Osteoarthritic Milieu Affects Adipose-Derived Mesenchymal Stromal Cells

The objective of this study was to define the effects of osteoarthritic (OA) milieu on good manufactured practice-adipose-derived mesenchymal stromal cells (GMP-ASC) that are commonly utilized in cell therapies. Two different OA milieu: OA synovial fluid (SF) and OA-conditioned medium (CM) from synoviocytes were used to treat GMP-ASC both in normoxia or hypoxia. GMP-ASC were tested for cell migration, proliferation, cytokine receptors expression (CXCR1, CXCR2, CXCR3, CXCR4, CXCR7, CCR1, CCR2, CCR3, CCR5, IL6R), and cytokines (CXCL8/IL8, CXCL10/IP10, CXCL12/SDF-1, CCL2/MCP1, CCL3/MIP1α, CCL4/MIP1β, CCL5/RANTES, IL6) release. Healthy SF was used as controls. We demonstrated that GMP-ASC show an increase in proliferation, migration, and modulation of CXCR1, CXCR3, CCR1, and CCR5 receptors in hypoxic condition. Moreover, GMP-ASC migration increased 15-fold when treated either with OA-SF or OA-CM compared with healthy SF both in normoxia and hypoxia. GMP-ASC treated in both OA milieu showed an increase in CXCR3, CCR3, and IL6R and a decrease in CCR1 and CCR2 receptors. In OA-SF, we detected higher amount of CXCL10/IP10 than in OA-CM, while CCL2/MCP1 and CCL4/MIP1β were higher in OA-CM compared with OA-SF. CXCL10/IP10 was the only chemokine of the OA milieu, which was down-modulated after treatment with GMP-ASC. In conclusion, we demonstrated specific effects of OA milieu on both GMP-ASC proliferation, migration, and cytokine receptor expression that were strictly dependent on the inflammatory and hypoxic environment. The use of characterized OA milieu is crucial to define the therapeutic effect of GMP-ASC and indicates that CXCL10/IP10–CXCR3 axis is partially involved in the GMP-ASC effect on synovial macrophages. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:336-347, 2020     

Immature Renal Dendritic Cells Recruit Regulatory CXCR6+ Invariant Natural Killer T Cells to Attenuate Crescentic GN

Immature renal dendritic cells (DCs) are protective early in murine crescentic GN, but the mechanisms underlying this protection are unknown. Here, depletion of DCs reduced the recruitment of invariant natural killer T (iNKT) cells, which attenuate GN, into the kidney in the early stage of experimental crescentic GN. More than 90% of renal iNKT cells expressed the chemokine receptor CXCR6, and renal DCs produced high amounts of the cognate ligand CXCL16 early after induction of nephritis, suggesting that renal DC-derived CXCL16 might attract protective CXCR6⁺ iNKT cells. Consistent with this finding, CXCR6-deficient mice exhibited less iNKT cell recruitment and developed nephritis that was more severe, similar to the aggravated nephritis observed in mice depleted of immature DCs. Finally, adoptive transfer of CXCR6-competent NKT cells ameliorated nephritis. Taken together, these results suggest an immunoprotective mechanism involving immature DCs, CXCL16, CXCR6, and regulatory iNKT cells, which might stimulate the development of new therapeutic strategies for GN.GN, as a disease category, is one of the leading causes of progressive renal failure leading to ESRD.¹ Among the different types of GN, crescentic GN is the most aggressive form with the worst prognosis. Crescentic GN represents a heterogeneous collection of disease entities, such as ANCA-associated GN and antiglomerular basement membrane (anti-GBM) nephritis.² Cell-mediated renal damage is, however, a fundamental characteristic of each form of crescentic GN.^3,4Nephrotoxic nephritis (NTN) is a well established murine model of crescentic GN.⁵ In this model, a sheep antiserum raised against kidney cortical components is injected into mice, resulting in activation of Th1 and Th17 cells in lymphatic organs. Because of their specificity, sheep Igs are preferentially deposited in the kidney, inducing tissue destruction by infiltrating activated Th1 and Th17 cells.^6–8 These cells subsequently activate intrarenal macrophages and drive neutrophil recruitment, causing renal tissue injury.⁹Dendritic cells (DCs) are professional antigen-presenting cells that play a pivotal role in the priming and activation of effector T cells, but under certain circumstances, they might also protect against an overwhelming inflammatory response by less well characterized mechanisms. Renal DCs form an extensive parenchymal network that spans the entire tubulointerstitium, ensuring complete surveillance of the kidney to protect it, for example, against infections ascending through the tubular system. Despite their absence from glomeruli, renal DCs are also involved in GN by capturing glomerular antigens or antigens filtered in glomeruli and presenting them to T effector cells, thereby triggering an inflammatory mononuclear infiltrate in the tubulointerstitium that drives renal disease.^10,11 It is important to note that renal DCs only exert this pathogenic effect when nephritis becomes chronic (DC maturation).¹² At steady state and in acute GN, they can also mediate anti-inflammatory and disease-attenuating effects.¹³ Generally, immature DCs are thought to induce immune tolerance, and this finding is thought to result from the absence of costimulatory signals.^14,15 It is unclear whether this tolerance causes the protective function of immature kidney DCs.Recent studies showed that renal DCs significantly contribute to the early production of chemokines and other inflammatory mediators, which regulates the recruitment of leukocytes into the inflamed kidney.¹⁶ We were, therefore, interested in studying whether renal DCs promote the infiltration of potentially anti-inflammatory leukocyte subsets in the early course of experimental GN (NTN) and characterizing the underlying mechanisms.     

