Search

SummaryThe lymph node (LN) is home to resident macrophage populations that are essential for immune function and homeostasis. The T cell paracortical zone is a major site of macrophage efferocytosis of apoptotic cells, but key factors controlling this niche are undefined. Here we show that fibroblastic reticular cells (FRCs) are an essential component of the LN macrophage niche. Macrophages co-localised with FRCs in human LNs, and murine single-cell RNA-sequencing revealed that most reticular cells expressed master macrophage regulator CSF1. Functional assays showed that CSF1R signalling was sufficient to support macrophage development. In the presence of LPS, FRCs underwent a mechanistic switch and maintained support through CSF1R-independent mechanisms. These effects were conserved between mouse and human systems. Rapid loss of macrophages and monocytes from LNs was observed upon genetic ablation of FRCs. These data reveal a critically important role for FRCs in the creation of the parenchymal macrophage niche within LNs.

Cellular immunotherapy is revolutionizing cancer treatment. However, autologous transplants are complex, costly, and limited by the number and quality of T cells that can be isolated from and expanded for re-infusion into each patient. This paper demonstrates a stromal support cell-free in vitro method for the differentiation of T cells from umbilical cord blood hematopoietic stem cells (HSCs). For each single HSC cell input, approximately 5 × 104 T cells were created with an initial five days of HSC expansion and subsequent T cell differentiation over 49 days. When the induced in vitro differentiated T cells were activated by cytokines and anti-CD3/CD28 beads, CD8+ T cell receptor (TCR) γδ+ T cells were preferentially generated and elicited cytotoxic function against ovarian cancer cells in vitro. This process of inducing de novo functional T cells offers a possible strategy to increase T cell yields, simplify manufacturing, and reduce costs with application potential for conversion into chimeric antigen receptor (CAR)-T cells for cancer immunotherapy and for allogeneic transplantation to restore immune competence.

Aim: To evaluate the efficacy of autologous adipose-derived mesenchymal stem cell (ADMSC) therapy on pain, function and disease modification in knee osteoarthritis. Methods: 30 participants with symptomatic knee osteoarthritis were randomized into three groups. Two treatment groups received intra-articular ADMSC therapy consisting of either a single injection (100 × 106 ADMSCs) or two injections (100 × 106 ADMSCs at baseline and 6 months). The third group served as control and continued conservative management. Results: No serious adverse events were observed. Both treatment groups receiving ADMSCs showed clinically significant pain and functional improvement at completion of follow-up at 12 months. Radiological analysis using the Magnetic Resonance Imaging Osteoarthritis Knee Score indicated modification of disease progression. Conclusion: Autologous ADMSC therapy appears to be a safe and effective therapy for knee osteoarthritis and may have the potential to prevent disease progression. Trial registration number: ACTRN12614000814673

Natural killer (NK) cells are potent innate immune system effector lymphocytes armed with multiple mechanisms for killing cancer cells. Given the dynamic roles of NK cells in tumor surveillance, they are fast becoming a next-generation tool for adoptive immunotherapy. Many strategies are being employed to increase their number and improve their ability to overcome cancer resistance and the immunosuppressive tumor microenvironment. These include the use of cytokines and synthetic compounds to bolster propagation and killing capacity, targeting immune-function checkpoints, addition of chimeric antigen receptors (CARs) to provide cancer specificity and genetic ablation of inhibitory molecules. The next generation of NK cell products will ideally be readily available as an “off-the-shelf” product and stem cell derived to enable potentially unlimited supply. However, several considerations regarding NK cell source, genetic modification and scale up first need addressing. Understanding NK cell biology and interaction within specific tumor contexts will help identify necessary NK cell modifications and relevant choice of NK cell source. Further enhancement of manufacturing processes will allow for off-the-shelf NK cell immunotherapies to become key components of multifaceted therapeutic strategies for cancer.

Chimeric antigen receptor (CAR) T cells have revolutionized blood cancer immunotherapy; however, their efficacy against solid tumors has been limited. A common mechanism of tumor escape from single target therapies is downregulation or mutational loss of the nominal epitope. Targeting multiple antigens may thus improve the effectiveness of CAR immunotherapies. We generated dual CAR-T cells targeting two tumor antigens: TAG-72 (tumor-associated glycoprotein 72) and CD47. TAG-72 is a pan-adenocarcinoma oncofetal antigen, highly expressed in ovarian cancers, with increased expression linked to disease progression. CD47 is ubiquitously overexpressed in multiple tumor types, including ovarian cancer; it is a macrophage “don’t eat me” signal. However, CD47 is also expressed on many normal cells. To avoid this component of the dual CAR-T cells killing healthy tissue, we designed a truncated CD47 CAR devoid of intracellular signaling domains. The CD47 CAR facilitates binding to CD47+ cells, increasing the prospect of TAG-72+ cell elimination via the TAG-72 CAR. Furthermore, we could reduce the damage to normal tissue by monomerizing the CD47 CAR. Our results indicate that the co-expression of the TAG-72 CAR and the CD47-truncated monomer CAR on T cells could be an effective, dual CAR-T cell strategy for ovarian cancer, also applicable to other adenocarcinomas., Graphical Abstract, Tumors often escape immunotherapy by mutational downregulation of the nominal target antigen. Use of CAR-T cells with dual antigen specificity can mitigate this problem. Adenocarcinomas expressing low levels of TAG-72 are not killed by single specificity TAG-72 CAR-T cells, but they are killed by dual CAR-T cells targeting TAG-72 and CD47.

