Clinical GATA2 haploinsufficiency results in immunodeficiency that evolves to leukemia. How GATA2 haploinsufficiency disrupts the functionality of hematopoietic stem/progenitor cells (HSCs/HSPCs) to facilitate pre-leukemia development is poorly defined. Using a hematopoietic-specific conditional mouse model of Gata2 haploinsufficiency, we identified pervasive defects in HSPC differentiation in young adult Gata2 haploinsufficient mice and perturbed HSC self-renewal following transplantation. These alterations aligned with deregulated global DNA damage responses and inflammatory cell signaling from Gata2 haploinsufficient HSCs. We also discovered genetic interplay between Gata2 and Asxl1, a secondary mutation leading to leukemia in GATA2 deficiency syndromes. HSCs from young adult compound Gata2/Asxl1 haploinsufficient mice were hyperproliferative, functionally compromised after transplantation, and displayed a broad pre-leukemia transcriptomic program. Thus, Gata2 haploinsufficiency triggers HSC genomic instability. Our data further suggest that secondary mutations like ASXL1 exploit this impaired HSC genomic integrity to nurture a pre-leukemic state in GATA2 haploinsufficiency syndromes.

PurposeThe study conducts a comparative analysis between two prominent methods for fabricating composites for bone scaffolds—the (solid) solvent method and the solvent-free (melting) method. While previous research has explored these methods individually, this study provides a direct comparison of their outcomes in terms of physicochemical properties, cytocompatibility, and mechanical strength. We also analyse their workflow and scalability potentials.Design/methodology/approachPolycaprolactone (PCL) and hydroxyapatite (HA) composites were prepared using solvent (chloroform) and melting (180°C) methods, then 3D-printed using an extrusion-based 3D printer to fabricate scaffolds (8 × 8 × 4 mm). Rheology, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), accelerated degradation, mechanical/compression test, wettability/contact angle, live/dead assay, and DNA quantification (Picogreen) assays were evaluated.FindingsThe study finds that scaffolds made via the solid solvent method have higher mechanical strength and degradation rate as compared to those from the melting method, while both methods ensure adequate cytocompatibility and homogenous hydroxyapatite distribution, supporting their use in bone tissue engineering.OriginalityThis research investigates the utility of chloroform as a solvent for PCL composite in a direct comparison with the melting method. It also highlights the differences in workflows between the two methods and their scalability implications, emphasizing the importance of considering workflow efficiency and the potential for automation in scaffold fabrication processes for bone tissue engineering applications.

Advancing bioprinted vascular grafts for clinical applications faces the challenge of obtaining sufficient functional endothelial cells and smooth muscle cells essential for the biofabrication of blood vessels. Accurate placement of these cells is crucial for optimal performance. Tissue engineering, particularly with adipose-derived stem cells (ADSCs), offers promising solutions.In this approach, ADSCs were cultivated and differentiated into endothelial cells (dECs) in vitro using VEGF-165 PODS® (Polyhedrin Delivery System), while smooth muscle cells (dSMCs) were differentiated in situ within the outer layer of the 3D bioprinted vessel using TGF-β1 PODS® with bioink VascuBiomatrixTM. The effect of PODS® on production of differentiated Endothelial Cells (dECs) and Smooth Muscle Cells (dSMCs) was validated through flow cytometry, immunocytochemical staining, and RT-PCR, using cell specific markers, as well as immunolabeling for extracellular collagen I and elastin. This confirmed that cells within the vessel walls retained their phenotype and secreted human extracellular matrix (ECM) components.Scanning electron microscopy (SEM) confirmed the vessels’ morphology and dimensions, and tensile testing and burst pressure tests assessed mechanical properties. In vivo compatibility was evaluated by blood hemocompatibility and the CAM (Ex Ovo chorioallantoic membrane) assay.Results confirmed the successful fabrication of a bilayer blood vessel structure with smooth muscle cells and an endothelial lining, exhibiting adequate physiological properties. Hemocompatibility and in vivo CAM assays demonstrated low platelet adhesion, improved biocompatibility, and angiogenic properties. These findings suggest that integrating ADSCs and bioink for 3D bioprinting provides a practical solution for fabricating functional small-diameter vascular grafts. This study advances vascular tissue engineering through the combination of stem cells, growth factor delivery systems, and bioprinting technology.