Cell-Assisted Lipotransfer for Cosmetic Breast Augmentation: Supportive Use of Adipose-Derived Stem/Stromal Cells

Background Lipoinjection is a promising treatment but has some problems, such as unpredictability and a low rate of graft survival due to partial necrosis. Methods To overcome the problems with lipoinjection, the authors developed a novel strategy known as cell-assisted lipotransfer (CAL). In CAL, autologous adipose-derived stem (stromal) cells (ASCs) are used in combination with lipoinjection. A stromal vascular fraction (SVF) containing ASCs is freshly isolated from half of the aspirated fat and recombined with the other half. This process converts relatively ASC-poor aspirated fat to ASC-rich fat. This report presents the findings for 40 patients who underwent CAL for cosmetic breast augmentation. Results Final breast volume showed augmentation by 100 to 200 ml after a mean fat amount of 270 ml was injected. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Cyst formation or microcalcification was detected in four patients. Almost all the patients were satisfied with the soft and natural-appearing augmentation. Conclusions The preliminary results suggest that CAL is effective and safe for soft tissue augmentation and superior to conventional lipoinjection. Additional study is necessary to evaluate the efficacy of this technique further. Partially presented at the 3rd annual meeting of the International Fat Applied Technology Society (IFATS), Charlottesville, Virginia, 11 September 2005, and the 8th annual meeting of Tissue Engineering Society International (TESI), Shanghai, China, 23 October 2005.     

联合培养VECs与ADSCs与部分脱蛋白生物骨体外构建组织工程骨的实验研究

目的本实验旨在研究体外血管内皮细胞和脂肪来源的联合培养体系在部分脱蛋白生物骨支架材料上的黏附、增殖,以及二者体外培养构建组织工程骨时移植入体内的最佳时机,为体内构建组织工程骨提供理论依据。方法取血管内皮细胞、脂肪干细胞和1：1联合培养细胞分别接种部分脱蛋白生物骨片,MTT法检测吸光度,评价三种不同细胞组在PDPBB生长情况,分析联合培养细胞在支架材料上增殖情况,扫描电镜观察不同细胞组在PDPBB生长情况。结果各细胞组在PDPBB上吸光度逐渐增加,第10天均达到高峰,1：1混合细胞组最高,各细胞组之间差异有统计学意义（P〈0．01）;10天可见联合培养细胞细胞组大量细胞与PDPBB附着,呈巢状分布。结论1：1混合细胞在PDPBB支架材料上的增殖优于单种细胞,10天为最佳移植时机。     