Light triggered release of an antibiotic from a click crosslinked hydrogel was developed by conjugating ciprofloxacin through a photo-cleavable linker to the hydrogel network structure. Upon irradiation of the hydrogel material with UV light (365 nm) at low intensity, native ciprofloxacin was released into the surrounding environment and could be detected by HPLC. The antimicrobial activity of the released compound on Staphylococcus aureus was demonstrated.

Cell transplantation holds considerable promise for end-stage liver diseases but identifying a suitable, transplantable cell type has been problematic. Here, we describe a novel type of mesenchymal stem cells (MSCs) from human adipose tissue. These cells are different from previously reported MSCs, they are in the euchromatin state with epigenetic multipotency, and express pluripotent markers MYC, KLF4, and GMNN. Most of the genes associated with germ layer specification are modified by H3K4me3 or co-modified by H3K4me3 and H3K27me3. We named this new type of MSCs as adult multipotent adipose-derived stem cells (M-ADSCs). Using a four-step nonviral system, M-ADSCs can be efficiently Induced into hepatocyte like cells with expression of hepatocyte markers, drug metabolizing enzymes and transporters, and the other basic functional properties including albumin (ALB) secretion, glycogen storage, detoxification, low-density lipoprotein intake, and lipids accumulation. In vivo both M-ADSCs-derived hepatoblasts and hepatocytes could form vascularized liver-like tissue, secrete ALB and express metabolic enzymes. Single-cell RNA-seq was used to investigate the important stages in this conversion. M-ADSCs could be converted to a functionally multipotent state during the preinduction stage without undergoing reprogramming process. Our findings provide important insights into mechanisms underlying cell development and conversion.

In this issue of Cell Stem Cell, Xu et al. (2019) demonstrate that editing iPSCs' major histocompatibility antigens may potentially provide a small set of universally compatible stem cell lines for therapies. However, these modifications may result in patient minor histocompatibility responses and deficiencies in their T cell response repertoire to infection and cancer.

Graft-versus-host disease (GVHD) and posttransplant immunodeficiency are frequently related complications of allogeneic hematopoietic transplantation. Alloreactive donor T cells can damage thymic epithelium, thus limiting new T-cell development. Although the thymus has a remarkable capacity to regenerate after injury, endogenous thymic regeneration is impaired in GVHD. The mechanisms leading to this regenerative failure are largely unknown. Here we demonstrate in experimental mouse models that GVHD results in depletion of intrathymic group 3 innate lymphoid cells (ILC3s) necessary for thymic regeneration. Loss of thymic ILC3s resulted in deficiency of intrathymic interleukin-22 (IL-22) compared with transplant recipients without GVHD, thereby inhibiting IL-22-mediated protection of thymic epithelial cells (TECs) and impairing recovery of thymopoiesis. Conversely, abrogating IL-21 receptor signaling in donor T cells and inhibiting the elimination of thymic ILCs improved thymopoiesis in an IL-22-dependent fashion. We found that the thymopoietic impairment in GVHD associated with loss of ILCs could be improved by restoration of IL-22 signaling. Despite uninhibited alloreactivity, exogenous IL-22 administration posttransplant resulted in increased recovery of thymopoiesis and development of new thymus-derived peripheral T cells. Our study highlights the role of innate immune function in thymic regeneration and restoration of adaptive immunity posttransplant. Manipulation of the ILC-IL-22-TEC axis may be useful for augmenting immune reconstitution after clinical hematopoietic transplantation and other settings of T-cell deficiency.

Background A prospective analysis of the effect of autologous adipose derived mesenchymal stem cell (MSC) therapy in the treatment of an osteochondral defect of the knee with early progressive osteoarthritis following unsuccessful surgical intervention of osteochondritis dissecans (OCD). Case presentation After failed conventional management of OCD a patient undergoes intra-articular MSC therapy. Patient outcome measures included the Numeric Pain Rating Scale (NPRS), the Western Ontario and McMaster Universities Arthritis Index (WOMAC) and the Knee Injury and Osteoarthritis Outcome Score (KOOS). Structural outcome was assessed using MRI with the novel technique of T2 mapping used to indicate cartilage quality. Following MSC therapy the patient reported improvement in pain and function as measured by NPRS, WOMAC and KOOS. Repeat MRI analysis showed regeneration of cartilage. MRI T2 mapping indicated hyaline like cartilage regrowth. Conclusion In this report, the use of MSCs, after unsuccessful conventional OCD management, resulted in structural, functional and pain improvement. These results highlight the need to further study the regenerative potential of MSC therapy. Trial registration Australian and New Zealand Clinical Trial Registry Number - ACTRN12615000258550 (Date registered 19/03/2015 – retrospectively registered).

