Journal of immunology and regenerative medicine最新文献

Introduction

Osteoclasts are bone-resorbing cells closely related to bone turnover, whereas different macrophage subtypes contribute to bone fracture healing. As osteoclasts and macrophages share the same hematopoietic origin, the difference between both cell types on osteoblast coupling, crosstalk extent and consequent bone formation remains poorly understood. This study compares the potential of primary cells that are routinely considered as osteoclast and macrophage cultures on their ability to support osteogenic differentiation of human mesenchymal stromal cells (hMSCs).

Methods

Human Peripheral Blood Mononuclear Cells (hPBMCs) were used to obtain macrophage or osteoclast cultures using appropriate stimulatory factors. With different seeding densities of hPBMCs, conditioned media from macrophage or osteoclast cultures were harvested for comparative evaluation of effects thereof on the osteogenic differentiation of hMSCs. Specific cytological staining was used to qualitatively evaluate macrophage and osteoclast cultures. Additionally, quantitative data on hMSC proliferation, osteogenic differentiation and mineralization were obtained via biochemical assays.

Results

Conditioned medium from osteoclast cultures obtained via low hPBMCs seeding densities, but not from high hPBMCs seeding densities or macrophages, stimulated hMSC osteogenic differentiation and mineralization. Upon cellular crosstalk, both pre-differentiated osteoclasts and non-polarized macrophages equally supported early hMSC osteogenic differentiation and mineralization, as confirmed by increased alkaline phosphatase levels within 7 days and increased calcium content within 14 days in comparison with undifferentiated controls. Initial hPBMCs seeding density strongly influences osteoclastogenesis and the paracrine effect of the resultant osteoclast population on the osteogenic differentiation of hMSCs. In addition, only in indirect coculture, macrophages provide similar stimulatory effects as pre-differentiated osteoclasts on the osteogenic differentiation of MSCs and mineralization.

Conclusion

Our results demonstrate stimulatory effects of osteoclast conditioned medium on hMSC osteogenic differentiation, depending on initial hPBMC seeding density. In addition, we show that osteoclast and macrophage cultures contain pools of polarized macrophages, which may be involved in the osteogenic effects. Our data provide insight into bone tissue engineering approaches by using multicellular interactions related to bone remodeling and healing for the in vitro modulation of osteogenic differentiation.

Introduction

The immune system is widely recognized as a crucial determinant in implant biocompatibility and tissue integration. In order to assess macrophage response to biomaterials, commonly used analysis techniques require the removal of cells from the material. This process inherently has a negative impact on the cells, but few studies have comprehensively compared different dissociation methods in terms of impact on cell yield, viability, phenotype and function.

Methods

Cell scraping, EDTA at 4 °C, EDTA at 37 °C, trypsin, Accutase and the PromoCell macrophage detachment solution were compared in terms of cell yield and viability. The effect of Accutase on cell surface antigen conservancy and cell functionality was investigated. Lastly, effect of Accutase detachment of macrophages from electrospun biomaterials was analyzed.

Results

The efficiency of common cell detachment protocols for human monocyte-derived macrophages (MDMs) varies significantly between enzymatic and non-enzymatic approaches. In terms of surface marker detection, we showed that enzymatic detachment not only selectively cleaves the M2 markers CD206 and CD163, but we also identified that this effect is variable across donors. Furthermore, we observed that the process of cell detachment impairs macrophage endocytic ability. Lastly, we tested the efficiency of enzymatic cell detachment on electrospun 3D matrices designed for tissue engineering.

Conclusion

Our results provide a thorough understanding of the advantages and disadvantages of commonly used cell dissociation methods and have important implications for studies investigating the macrophage response to biomaterials.

Objectives

Natural killer (NK) cells are an attractive and potent tool for cancer immunotherapy, however, ex vivo expansion of NK cells is required to achieve therapeutic cell dosages. As such, this review will discuss recent NK cell manufacturing methods applied in clinical trials for expanded NK cells for the treatment of solid tumors, as well as investigational NK cell manufacturing protocols. Given the unique challenges posed by the solid tumor microenvironment, the main objective of this review is to highlight key biological mechanisms associated with tumor homing and infiltration of NK cells and how manufacturing methods impact these functions.