Metabolic Rescue of Obese Adipose-Derived Stem Cells by Lin28/Let7 Pathway

Adipose-derived stem cells (ASCs) are promising candidates for autologous cell-based regeneration therapies by virtue of their multilineage differentiation potential and immunogenicity; however, relatively little is known about their role in adipose tissue physiology and dysfunction. Here we evaluated whether ASCs isolated from nonobese and obese tissue differed in their metabolic characteristics and differentiation potential. During differentiation to mature adipocytes, mouse and human ASCs derived from nonobese tissues both increased their insulin sensitivity and inhibition of lipolysis, whereas obese-derived ASCs were insulin-resistant, showing impaired insulin-stimulated glucose uptake and resistance to the antilipolytic effect of insulin. Furthermore, obese-derived ASCs showed enhanced release of proinflammatory cytokines and impaired production of adiponectin. Interestingly, the delivery of cytosol from control ASCs into obese-derived ASCs using a lipid-based, protein-capture methodology restored insulin sensitivity on glucose and lipid metabolism and reversed the proinflammatory cytokine profile, in part due to the restoration of Lin28 protein levels. In conclusion, glucose and lipid metabolism as well as maturation of ASCs is truncated in an obese environment. The reversal of the altered pathways in obese cells by delivery of normal subcellular fractions offers a potential new tool for cell therapy.Adipose tissue is now recognized as an important endocrine and metabolic organ that, when accumulated in excess, increases the risk of chronic diseases such as diabetes, stroke, and arterial hypertension (1). Recently, new mechanisms that control the obesity phenotype have been identified such as the equilibrium between white and brown adipose tissue, the localization of adipose mass (visceral vs. ventral), and the presence of adipose stem cells (ASCs) and mesenchymal stem cells (MSCs) (1–4). Although the relative importance of fat tissue type and localization are being actively unraveled, the role of stem cells in adipose tissue physiology and dysfunction is still poorly understood.Adult stem cells are multipotent cells that contribute to the homeostasis of various organs, including adipose tissue. ASCs are a class of MSCs localized in adipose tissue that have attracted increasing interest because of their potential to differentiate into adipogenic, osteogenic, chondrogenic, and other mesenchymal lineages (5–8). Other clinically attractive properties attributed to ASCs include proangiogenic and anti-inflammatory actions (9–11). Moreover, depending on the environmental conditions, ASCs can be beneficial or detrimental to health. ASCs thus represent a possible target for therapies aimed at modulating the response of the body to obesity and diabetes as well as a potential tool for regenerative medicine.Adipocytes are central to the control of energy balance and lipid homeostasis (12). In response to prolonged obesity, adipocytes become hypertrophic, and new adipocytes are required to counter the metabolic dysfunction of the hypertrophic cells (13,14). It has been postulated that the adipose tissue depots of obese individuals have already committed all of their stem cell reserves to the adipocyte lineage and, therefore, have no capacity to generate new adipocytes (15–17).In this study, we demonstrate that the differentiation of mouse and human adipose MSCs into mature well-functioning adipocytes is truncated in an obese environment, resulting in impaired metabolic function. We also validate a novel approach to restore normal adipocyte metabolic responsiveness in obese-derived stem cells by cytosolic transfer.     

Activation of Natural Killer Cells and Macrophages by Porcine Endothelial Cells Augments Specific T-Cell Xenoresponse

The rejection of xenografts is characterized by infiltration of monocytes and natural killer (NK) cells into the graft, suggesting an important role for the innate immune system in xenorecognition. In this study, purified human NK or T cells were cocultured with porcine endothelial cells, and cytokines were analyzed by ELISA and intracellular FACS. We demonstrated a vigorous human anti-porcine xenoresponse that was associated with a strong T-cell proliferation against porcine endothelial cells. Limiting dilution cloning and T-cell receptor (TCR) Vbeta gene usage revealed a low number of xenoreactive T-cell precursors. We demonstrated that xenogeneic porcine but not allogeneic human endothelial cells induced the early production of interferon (IFN)-gamma by human NK cells but not by CD3+ T cells. Porcine xenoantigen-induced IFN-gamma production was only partially dependent on IL-12. Blocking IL-12 with neutralizing antibodies or by depletion of human macrophages partially decreased IFN-gamma production by CD56+ NK cells. Three-color flow cytometry revealed that IL-12 was produced through a species-specific activation of human macrophages by porcine endothelial cells. Our results indicate that the direct activation of NK cells and macrophages by porcine endothelial cells provides a unique pathway of xenorecognition that augments downstream specific T-cell immunity and represents a powerful effector mechanism in xenograft rejection.     