Although forkhead-box n1 (Foxn1) is a critical thymic epithelial cell regulator in thymus organogenesis, its association with epithelial differentiation and homeostasis in the postnatal and aged thymic microenvironment remains conflicting. Consequently, we have generated a Foxn1eGFP/+ knock-in mouse model that allows for refined investigation of the aging thymic epithelium. This reporter line differs from those previously published in that concomitant expression of enhanced green fluorescent protein enables live cell sorting of Foxn1+ cell populations. Our heterozygotes did not exhibit haploinsufficiency, with Foxn1 expression resembling that of wild-type mice. Comparative analysis between Foxn1 and enhanced green fluorescent protein at both the transcriptional and translational levels revealed co-localization, with progressive down-regulation observed predominantly in the aging cortical epithelium. Supplementation with bone morphogenetic protein (Bmp)-4 enhanced Foxn1 expression and colony forming efficiency in both embryonic and adult progenitor 3D cultures. Strikingly, selective maintenance of immature cortical and medullary epithelial cells was observed which is consistent with the higher Bmp receptor 2 expression levels seen in these progenitor populations. This study demonstrates the significance of our mouse model in unraveling the role of this master regulator in thymus development, homeostasis and aging, providing a faithful reporter system for phenotypic and functional investigations.

Background & Aim The clinical impact of Chimeric Antigen Receptor T cell (CAR-T) technologies on hematological malignancies have revolutionized cancer treatment. However, current autologous CAR-T therapies face major roadblocks for mass adoption. These include high-cost, patient-specific manufacturing, inconsistent CAR-T yield and function due to inherently depleted patient immune systems, and life-threatening adverse events. NK cell therapies have the potential to overcome at least some of these deficiencies. NK cells utilize multiple anti-cancer receptors without risk of graft versus host disease. However, they have reduced longevity in vivo, which may necessitate multiple infusions, increasing the risk of their rejection by the patient. Both CAR-T and CAR-NK cells are subject to checkpoint blockades and other mechanisms of immunosuppression that diminish their killer function in vivo. To address these key deficiencies, we developed ‘off-the-shelf’, genetically-enhanced CAR-NK cells via induced pluripotent stem cells (iPSCs). Utilization of iPSCs as a renewable source for NK cells allows for consistent, precisely-defined immunotherapy and the capacity to gene-edit in multiple anti-cancer modes of action, which we have taken advantage of. The CAR-iNK cells can be cryopreserved, delivered on-demand for each patient, and crucially enable a major reduction in manufacturing cost. Methods, Results & Conclusion Our manufacturing system is highly scalable, completely xeno- and feeder-free, yielding ∼105 iNK cells from a single iPSC in less than 30 days. The iPSCs are gene-edited to carry a CAR (specific for the adenocarcinoma neoantigen TAG72) and deleted of immune suppression gene(s) to enhance NK longevity and efficacy. Unlike nearly all current allogeneic NK therapies, our iPSCs are derived from rare triple homozygous HLA donors, reducing the risk of host-mounted rejection of these iNK cells. The manufactured CAR-iNK cells display potent, on-target cytotoxic functionality against multiple ovarian cancer cell lines in vitro but do not kill cells from fresh healthy tissue. They are presently being evaluated in vivo. Our process provides a near limitless, on-demand supply of standardized ‘off-the-shelf’ CAR-iNK cells, with potential application to a variety of cancers, sufficient to treat many patients using a single manufactured product.

Osteoarthritis is a common condition that causes joint pain and stiffness that affects both humans and dogs. In Australia, allogeneic canine adipose-derived mesenchymal stem cells for therapy have been commercially available since 2010. In this report, we describe the outcome of the treatment of two hundred and three dogs diagnosed with degenerative arthritis with severe chronic pain in joints causing lameness at walk, reduced mobility, and functional disability. Posttreatment assessment data after 10 weeks revealed significant improvement (p<0.007) of the symptoms: pain reduction, improvement of mobility, and increased daily activity as measured as quality of life score. Ninety percent of young dogs (