Key findings

For efficient adoptive NK cell therapy for the treatment of solid malignancies, NK cells need to extravasate from the blood stream, migrate through the tumor extracellular matrix, lyse cancer cells, activate other immune cells, and persist in the blood stream. The NK cell manufacturing process is complex, with each parameter influencing the expansion rate, and final NK cell number, purity, phonotype, and cytotoxicity. Many investigational and clinical NK cell manufacturing protocols generate high numbers of NK cells with greater cytotoxicity than freshly isolated NK cells. The expression of factors related to homing and migration in NK cells after ex vivo expansion is largely overlooked, but the few studies which have explored this indicate manufacturing processes can affect these critical mechanisms.

Conclusion

The current manufacturing protocols can generate high numbers of NK cells with increased cytotoxic functions, however understanding the effect of expansion on factors related to NK cell homing and migration is also important for treating solid malignancies. Furthermore, to progress the field of expanded NK cells for the treatment of solid tumors, improving “off-the-shelf” NK cell therapies and standardizing the manufacturing protocols and release criteria should be prioritized.

Teratoma formation remains a safety concern in therapeutic cells derived from human-induced pluripotent stem cells (hiPSCs). Residual Teratoma forming cells are present in small numbers in differentiated hiPSC cultures and yet are of significant roadblock to the manufacturing and clinical translation of stem cell therapies. Rare cells are often difficult to remove with standard flow cytometry or magnetic bead sorting techniques. Here, we first characterized time-dependent expression of a teratoma marker, stage-specific embryonic antigen (SSEA)-5, which binds the H type-1 glycan during neural differentiation of hiPSCs. The fraction of cells SSEA-5+ remained high at 97% on day 3, dropped to 70% on day 4, 40% by day 6, and down to 1% on day 12 of differentiation, indicating successful differentiation. We engineered a microfluidic geometrically enhanced differential immunocapture (GEDI) technology to remove SSEA-5+ rare cells from hiPSC-derived neural progenitor cells (hiPSC-NPCs). The GEDI chip removed more than 95% of teratoma-forming cells and presents a facile tool to potentially functionalize with multiple antibodies and robustly enhance hiPSC-derived cell population safety prior to therapeutic transplantation. The approach is potentially amenable to generate a wide variety of high-quality therapeutic cells and can be integrated within the pipeline of cell manufacturing to improve patient safety and reduce the cost of manufacturing through early removal of undesirable cell types.

The polarization of monocytes into macrophages that possess anti-inflammatory and pro-angiogenic properties could provide a novel therapeutic strategy for patients who are at a high risk for developing heart failure following myocardial infarction (MI). Here in, we describe a novel method of “educating” monocytes into a distinct population of macrophages that exhibit anti-inflammatory and pro-angiogenic features through a 3-day culture on fibronectin-rich cardiac matrix (CX) manufactured using cultured human cardiac fibroblasts. Our data suggest that CX can educate monocytes into a unique macrophage population termed CX educated macrophages (CXMq) that secrete high levels of VEGF and IL-6. In vitro, CXMq also demonstrate the ability to recruit mesenchymal stromal cells (MSC) with known anti-inflammatory properties. Selective inhibition of fibronectin binding to αVβ3 surface integrins on CXMq prevented MSC recruitment. This suggests that insoluble fibronectin within CX is, at least in part, responsible for CXMq conversion.

In clinical islet transplantation, the engrafted islet cell mass is a critical factor for maintaining graft function and transplant outcome. This review dissects components that contribute to an acute innate immune response, including ischemia-reperfusion injury, damage-associated molecular patterns, islet-derived cytokines (isletokines), and an instant blood-mediated inflammatory reaction. The cyclical amplification of immune cell response and islet inflammation can occur at each stage of the transplant process. As such, strategies to improve islet transplantation include minimizing islet and immune cell cytokine production prior to procurement; suppressing the pretransplant islet immune inflammatory response; inhibiting the instant blood-mediated inflammatory reaction in the liver; preventing an innate immune response by encapsulation and islet surface modification; targeting cytokine/chemokine secretion and innate immune cell infiltration; considering the optimal site for islet engraftment; blocking cytokine signaling in islets; and implementing anti-inflammatory strategies to improve islet transplantation. A large body of evidence indicates that targeting immune components at multiple steps throughout the transplant procedure can improve overall islet transplant outcomes.