人骨髓基质细胞接种珊瑚构建组织工程骨

目的：观察体外培养的人骨髓基质细胞与珊瑚复合物植入体内后的成骨能力。方法：穿刺抽吸入髂骨区骨髓基质细胞，体外培养扩增、诱导，将其与珊瑚复合后植入裸鼠体内，以单纯珊瑚作为对照。术后4、8周取材，通过大体、组织学、扫描电镜观察植入物体内成骨情况。结果：术后4周，复合物中有少量新骨形成；术后8周，复合物中出现大量成熟骨组织。而对照组无骨组织形成。结论：人骨髓基质细胞复合珊瑚在无免疫动物体内具有成骨能力，穿刺抽吸的人骨髓基质细胞可作为骨组织工程的种子细胞。     

Expression of BMP-2 by Rat Bone Marrow Stromal Cells in Culture

To investigate the role of bone morphogenetic protein (BMP-2) in ossifying rat bone marrow stromal cell cultures, we determined the population of fibroblast-like stromal cells that expressed BMP-2 immunocytochemically (anti-rhBMP-2 monoclonal antibody), and compared that to alkaline phosphatase (AP) and collagen synthesis formed in culture over a 4-week period in control and dexamethasone-supplemented mineralizing media. In control media, the percentage of BMP-2-positive stromal cells (BMP-2⁺) increased from 12 to 25% within the first 4 days of culture. In mineralizing media, the level of BMP-2⁺ cells was significantly increased (43–44%). The intensity of immunostaining gradually increased with time. The levels of AP were undetectable at 1 week in both control and mineralizing media, but increased gradually over the next 2 weeks and peaked at 3 weeks. ALP levels were significantly greater in cultures grown in mineralizing medium (P < 0.05 at 3 weeks, P < 0.01 at 4 weeks). Collagen synthesis peaked and was significantly greater at 3 weeks (P < 0.05) in cultures grown in mineralizing medium. The levels of AP and collagen synthesis most closely reflected the changes in the percentage of BMP-2⁺ cells from 7 to 28 days. Though these changes may reflect a primary action of BMP-2 on marrow osteoprogenitor-like stromal cells, they do not exclude a mechanism that involves the induction of other members of the BMP family known to stimulate AP and collagen synthesis. We conclude that BMP-2 expression in cultures of fibroblast-like marrow stromal cells is enhanced when those cells are induced to become osteoblasts by exposure to dexamethasone. Received: 30 October 1997 / Accepted: 24 June 1998     

脂肪干细胞通过旁分泌作用改善异种脱细胞真皮基质体内植入效果的机制研究

目的 探讨脂肪干细胞(ADSCs)在异种脱细胞真皮基质(HADM)微环境中能否有效发挥其旁分泌功能,从而改善HADM体内植入的效果。方法 将ADSCs分别接种于HADM及培养皿上。分别收集两组细胞的总RNA,通过q RT-PCR检测ADSCs旁分泌基因的表达。将ADSCs预处理前后的HADM分别植入大鼠体内,4周后取材行组织学观察。结果 在HADM上培养的ADSCs与在普通培养皿上培养的ADSCs相比,其促血管形成因子(VEGF、HGF、bFGF)、炎症调节因子(TGF-β、i NOS、TSG-6、COX-2)及干性因子(SOX-2)均有所上调。经ADSC体外预培养后的HADM与未经处理的HADM相比,体内植入后表现出更好的血管化能力且炎症反应也更低。结论 HADM能为ADSCs发挥旁分泌作用提供合适的微环境,且HADM经过ADSCs体外预培养,其血管化程度更高,而炎症反应则得以缓解及局限化。     

Prevention of Chronic Rejection with Immunoregulatory Cells Induced by Intrathymic Immune Modulation with Class I Allopeptides

Intrathymic immune modulation with RT1.A^a allopeptides in the PVG.R8‐to‐PVG.1 U rat strain combination leads to long‐term survival of cardiac allografts. This regimen, however, does not induce transplantation tolerance, since most long‐surviving allografts undergo chronic rejection. We investigated recipients with chronic rejection for donor‐specific immune nonresponsiveness and immunoregulatory cells as possible mechanisms responsible for long‐term graft survival. There was a significant reduction in the proliferative response of T cells from long‐term allograft recipients to donor alloantigens as compared with that of naïve T cells. Adoptive transfer of splenocytes from intrathymically manipulated primary long‐term graft survivors into minimally irradiated secondary hosts resulted in indefinite survival of > 80% of allografts, providing evidence for immunoregulatory cells. Secondary recipients had total absence of donor‐reactive cellular and humoral responses. Immunoregulation was also transferable from secondary to tertiary graft recipients. More importantly, there was a significant reduction in the incidence of chronic rejection in secondary hosts (> 85%) and complete prevention of acute and chronic rejection in tertiary hosts. This study demonstrates that intrathymic immunomodulation with class I allopeptides results in the generation of immunoregulatory cells that do not block chronic rejection in primary hosts where they develop, but prevent both acute and chronic allograft rejection when adoptively transferred into secondary and tertiary recipients.     