Summary Mechanisms underlying age-related defects within lymphoid-lineages remain poorly understood. We previously reported that sex steroid ablation (SSA) induced lymphoid rejuvenation and enhanced recovery from hematopoietic stem cell (HSC) transplantation (HSCT). We herein show that, mechanistically, SSA induces hematopoietic and lymphoid recovery by functionally enhancing both HSC self-renewal and propensity for lymphoid differentiation through intrinsic molecular changes. Our transcriptome analysis revealed further hematopoietic support through rejuvenation of the bone marrow (BM) microenvironment, with upregulation of key hematopoietic factors and master regulatory factors associated with aging such as Foxo1. These studies provide important cellular and molecular insights into understanding how SSA-induced regeneration of the hematopoietic compartment can underpin recovery of the immune system following damaging cytoablative treatments. These findings support a short-term strategy for clinical use of SSA to enhance the production of lymphoid cells and HSC engraftment, leading to improved outcomes in adult patients undergoing HSCT and immune depletion in general., Graphical Abstract, Highlights • Sex steroid ablation (SSA) increases number of hematopoietic stem cells (HSCs) • SSA enhances reconstitution potential and self-renewal of HSCs • SSA reverses the age-associated decline in Foxo1 expression by hematopoietic niche • There is an increase in niche expression of hematopoiesis-associated factors after SSA, Age-related declines in immune function underlie a wide range of diseases and impaired capacity to recover adaptive immunity following therapeutic immune depletion. Increasing evidence implicates a functional decline in hematopoietic stem cells (HSCs). Here, Chidgey, Dudakov, and colleagues demonstrate that reversal of HSC aging by blocking the immunosuppressive actions of sex steroids involves mechanisms both intrinsic and extrinsic to HSCs. These findings will enable progress in understanding bone marrow and HSC aging and in developing strategies for immune regeneration.

Damage to the enteric nervous system (ENS) associated with intestinal inflammation may underlie persistent alterations to gut functions, suggesting that enteric neurons are viable targets for novel therapies. Mesenchymal stem cells (MSCs) offer therapeutic benefits for attenuation of neurodegenerative diseases by homing to areas of inflammation and exhibiting neuroprotective, anti-inflammatory, and immunomodulatory properties. In culture, MSCs release soluble bioactive factors promoting neuronal survival and suppressing inflammation suggesting that MSC-conditioned medium (CM) provides essential factors to repair damaged tissues. We investigated whether MSC and CM treatments administered by enema attenuate 2,4,6-trinitrobenzene-sulfonic acid (TNBS)-induced enteric neuropathy and motility dysfunction in the guinea pig colon. Guinea pigs were randomly assigned to experimental groups and received a single application of TNBS (30 mg/kg) followed by 1 × 106 human bone marrow-derived MSCs, 300 μl CM, or 300 μl unconditioned medium 3 h later. After 7 days, the effect of these treatments on enteric neurons was assessed by histological, immunohistochemical, and motility analyses. MSC and CM treatments prevented inflammation-associated weight loss and gross morphological damage in the colon; decreased the quantity of immune infiltrate in the colonic wall ( P < 0.01) and at the level of the myenteric ganglia ( P < 0.001); prevented loss of myenteric neurons ( P < 0.05) and damage to nerve processes, changes in ChAT, and nNOS immunoreactivity ( P < 0.05); and alleviated inflammation-induced colonic dysmotility (contraction speed; P < 0.001, contractions/min; P < 0.05). These results provide strong evidence that both MSC and CM treatments can effectively prevent damage to the ENS and alleviate gut dysfunction caused by TNBS-induced colitis.

Mixed hematopoietic chimerism is a powerful means of generating donor-specific tolerance, allowing long-term graft acceptance without lifelong dependence on immunosuppressive drugs. To avoid the need for whole body irradiation and associated side effects, we utilized a radiation-free minimal conditioning regime to induce long-term tolerance across major histocompatibility barriers. We found that low-dose busulfan, in combination with host T cell depletion and short-term sirolimus-based immunosuppression, facilitated efficient donor engraftment. Tolerance was achieved when mice were transplanted with whole or T cell-depleted bone marrow, or purified progenitor cells. Tolerance induction was associated with an expansion in regulatory T cells and was not abrogated in the absence of a thymus, suggesting a dominant or compensatory peripheral mode of tolerance. Importantly, we were able to generate durable chimerism and tolerance to donor skin grafts in both young and aged mice, despite age-related thymic atrophy and immune senescence. Clinically, this is especially relevant as the majority of transplant recipients are older patients whose immune recovery might be dangerously slow and would benefit from radiation-free minimal conditioning regimes that allow efficient donor engraftment without fully ablating the recipient immune system.

Summary Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1GFP/w line as a readout, we developed a reproducible protocol for generating FOXN1-GFP+ thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-β4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1+ TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1+ TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine., Highlights • FOXN1-GFP reporter hESC lines were generated • KGF promotes the proliferation of FOXN1-GFP+ cells • FOXN1-GFP+ cells express TEC-associated genes • ITGB4, HLA-DR, and EpCAM can be used to purify FOXN1+ TEC progenitors (219), Stanley and colleagues generated human embryonic stem cell (hESC) reporter lines in which sequences encoding GFP were inserted into the FOXN1 locus, enabling the isolation of viable thymic progenitors from in vitro differentiation cultures. These reporter lines were used to identify integrin-β4, HLA-DR, and EpCAM as markers of human pluripotent stem cell-derived FOXN1+ thymic epithelial progenitors.