Zeb1, a zinc finger E-box binding homeobox epithelial–mesenchymal (EMT) transcription factor, acts as a critical regulator of hematopoietic stem cell (HSC) self-renewal and multi-lineage differentiation. Whether Zeb1 directly regulates the function of multi-potent progenitors primed for hematopoietic lineage commitment remains ill defined. By using an inducible Mx-1 Cre conditional mouse model where Zeb1 was genetically engineered to be deficient in the adult hematopoietic system (hereafter Zeb1−/−), we found that the absolute cell number of immunophenotypically defined lympho-myeloid primed progenitors (LMPPs) from Zeb1−/− mice was reduced. Myeloid- and lymphoid-biased HSCs in Zeb1−/− mice were unchanged, implying that defective LMPP generation from Zeb1−/− mice was not directly caused by an imbalance of lineage-biased HSCs. Functional analysis of LMPP from Zeb1−/− mice, as judged by competitive transplantation, revealed an overall reduction in engraftment to hematopoietic organs over 4 weeks, which correlated with minimal T-cell engraftment, reduced B-cell and monocyte/macrophage engraftment, and unperturbed granulocyte engraftment. Thus, Zeb1 regulates LMPP differentiation potential to select lympho-myeloid lineages in the context of transplantation.

Chronic Kidney Disease (CKD) is a major cause of morbidity and mortality characterized by progressive renal fibrosis, and in extreme cases, renal failure. Human CKD models that replicate the biological complexity of the kidney and CKD are lacking and will be invaluable in identifying drugs to revert and/or prevent fibrosis. To address this unmet need, we developed 3D renal organoids where human induced pluripotent stem cells (hiPSCs) were differentiated to renal progenitors within a renal extracellular matrix (rECM) gel, based on the premise that an rECM could recreate the renal niche to facilitate hiPSC-derived renal progenitor generation. We used mouse kidneys as a source of rECM and identified that superior detergent-mediated decellularization of mouse kidneys was achieved with a combination of 0.5% w/v Sodium Dodecyl Sulphate and 1% v/v Triton-X and mechanical agitation for 60 h. HiPSCs that underwent specification to become metanephric mesenchyme (MM) were subsequently cultured within the rECM gel and, notably, mesenchymal to epithelial transition (MET) was observed, as judged by expression of nephron markers K-cadherin, Nephrin and WT1. These data demonstrate a role for rECM gel in developing human renal organoids from hiPSCs, which will aid the further development of a human disease model for renal fibrosis.

Objective.Repair of nerve gap injuries can be achieved through nerve autografting, but this approach is restricted by limited tissue supply and donor site morbidity. The use of living nerve allografts would provide an abundant tissue source, improving outcomes following peripheral nerve injury. Currently this approach is not used due to the requirement for systemic immunosuppression, to prevent donor-derived cells within the transplanted nerve causing an immune response, which is associated with severe adverse effects. The aim of this study was to develop a method for delivering immunosuppression locally, then to test its effectiveness in reducing the immune response to transplanted tissue in a rat model of nerve allograft repair.Approach.A coaxial electrospinning approach was used to produce poly-ϵ-caprolactone fibre sheets loaded with the immunosuppressant tacrolimus. The material was characterised in terms of structure and tacrolimus release, then testedin vivothrough implantation in a rat sciatic nerve allograft model with immunologically mismatched host and donor tissue.Main results.Following successful drug encapsulation, the fibre sheets showed nanofibrous structure and controlled release of tacrolimus over several weeks. Materials containing tacrolimus (and blank material controls) were implanted around the nerve graft at the time of allograft or autograft repair. The fibre sheets were well tolerated by the animals and tacrolimus release resulted in a significant reduction in lymphocyte infiltration at 3 weeks post-transplantation.Significance.These findings demonstrate proof of concept for a novel nanofibrous biomaterial-based targeted drug delivery strategy for immunosuppression in peripheral nerve allografting.

Treatment options for neurodegenerative conditions such as Parkinson's disease have included the delivery of cells which release dopamine or neurotrophic factors to the brain. Here, we report the development of a novel approach for protecting cells after implantation into the central nervous system (CNS), by developing dual-layer alginate beads that encapsulate therapeutic cells and release an immunomodulatory compound in a sustained manner. An optimal alginate formulation was selected with a view to providing a sustained physical barrier between engrafted cells and host tissue, enabling exchange of small molecules while blocking components of the host immune response. In addition, a potent immunosuppressant, FK506, was incorporated into the outer layer of alginate beads using electrosprayed poly-ε-caprolactone core-shell nanoparticles with prolonged release profiles. The stiffness, porosity, stability and ability of the alginate beads to support and protect encapsulated SH-SY5Y cells was demonstrated, and the release profile of FK506 and its effect on T-cell proliferation in vitro was characterized. Collectively, our results indicate this multi-layer encapsulation technology has the potential to be suitable for use in CNS cell delivery, to protect implanted cells from host immune responses whilst providing permeability to nutrients and released therapeutic molecules.