Phagocytosis of Different Particulate Dermal Filler Substances by Human Macrophages and Skin Cells

BACKGROUND: Foreign substances have been introduced into the human body with varying degrees of success. Polymethylmethacrylate (PMMA) microspheres of different sizes recently have been manufactured for use as a filler substances in the skin and other organs. OBJECTIVE: To establish whether the size of PMMA microspheres determines whether various cell types initiate phagocytosis. METHODS: The capacity of three different cell lines-U-937 cells, XS 106 and XS 52 Langerhans cells, and HaCaT keratinocytes-to phagocytose microspheres of varying sizes was examined using light and confocal microscopy as well as fluorescence-activated cell sorter (FACS) analysis. Tumor necrosis factor (TNF)-alpha secretion was also determined. RESULTS: The U-937 cells, keratinocytes, and Langerhans cells could phagocytose PMMA particles of 20 microm or smaller. Microspheres larger than 20 microm were not ingested by any of the cells. CONCLUSION: Microspheres larger than 20 microm have a lower likelihood of being phagocytosed. Thus this study suggests that microspheres 40-50 microm in diameter are less likely to initiate an inflammatory reaction when injected into the dermis and subdermis as a filler substance. On the other hand, microparticles made of silicone and polymethacrylate were phagocytosed, possibly because of their different structure.     

Targeting of Mesenchymal Stromal Cells by Cre‐Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells

The targeting specificity of tissue‐specific Cre‐recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn‐Cre and Dmp1‐Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high‐resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12^gfp mice to identify Cxcl12‐abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn‐Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1‐Cre also targets a subset of CAR cells, in which expression of osteoblast‐lineage genes is enriched. Finally, we introduce a new tissue‐specific Cre‐recombinase, Tagln‐Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn‐Cre and Dmp1‐Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research.     

SDF-1-CXCR4/CXCR7信号对脂肪干细胞促血管新生能力的影响

目的 研究SDF-1对ADSCs体外促血管新生能力的影响,并探讨CXCR4、CXCR7在其中的作用。方法 体外培养ADSCs,检测其CXCR4、CXCR7的表达;SDF-1刺激ADSCs,并分别加入CXCR4、CXCR7封闭抗体,检测ADSCs-CM中VEGF、HGF、β-FGF含量,及其对h UVECs微血管形成的影响。结果 成功培养ADSCs;ADSCs体外培养传代过程中CXCR4、CXCR7表达持续下降;SDF-1刺激上调CXCR4、CXCR7表达,并提高ADSCs对血管活性因子的分泌,及促进h UVEC微血管的形成;封闭CXCR4、CXCR7均可显著抑制SDF-1的促进作用。结论 体外环境下,SDF-1可上调ADSCs中CXCR4、CXCR7表达,并通过SDF-1-CXCR4/CXCR7两条通路提高ADSCs的促血管新生能力。     

Schwann Cells Contribute to Alveolar Bone Regeneration by Promoting Cell Proliferation

The plasticity of Schwann cells (SCs) following nerve injury is a critical feature in the regeneration of peripheral nerves as well as surrounding tissues. Here, we show a pivotal role of Schwann cell-derived cells in alveolar bone regeneration through the specific ablation of proteolipid protein 1 (Plp)-expressing cells and the transplantation of teased nerve fibers and associated cells. With inducible Plp specific genetic tracing, we observe that Plp⁺ cells migrate into wounded alveolar defect and dedifferentiate into repair SCs. Notably, these cells barely transdifferentiate into osteogenic cell lineage in both SCs tracing model and transplant model, but secret factors to enhance the proliferation of alveolar skeletal stem cells (aSSCs). As to the mechanism, this effect is associated with the upregulation of extracellular matrix (ECM) receptors and receptor tyrosine kinases (RTKs) signaling and the downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase–protein kinase B (PI3K-Akt) pathway. Collectively, our data demonstrate that SCs dedifferentiate after neighboring alveolar bone injury and contribute to bone regeneration mainly by a paracrine function. © 2022 American Society for Bone and Mineral Research (ASBMR).