Context: Articular cartilage possesses poor natural healing mechanisms, and a variety of non-cell-based and cell-based treatments aim to promote regeneration of hyaline cartilage. Data Sources: A review of the literature to December 2013 using PubMed with search criteria including the keywords stem cell, cell therapy, cell transplantation, cartilage, chondral, and chondrogenic. Study Selection: Forty-five articles were identified that employed local mesenchymal stem cell (MSC) therapy for joint disorders in humans. Nine comparative studies were identified, consisting of 3 randomized trials, 5 cohort studies, and 1 case-control study. Study Type: Clinical review. Level of Evidence: Level 4. Data Extraction: Studies were assessed for stem cell source, method of implantation, comparison groups, and concurrent surgical techniques. Results: Two studies comparing MSC treatment to autologous chondrocyte implantation found similar efficacy. Three studies reported clinical benefits with intra-articular MSC injection over non-MSC controls for cases undergoing debridement with or without marrow stimulation, although a randomized study found no significant clinical difference at 2-year follow-up but reported better 18-month magnetic resonance imaging and histologic scores in the MSC group. No human studies have compared intra-articular MSC therapy to non-MSC techniques for osteoarthritis in the absence of surgery. Conclusion: Mesenchymal stem cell–based therapies appear safe and effective for joint disorders in large animal preclinical models. Evidence for use in humans, particularly, comparison with more established treatments such as autologous chondrocyte implantation and microfracture, is limited.

Velardi et al. show that sex steroids regulate thymopoiesis by directly modulating Notch signaling, and provide a novel clinical strategy to boost immune regeneration., Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.

Background & Aim Background Osteoarthritis is a degenerative condition and a leading cause of pain and disability world wide. Current medical treatments are aimed at symptomatic control and do not achieve disease modification. Surgical interventions including total joint arthroplasty can be unpredictable. Pre-clinical and early clinical trials have shown initial evidence of safety and efficacy of mesenchymal stem cell (MSC) therapies. Aim To evaluate the efficacy of autologous adipose derived mesenchymal stem cell (AdMSC) therapy on pain, function and disease modification in symptomatic knee osteoarthritis (OA). Methods, Results & Conclusion Methods 30 participants with symptomatic knee osteoarthritis were recruited via website and general practice advertisements. Eligibility criteria included radiological confirmed Grade II-III knee OA according to Kellgren Lawrence criteria, age ≥18, and a previous conservative management program. Exclusion criteria included a history of cancer, an atypical pain disorder and pregnancy. Subjects were randomised into 3 groups of 10 participants. The 2 treatment groups received intra-articular autologous AdMSC therapy consisting of either a single injection of 100 × 106 AdMSCs or two injections of 100 × 106 AdMSCs (baseline and again at 6 months). The control group continued conservative management. Participants were not blinded. Outcome measures were recorded over 12 months. Primary and secondary outcome measures: Primary outcome aims of this trial were pain and functional changes as measured by the numeric pain rating scale (NPRS), the Knee Injury and Osteoarthritis Outcome Score and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). A secondary objective was to assess structural change using the semi-quantitative MRI Osteoarthritis Knee Score (MOAKS). Results Participants in the treatment groups receiving a single or two injection protocol showed statistical and clinically significant pain and functional improvement in comparison to control at completion of follow-up at 12 months. Radiological analysis indicated modification of disease progression in both treatment groups with the two-injection protocol achieving the greatest effect. No serious AEs were observed. Conclusion Autologous AdMSC therapy resulted in significant improvements in pain and function and also resulted in structural stabilisation. AdMSC therapy appears to be a safe and effective therapy for symptomatic knee osteoarthritis and has the potential to prevent disease progression.

Hassall’s corpuscles are concentric clusters of keratinized epithelial cells located within the thymic medulla of humans and guinea pigs but are scant in mouse and rat. They are considered to be the terminally differentiated stages of medullary thymic epithelial cells (mTECs) but the mechanisms of their origin are unclear. We have previously deleted the TGF-β type II receptor (TGFβRII) specifically in mouse TECs and reported that these mice have mitigated thymic involution and exhibit earlier reconstitution post-irradiation. In this study, we analyzed the differentiation of mTECs in the TGFβRII-knockout mice. Interestingly, the TGFβRII-knockout mice display enhanced development of Hassall’s corpuscles. The expression of Aire, stromal-cell-derived factor 1 and thymic stromal lymphopoietin in the thymi of the TGFβRII-knockout mice was similar to that previously reported for the human thymus. In addition, the putative epithelial progenitor markers MTS20 and MTS24 labeled Hassall’s corpuscles in normal mice, but the extent and intensity of this staining were greatly enhanced in Hassall’s corpuscles of the TGFβRII-knockout mice. The phosphorylated forms of ERK and JNK were also found in Hassall’s corpuscles of the TGFβRII-knockout mice. Taken together, we suggest that TGFβRII-mediated signaling in TECs inhibits their development into Hassall’s corpuscles in mice.