Background: The effect of demineralized bone matrix (DBM), bone marrow–derived mesenchymal stromal cells (BMSCs), and platelet-rich plasma (PRP) on bone tunnel healing in anterior cruciate ligament reconstruction (ACLR) has not been comparatively assessed. Hypothesis: These orthobiologics would reduce tunnel widening, and the effects on tunnel diameter would be correlated with tunnel wall sclerosis. Study Design: Controlled laboratory study. Methods: A total of 20 sheep underwent unilateral ACLR using tendon allograft and outside-in interference screw fixation. The animals were randomized into 4 groups (n = 5 per group): Group 1 received 4mL of DBM paste, group 2 received 10 million BMSCs in fibrin sealant, group 3 received 12 mL of activated leukocyte-poor platelet-rich plasma, and group 4 (control) received no treatment. The sheep were euthanized after 12 weeks, and micro-computed tomography scans were performed. The femoral and tibial tunnels were divided into thirds (aperture, midportion, and exit), and the trabecular bone structure, bone mineral density (BMD), and tunnel diameter were measured. Tunnel sclerosis was defined by a higher bone volume in a 250-µm volume of interest compared with a 4-mm volume of interest surrounding the tunnel. Results: Compared with the controls, the DBM group had a significantly higher bone volume fraction (bone volume/total volume [BV/TV]) (52.7% vs 31.8%; P = .020) and BMD (0.55 vs 0.47 g/cm3; P = .008) at the femoral aperture and significantly higher BV/TV at femoral midportion (44.2% vs 32.9%; P = .038). There were no significant differences between the PRP and BMSC groups versus controls in terms of trabecular bone analysis or BMD. In the controls, widening at the femoral tunnel aperture was significantly greater than at the midportion (46.7 vs 41.7 mm2; P = .034). Sclerosis of the tunnel was common and most often seen at the femoral aperture. In the midportion of the femoral tunnel, BV/TV ( r = 0.52; P = .019) and trabecular number ( r S = 0.50; P = .024) were positively correlated with tunnel widening. Conclusion: Only DBM led to a significant increase in bone volume, which was seen in the femoral tunnel aperture and midportion. No treatment significantly reduced bone tunnel widening. Tunnel sclerosis in the femoral tunnel midportion was correlated significantly with tunnel widening. Clinical Relevance: DBM might have potential clinical use to enhance healing in the femoral tunnel after ACLR.

Zeb1, a zinc finger E-box binding homeobox epithelial-mesenchymal transition (EMT) transcription factor, confers properties of “stemness,” such as self-renewal, in cancer. Yet little is known about the function of Zeb1 in adult stem cells. Here, we used the hematopoietic system as a well-established paradigm of stem cell biology to evaluate Zeb1-mediated regulation of adult stem cells. We employed a conditional genetic approach using the Mx1-Cre system to specifically knock out (KO) Zeb1 in adult hematopoietic stem cells (HSCs) and their downstream progeny. Acute genetic deletion of Zeb1 led to rapid-onset thymic atrophy and apoptosis-driven loss of thymocytes and T cells. A profound cell-autonomous self-renewal defect and multilineage differentiation block were observed in Zeb1-KO HSCs. Loss of Zeb1 in HSCs activated transcriptional programs of deregulated HSC maintenance and multilineage differentiation genes and of cell polarity consisting of cytoskeleton-, lipid metabolism/lipid membrane–, and cell adhesion–related genes. Notably, epithelial cell adhesion molecule (EpCAM) expression was prodigiously upregulated in Zeb1-KO HSCs, which correlated with enhanced cell survival, diminished mitochondrial metabolism, ribosome biogenesis, and differentiation capacity and an activated transcriptomic signature associated with acute myeloid leukemia (AML) signaling. ZEB1 expression was downregulated in AML patients, and Zeb1 KO in the malignant counterparts of HSCs — leukemic stem cells (LSCs) — accelerated MLL-AF9– and Meis1a/Hoxa9-driven AML progression, implicating Zeb1 as a tumor suppressor in AML LSCs. Thus, Zeb1 acts as a transcriptional regulator in hematopoiesis, critically coordinating HSC self-renewal, apoptotic, and multilineage differentiation fates required to suppress leukemic potential in AML.

