immature CD8α(+) DCs were prepared from C57BL/6 (H-2(b)) bone marrow cells by using a cytokine cocktail. On the 3rd day of culture, CD8α(+) DCs were pulsed by allogeneic (Balb/c, H-2(d)) EL9611 leukemia antigen, or RM-1 syngeneic prostate cancer antigen, with the concentration series of 0, 2.5, 5.0, 10.0, 20.0 µg/mL, respectively, then antigen-loaded immature CD8α(+) DCs were co-cultured with syngeneic T cells according to the DC/T ratio of 1:1, 2:1 and 4:1. T cell proliferation was measured by MTT assay. Cytokines including interferon gamma (IFN-γ) and interleukin-10 (IL-10) in CD8α(+) DCs and T co-culture supernatant were detected by using ELISA. Cytotoxic effect of antigen-specific T cells was tested by LDH release assay. Conventional mature DCs (mDCs) induced from C57BL/6 (H-2(b)) bone marrow cells by using granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) served as a control. The results showed that the proliferative activity of T cells stimulated by CD8α(+) DCs loaded with allogeneic or syngeneic tumor antigen was augmented with the CD8α(+) DC/T ratio increased (P<0.05). When antigen concentration ≤ 5 µg/mL and CD8α(+) DC/T ratio ≤ 2:1, the ability of CD8α(+) DCs to stimulate T cell proliferation was higher than mDC control in allogeneic tumor antigen-pulsed groups (P<0.05), but not in syngeneic tumor antigen-pulsed groups (P>0.05). The level of IFN-γ and IL-10 in CD8α(+) DCs and T cell co-culture supernatant were increased in both allogeneic and syngeneic antigen-pulsed groups (P<0.05), and the cytokine level was higher in allogeneic antigen-pulsed groups than in syngeneic antigen groups when the CD8α(+) DC/T was 1:1 or 2:1 (P<0.05). There existed a negative correlation between the level of IL-10 and T cell proliferation. T cell cytotoxicity assay showed that when CD8α(+) DCs were pulsed with allogeneic tumor antigen, the maximal T cell killing efficiency could reach (100±7.7)%, whereas syngeneic tumor antigen-pulsed group had only (65.0±3.4)%. It was concluded that syngeneic and allogeneic tumor antigen-pulsed immature CD8α(+) DCs could stimulate T cells to exert the GVT effect in vitro, and the GVT effect was more obvious with allogeneic tumor antigen than with syngeneic tumor antigen. The optimal condition was low allogeneic tumor antigen pulsation (≤ 5 µg/mL) and low CD8α(+) DC/T ratio (1:1 and 2:1).

Engineering calcium deposits on polycaprolactone scaffolds for intravascular applications using primary human osteoblasts.

Total atherosclerotic occlusions often include significant calcium deposits. Current animal models do not mimic the pathology of gradual occlusion of arteries and lack cell-mediated calcium. The primary goal of this project was to establish an animal model incorporating these features into chronic total occlusions, using biodegradable scaffolds. As the first step, this study sought to determine the optimal dosage of TGF-ß1 on polycaprolactone (PCL) scaffolds cultured with primary human osteoblasts (HOBs) to effectively induce in vitro calcification. HOBs were cultured in TGF-ß1 and dexamethsaone (Dex)-supplemented medium in well plates. Calcium in the cultures was visualized using alizarin red. The highest calcification was observed in groups with both TGF-ß1 (0.02 ng/ml) and Dex (10(-10) M) in the medium. Next, HOBs were cultured on PCL scaffolds with different loadings of TGF-ß1: 0 (control), 5, 10, 50 and 100 ng. These cultures were performed with or without Dex (10(-10) M) in the medium. DNA content, ALP activity and the amount and distribution of calcium were examined at 7, 14, 21 and 28 days. TGF-ß1 appeared to have an inhibitory effect on scaffold calcification when grown in Dex-supplemented medium. When cultured without Dex, the lower amount of TGF-ß1 loading (5 ng) showed the most calcification, high DNA synthesis and high ALP activity on scaffolds. This study demonstrates the potential of implanting a PCL-HOB construct in an animal artery to establish a model of atherosclerotic occlusion with calcification.

Tumor Antigen-pulsed CD8α+ Dendritic Cells Induce T Cell-mediated Graft-versus-tumor Effect In Vitro / 华中科技大学学报（医学）（英德文版）