During early thymocyte development, coordinated JAK/STAT5 and SFK/pre-TCR signaling is critical for T cell lineage commitment and αβ versus γδ specification. Wiede et al. show a role for the tyrosine phosphatase PTPN2 in attenuating SRC family kinase LCK and STAT5 signaling to regulate αβ and γδ T cell development., In the thymus, hematopoietic progenitors commit to the T cell lineage and undergo sequential differentiation to generate diverse T cell subsets, including major histocompatibility complex (MHC)–restricted αβ T cell receptor (TCR) T cells and non–MHC-restricted γδ TCR T cells. The factors controlling precursor commitment and their subsequent maturation and specification into αβ TCR versus γδ TCR T cells remain unclear. Here, we show that the tyrosine phosphatase PTPN2 attenuates STAT5 (signal transducer and activator of transcription 5) signaling to regulate T cell lineage commitment and SRC family kinase LCK and STAT5 signaling to regulate αβ TCR versus γδ TCR T cell development. Our findings identify PTPN2 as an important regulator of critical checkpoints that dictate the commitment of multipotent precursors to the T cell lineage and their subsequent maturation into αβ TCR or γδ TCR T cells.

Although forkhead-box n1 (Foxn1) is a critical thymic epithelial cell regulator in thymus organogenesis, its association with epithelial differentiation and homeostasis in the postnatal and aged thymic microenvironment remains conflicting. Consequently, we have generated a Foxn1

Osteoarthritis is a leading cause of pain and disability across the world. With an aging population its prevalence is likely to further increase. Current accepted medical treatment strategies are aimed at symptom control rather than disease modification. Surgical options including joint replacement are not without possible significant complications. A growing interest in the area of regenerative medicine, led by an improved understanding of the role of mesenchymal stem cells in tissue homeostasis and repair, has seen recent focused efforts to explore the potential of stem cell therapies in the active management of symptomatic osteoarthritis. Encouragingly, results of pre-clinical and clinical trials have provided initial evidence of efficacy and indicated safety in the therapeutic use of mesenchymal stem cell therapies for the treatment of knee osteoarthritis. This paper explores the pathogenesis of osteoarthritis and how mesenchymal stem cells may play a role in future management strategies of this disabling condition.

The thymus plays a critical role in maintaining immune well-being, but paradoxically undergoes progressive age-related atrophy. Many efforts have been made to deepen our understanding of its biology, with future therapies aimed at re-establishing T cell production in immunocompromised patients. This includes the elderly, and patients undergoing cytoablative treatments or chronically infected with immunotropic viruses. Here we discuss major pre-clinical approaches to thymic regeneration, as a representation of the strategies potentially able to be utilised therapeutically. We outline thymic physiology and development to assist in understanding the rationale behind each regenerative strategy – broadly, reactivation of endogenous thymic epithelial progenitor cells, de novo generation of thymic epithelium, and the facilitating effects on these of bioengineering. Continued advancement in these approaches may lead to their clinical translation for the recovery of immune competence in the aged or immune deficient, and tolerance applications in the developing field of stem cell therapeutics.

Introduction: Side effects of psychotropic medication often lead to rapid weight gain, having detrimental effects on forensic mental health patients' health, wellbeing, occupational performance and quality of life. Virtual reality technology could provide novel environments and motivating forums for exercise, which are otherwise unavailable to patients in such secure settings. This exploratory study aimed to evaluate the use of the Nintendo Wii Fit in changing engagement in physical activity for patients at risk of obesity at a secure hospital. Method: Two participants used a Wii Fit for 8 weeks in individual or group sessions. A mixed methods approach was taken, because participants' use of the Wii Fit was compared with their attitudes towards it (reported during interviews) and their daily physical activity levels (measured using an accelerometer). Researcher field notes were also used to gather contextual data. Findings: Participants played Wii Fit up to four times a week in sessions ranging from 7 to 127 minutes. When using the Wii Fit, participants increased their overall time spent actively moving their bodies in physical activity, as measured by the accelerometer. Using the Wii Fit also changed participants' attitudes towards exercise as they realised that it could be ‘fun’ and ‘challenging’, especially if staff members also participated. Conclusion: The Wii Fit encouraged patients to attempt physical activities and to learn about their bodily response to exercise. It provided a meaningful occupational intervention in a secure setting and demonstrated a potential use of the technology in mental health settings.