Accurate determination of cellular subsets that secrete particular cytokine(s) is a significant parameter for functional characterization of an immunological response. The present study was conducted to develop a method for simultaneous measurement of intracellular cytokine positive CD4 and CD8 positive T lymphocytes in a single tube, with a no-wash protocol. We report here the development of a simplified, rapid procedure for precise enumeration of cytokine positive T lymphocytes using BD Trucount tubes. This single step protocol for accurate enumeration of cytokine positive T lymphocytes, will allow for better characterization of immune cell phenotype and function.

Lizards are the closest relatives of mammals capable of tail regeneration, but the specific determinants of amniote regenerative capabilities are currently unknown. Macrophages are phagocytic immune cells that play a critical role in wound healing and tissue regeneration in a wide range of species. We hypothesize that macrophages regulate the process of lizard tail regeneration, and that comparisons with mammalian cell populations will yield insight into the role phagocytes play in determining an organism's regenerative potential. Single cell RNA sequencing (scRNAseq) was used to profile lizard immune cells and compare with mouse counterparts to contrast cell types between the two species. Treatment with clodronate liposomes effectively inhibited lizard tail stump tissue ablation and subsequent regeneration, and scRNAseq was used to profile changes in lizard immune cell populations resulting from tail amputation as well as identifying specific cell types affected by clodronate treatment. ScRNAseq analysis of lizard bone marrow, peripheral blood, and tissue-resident phagocyte cell populations was used to trace marker progression during macrophage differentiation and activation. These results indicated that lizard macrophages are recruited to tail amputation injuries faster than mouse populations and express high levels of matrix metalloproteinases (MMPs). In turn, treatment with MMP inhibitors inhibited lizard tail regeneration. These results provide single cell sequencing data sets for evaluating and comparing lizard and mammalian immune cell populations, and identifying macrophage populations that are critical regulators of lizard tail regrowth.

Tissue repair and maintenance in adult organisms is dependent on the interactions between stem cells (SCs) and constituent cells of their microenvironment, or niche. Accumulating evidence suggests that immune cells, specifically Foxp3⁺ CD4⁺ Regulatory T cells (Tregs), play an important role as a regulator of the SC niche. Undisputedly, Tregs are the major immunosuppressive lineage of the CD4⁺ T cell compartment, and reside within numerous secondary lymphoid organs, where they exert their functions. These cells are also specialised in facilitating protective functions specific to their tissue of residence. In this review, we discuss the emerging concepts supporting the SC-regulatory functions of tissue-resident Tregs, during both the steady-state and SC-mediated regeneration. We highlight the skin, intestines, and lung as model organs which are subject to recurrent microinjury,exposure to microbiota, and constantly replenished by resident stem cell populations. An in-depth understanding of the biology of the Treg-SC axis will inform ongoing immunotherapeutic endeavours to target specific subpopulations of tissue-resident Tregs.

Decellularized tissues and organs from animal sources are widely used in regenerative medicine and tissue engineering. However, in tendon tissue engineering there is limitation not only in terms of tissue source -allografts and autografts- but also standardization of decellularization techniques. The goal of this study is to decellularize porcine Achilles tendon to be used as an off-the-shelf product for tendon reconstruction. We describe a novel, mild decellularization strategy which retains the biochemical and biomechanical characteristics of native tendon upon decellularization. We further show that decellularized tendon sections in vitro induce tenogenic differentiation in stem cells and anti-inflammatory response in naïve macrophages. Upon implantation in an Achilles tendon defectin rabbits, we observed that decellularized tendons integrated with the host tissue without signs of tissue rejection or encapsulation. Together, we demonstrate that decellularized tendons produced with our new protocol bear a great potential for tendon tissue regeneration.