The graft-versus-tumor (GVT) effect of T cells induced by tumor antigen-pulsed CD8α+dendritic cells (DCs) in vitro was investigated in this study.Immature CD8α+ DCs were prepared from C57BL/6 (H-2b) bone marrow cells by using a cytokine cocktail.On the 3rd day of culture,CD8α- DCs were pulsed by allogeneic (Balb/c,H-2d) EL9611 leukemia antigen,or RM-1 syngeneic prostate cancer antigen,with the concentration series of 0,2.5,5.0,10.0,20.0 μg/mL,respectively,then antigen-loaded immature CD8α+ DCs were co-cultured with syngeneic T cells according to the DC/T ratio of 1∶1,2∶1and 4∶1.T cell proliferation was measured by MTT assay.Cytokines including interferon gamma (IFN-γ)and interleukin-10 (IL-10) in CD8α+ DCs and T co-culture supernatant were detected by using ELISA.Cytotoxic effect of antigen-specific T cells was tested by LDH release assay.Conventional mature DCs　(mDCs) induced from C57BL/6 (H-2b) bone marrow cells by using granulocyte-macrophage colony　stimulating factor (GM-CSF) and interleukin-4 (IL-4) served as a control.The results showed that the　proliferative activity of T cells stimulated by CD8α+ DCs loaded with allogeneic or syngeneic tumor antigen was augmented with the CD8α+ DC/T ratio increased (P＜0.05).When antigen concentration ≤ 5μg/mL and CD8α+ DC/T ratio ≤ 2∶1,the ability of CD8α+ DCs to stimulate T cell proliferation was　higher than mDC control in allogeneic tumor antigen-pulsed groups (P＜0.05),but not in syngeneic tumor antigen-pulsed groups (P＞0.05).The level of IFN-γ and IL-10 in CD8α+DCs and T cell co-culture　supernatant were increased in both allogeneic and syngeneic antigen-pulsed groups (P＜0.05),and the　cytokine level was higher in allogeneic antigen-pulsed groups than in syngeneic antigen groups when　the CD8α+DC/T was 1∶1 or 2∶1 (P＜0.05).There existed a negative correlation between the level of　IL-10 and T cell proliferation.T cell cytotoxicity assay showed that when CD8α+ DCs were pulsed with　allogeneic tumor antigen,the maximal T cell killing efficiency could reach (100±7.7)％,whereas syngeneic tumor antigen-pulsed group had only (65.0±3.4)％.It was concluded that syngeneic and allogeneic tumor antigen-pulsed immature CD8α+ DCs could stimulate T cells to exert the GVT effect in vitro,and the GVT effect was more obvious with allogeneic tumor antigen than with syngeneic tumor antigen.The optimal condition was low allogeneic tumor antigen pulsation (≤ 5 μg/mL) and low CD8α+ DC/T　ratio (1∶1 and 2∶1).

Site-specific modification of anti-angiogenesis peptide HM-3 by polyethylene glycol molecular weight of 20 kDa.

HM-3, an RGD modified endostatin-derived polypeptide, is a potent angiogenesis inhibitor synthesized in our laboratory. Its robust inhibitory effects on endothelial cell migration and tumour growth have been demonstrated by in vivo and in vitro activity assays. However, the drug has relatively short half-life in vivo. For the purpose of prolonging HM-3 half-life and retaining the safety and efficacy of the peptide, the study chose methoxy-polyethylene glycol-Succinimidyl Carbonate (SC-mPEG, molecular weight 20 kDa, named SC-mPEG(20k)) to specifically modify its N terminus. Compared with HM-3, the site-specific mono-PEGylated peptide PEG(20k)-HM-3 was shown the same activity in the inhibition of B16F10 tumour in vivo (the inhibitory effect of PEG(20k)-HM-3, HM-3 and Taxol were 44.35, 39.68%, respectively), while the frequency of drug-administering reduced from twice a day to once every 3 days. Its rate of in vitro degradation in serum was markedly reduced (72.78% could still be detected after 132 h). Histochemistry and immunohistochemistry analysis showed that both HM-3 and PEG(20k)-HM-3 induced large areas of continuous necrosis within tumours and significantly reduced the vessel density compared to control. It might be a breakthrough in PEG modification field to modify a small peptide with a large PEG and reach a good result.

Effects of surface-modified scaffolds on the growth and differentiation of mouse adipose-derived stromal cells.

PURPOSE: Adipose-derived stromal cells (ADSCs) have been shown to increase angiogenesis in ischemic tissue. Maintaining cell survival and facilitating angiogenesis in ischemic tissue, however, continues to be the major challenge of ADSCs implantation. Recently, bioengineered scaffolds were introduced to support and facilitate cell culture and differentiation. The effects of a surface modified three-dimensional (3D) scaffold on ADSC function have not been investigated. Accordingly, the objective of this study was to determine the influence of a gas-plasma treated scaffold on ADSC growth, differentiation into endothelial cell, and angiogenic gene expression. METHODS: Freshly isolated mouse ADSCs were characterized by flow cytometry and cultured into wells containing gas-plasma treated scaffolds, non-treated scaffolds, or control wells. Either endothelial growth media or differentiation media was used to alter cell environment. After 3 and 6 days, cell proliferation was analyzed. VEGF concentration in the medium was measured by ELISA. Gene expression was quantified by real-time PCR for VEGF receptor-2 (KDR), cyclooxygenase-2 (COX-2) and matrix metalloproteinases-2 (MMP-2). RESULTS: ADSCs expressed stem/endothelial progenitor markers CD34 and CD133 and endothelial cell marker CD31. ADSCs grew in the 3D scaffold. Cells grown on gas-plasma treated scaffolds displayed significantly increased expression of VEGF, COX-2, and MMP-2 when grown in differentiation but not growth media. When cultured in endothelial growth media, VEGF secretion and the expression of KDR, COX-2 and MMP-2 were lower in 3D scaffolds than controls. CONCLUSIONS: This study suggests that 3D scaffolds, especially gas-plasma treated scaffolds, support ADSC growth and support differentiation into endothelial cells.