Negative selection plays a key role in the clonal deletion of autoreactive T cells in the thymus. However, negative selection is incomplete; as high numbers of autoreactive T cells can be detected in normal individuals, mechanisms that regulate negative selection must exist. In this regard, we previously reported that CD24, a GPI-anchored glycoprotein, is required for thymic generation of autoreactive T lymphocytes. The CD24-deficient 2D2 TCR transgenic mice (2D2+CD24−/−), whose TCR recognizes myelin oligodendrocyte glycoprotein (MOG), fail to generate functional 2D2 T cells. However, it was unclear if CD24 regulated negative selection, and if so, what cellular mechanisms were involved. Here, we show that elimination of MOG or Aire gene expression in 2D2+CD24−/− mice — through the creation of 2D2+CD24−/−MOG−/− or 2D2+CD24/∼Aire−/−mice — completely restores thymic cellularity and function of 2D2 T cells. Restoration of CD24 expression on DCs, but not on thymocytes also partially restores 2D2 T-cell generation in 2D2+CD24−/− mice. Taken together, we propose that CD24 expression on thymic antigen-presenting cells (mTECs, DCs) down-regulates autoantigen-mediated clonal deletion of autoreactive thymocytes.

Commercial purity titanium with an average grain size in the low sub-micron range was produced by equal channel angular pressing (ECAP). Attachment of human bone marrow-derived mesenchymal stem cells (hMSCs) to the surface of conventional coarse grained and ECAP-modified titanium was studied. It was demonstrated that the attachment and spreading of hMSCs in the initial stages (up to 24h) of culture was enhanced by grain refinement. Surface characterization by a range of techniques showed that the main factor responsible for the observed acceleration of hMSC attachment and spreading on titanium due to grain refinement in the bulk is the attendant changes in surface topography on the nanoscale. These results indicate that, in addition to its superior mechanical properties, ECAP-modified titanium possesses improved biocompatibility, which makes it to a potent candidate for applications in medical implants.

Brain and spinal cord injuries present significant therapeutic challenges. The treatments available for these conditions are largely ineffective, partly due to limitations in directly targeting the therapeutic agents to sites of pathology within the central nervous system (CNS). The use of stem cells to treat these conditions presents a novel therapeutic strategy. A variety of stem cell treatments have been examined in animal models of CNS trauma. Many of these studies have used stem cells as a cell-replacement strategy. These investigations have also highlighted the significant limitations of this approach. Another potential strategy for stem cell therapy utilises stem cells as a delivery mechanism for therapeutic molecules. This review surveys the literature relevant to the potential of mesenchymal stem cells for delivery of therapeutic agents in CNS trauma in humans.

T cell development is a complex and tightly regulated process involving reciprocal interactions between the thymic stroma and differentiating thymocytes. Normal thymic function is critical for immunity and microenvironmental defects predispose to dysregulation in the T cell compartment. Thymic structure and function are also severely damaged by chemotherapy and pre-transplant conditioning. Furthermore, poor immune competence with ageing is closely linked to thymic atrophy. Overcoming such thymic defects would have immediate application in many diseases, especially the recovery of cancer patients from cytotoxic treatment. Reversing the thymus ageing process via inhibition of atrophic factors such as sex steroids or administration of thymopoietic growth factors is one possible approach. Moreover, it is becoming clear a common thymic epithelial progenitor exists, raising the possibility for de novo thymus generation using emerging stem cell and tissue engineering technologies. Achievement of this goal will open up many avenues for the application of thymus-based immune rejuvenation and manipulation.

We have previously established a chamber model of tissue engineering that promotes de novo angiogenesis and vascularization of engrafted cells and tissues when combined with an extracellular matrix. Here we demonstrate that the mouse chamber (MC) model can sustain ectopic grafts of murine fetal thymus lobes and, to a limited degree, human pediatric thymus tissue, resulting in de novo T-cell production. Silicone chambers containing Matrigel((R)) and thymus tissues were placed around exposed epigastric vessels and the ends sealed with bone wax, before implantation into the inguinal fat pad of athymic Balb/c(nu/nu) (nude) mice. Murine, embryonic day 15 (E15) thymus grafts were found to be well vascularized and viable within the MC upon harvest at week 11. In contrast, engraftment of both adult murine and pediatric human thymus tissue was limited, with only one out of the seven human thymus grafts sustaining mature, murine-derived T-cell development. Increased CD4(+) and CD8(+) T-cell numbers were observed in the peripheral blood of nude mice within 2 weeks after E15 thymus-MC grafts (n = 8), compared with nude control mice. Peripheral blood T-cell percentage and subset distribution were comparable to mice receiving conventional thymus kidney capsule grafts. T-cell function of both kidney capsule- and MC-E15 thymus grafts was established via successful rejection of major histocompatibility complex (MHC)-mismatched skin grafts. Sustained growth of fetal thymus tissue in the MC provides an alternative model for the study of thymopoiesis and related applications of T-cell-mediated immunity.

Age-related thymic involution causes a decreased output of thymocytes from the thymus, thereby resulting in impairment of T cell-mediated immunity. While alterations in the T cell and non-haematopoietic stromal compartments have been described, the effects of thymic involution on thymic dendritic cells (DC) are not clearly known. Thymic DC play an essential role in shaping T cell-mediated immune responses by deleting self-reactive thymocytes to establish central tolerance and by inducing regulatory T-cell (Treg) development. It is therefore important to assess the prevalence of and alterations to thymic DC with age, as this may impact on their function. We assessed the numbers and proportions of the three distinct subsets of thymic DC in ageing mice, and showed that these subsets are differentially regulated. This is expected as thymic DC subsets have different origins of development. We further assessed the responses of thymic DC in a regenerative environment, such as that induced by sex-steroid ablation (SSA), and clearly showed that, consistent with global thymus regrowth, all three DC populations increased in numbers and regained their relative proportions to thymocytes after an initial lag period. These findings are important for the clinical translation of thymic regenerative approaches, and indicate that SSA facilitates the maintenance of critical processes such as negative selection and Treg induction through promoting thymic DC regeneration.

Cytotoxic antineoplastic therapy is widely used in the clinic as a treatment for malignant diseases. The treatment itself, however, leads to long-term depletion of the adaptive immune system, which is more pronounced in older patients, predominantly due to thymic atrophy. We and others have previously shown that withdrawal of sex steroids is able to regenerate the aged thymus and enhance recovery from autologous and allogeneic hematopoietic stem cell transplant. In this study we have examined the effects of sex steroid ablation (SSA) on the recovery of lymphopoiesis in the bone marrow (BM) and thymus following treatment with the chemotherapeutic agent cyclophosphamide (Cy) in middle-aged and old mice. Furthermore, we have also examined the impact of this regeneration on peripheral immunity. SSA enhanced the recovery of BM resident hematopoietic stem cells and lymphoid progenitors and promoted lymphopoiesis. Interestingly, Cy alone caused a profound increase in the recently described common lymphoid progenitor 2 (CLP-2) population in the BM. In the thymus, SSA caused a profound increase in cellularity as well as all intrathymic T-lineage progenitors including early T-lineage progenitors (ETPs) and non-canonical T cell progenitors such as the CLP-2. We also found that these transferred into numerical increases in the periphery with enhanced B and T cell numbers. Furthermore, these lymphocytes were found to have an enhanced functional capacity with no perturbation of the TCR repertoire. Taken together, these results provide the basis for the use of SSA in the clinic to enhance treatment outcomes from cytotoxic antineoplastic therapy.

The ability of stem cells to differentiate into various different cell types holds great promise for the treatment of irreversible tissue damage that occurs in many debilitating conditions. With stem cell research advancing at a tremendous pace, it is becoming clear that one of the greatest hurdles to successful stem cell-derived therapies is overcoming immune rejection of the transplant. Although the use of immunosuppressive drugs can decrease the incidence of acute graft rejection, the burden of problems associated with prolonged immunosuppression must be reduced. Strategies inducing specific immunological tolerance complemented by enhanced immune function will bring stem cell therapies closer to reality.

Chronic thymus involution associated with aging results in less efficient T-cell development and decreased emigration of naïve T cells to the periphery. Thymic decline in the aged is linked to increased morbidity and mortality in a wide range of clinical settings. Negative consequences of these effects on global health make it of paramount importance to understand the mechanisms driving thymic involution and homeostatic processes across the lifespan. There is growing evidence that thymus tissue is plastic and that the involution process might be therapeutically halted or reversed. We present here progress on the exploitation of thymosuppressive and thymostimulatory pathways using factors such as keratinocyte growth factor, interleukin 7 or sex steroid ablation for therapeutic thymus restoration and peripheral immune reconstitution in adults.

A significant decline in immune function is characteristic of aging. Along with the involution of the thymus and associated impaired architecture, which contributes to profound loss of naive T cell production, there are also significant declines in B cell development and the progenitors that support lymphopoiesis. These collectively lead to a reduced peripheral immune repertoire, increase in opportunistic infections, and limited recovery following cytoablation through chemo- or radiotherapy. We have previously shown that sex steroid ablation (SSA) causes a major reversal of age-related thymic atrophy and improves recovery from hematopoietic stem cell transplant. This study focused on the impact of SSA on the B cell compartment and their progenitors in middle-aged and cyclophosphamide-treated mice. In both models, SSA enhanced the number of lymphoid progenitors and developing B cells in the bone marrow (BM) as well as reversing age-related defects in the cycling kinetics of these cells. Enhanced BM lymphopoiesis was reflected in the periphery by an increase in recent BM emigrants as well as immature and mature plasma cells, leading to an enhanced humoral response to challenge by hepatitis B vaccine. In conclusion, SSA improves lymphoid progenitor and B cell recovery from age- and chemotherapy-induced immunodepletion, complimenting the effects on T cells. Since SSA has been achieved clinically for over 25 years, this provides a novel, rational basis for approaching the need for immune recovery in many clinical conditions.