The effect of bone marrow mesenchymal stromal cells (BMSCs) and platelet-rich plasma (PRP) on tendon allograft maturation in a large animal anterior cruciate ligament (ACL) reconstruction model was reported for the first time. It was hypothesised that compared with non-augmented ACL reconstruction, BMSCs and PRP would enhance graft maturation after 12 weeks and this would be detected using magnetic resonance imaging (MRI). Fifteen sheep underwent unilateral tendon allograft ACL reconstruction using aperture fixation and were randomised into three groups (n = 5). Group 1 received 10 million allogeneic BMSCs in 2 ml fibrin sealant; Group 2 received 12 ml PRP in a plasma clot injected into the graft and bone tunnels; and Group 3 (control) received no adjunctive treatment. At autopsy at 12 weeks, a graft maturation score was determined by the sum for graft integrity, synovial coverage and vascularisation, graft thickness and apparent tension, and synovial sealing at tunnel apertures. MRI analysis (n = 2 animals per group) of the signal–noise quotient (SNQ) and fibrous interzone (FIZ) was used to evaluate intra-articular graft maturation and tendon–bone healing, respectively. Spearman’s rank correlation coefficient (r) of SNQ, autopsy graft maturation score and bone tunnel diameter were analysed. The BMSC group (p = 0.01) and PRP group (p = 0.03) had a significantly higher graft maturation score compared with the control group. The BMSC group scored significantly higher for synovial sealing at tunnel apertures (p = 0.03) compared with the control group. The graft maturation score at autopsy significantly correlated with the SNQ (r = − 0.83, p < 0.01). The tunnel diameter of the femoral tunnel at the aperture (r = 0.883, p = 0.03) and mid-portion (r = 0.941, p = 0.02) positively correlated with the SNQ. BMSCs and PRP significantly enhanced graft maturation, which indicates that orthobiologics can accelerate the biologic events in tendon allograft incorporation. Femoral tunnel expansion significantly correlated with inferior maturation of the intra-articular graft. The clinical relevance of this study is that BMSCs and PRP enhance allograft healing in a translational model, and biological modulation of graft healing can be evaluated non-invasively using MRI.

Immunological tolerance refers to the ability of not reacting by immunological defense reactions when they are not needed. It does not mean a mere lack of response but rather continuous active controlling and modifying of initiated responses towards tolerance. While tolerance is primarily a property of the adaptive immune system, it is also influenced by the innate immune system. The importance of tolerance is emphasized in the intestinal immune system, where foreign structures are present in abundance. In fact, orally administered antigens may in the future hopefully be utilized for the treatment of diseases caused by harmful immune responses.

Subversion of transcription factor (TF) activity in hematopoietic stem/progenitor cells (HSPCs) leads to the development of therapy-resistant leukemic stem cells (LSCs) that drive fulminant acute myeloid leukemia (AML). Using a conditional mouse model where zinc-finger TF Gata2 was deleted specifically in hematopoietic cells, we show that knockout of Gata2 leads to rapid and complete cell-autonomous loss of adult hematopoietic stem cells. By using short hairpin RNAi to target GATA2, we also identify a requirement for GATA2 in human HSPCs. In Meis1a/Hoxa9-driven AML, deletion of Gata2 impedes maintenance and self-renewal of LSCs. Ablation of Gata2 enforces an LSC-specific program of enhanced apoptosis, exemplified by attenuation of anti-apoptotic factor BCL2, and re-instigation of myeloid differentiation-which is characteristically blocked in AML. Thus, GATA2 acts as a critical regulator of normal and leukemic stem cells and mediates transcriptional networks that may be exploited therapeutically to target key facets of LSC behavior in AML.

Recently, adipose-derived stem cells (ASCs), obtained from fresh human lipoaspirate, have shown promise as immunomodulatory agents having demonstrated immunosuppressive functionality both in vitro and in vivo. A number of researchers have described the isolation of ASCs through the enzymatic digestion of fat samples, followed by a number of purification steps, involving centrifugation and filtration. Here, we utilize a standard isolation technique, with the added purification of putative ASCs by fluorescence activated cell sorting (FACS). ASCs are an extremely valuable resource in clinical applications, including reconstruction, regeneration, and investigations into immune activity. This method could be used to isolate and purify ASCs for such downstream applications.

To make personalised medicine a reality, there is a significant need for cost-effective methods that enable the rapid selection of optimal nutrient intake and/or disease treatment with a minimum of side effects. In this perspective, we briefly discuss the potential of merging the advances of microfluidic devices, organoid structures and advanced analytical methods to progress towards a personalised in vitro platform for health and wellbeing. (c) 2019 Society of Chemical Industry

GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality. Loss and gain of GATA2 expression has been implicated in myelodysplastic syndrome and acute myeloid leukemia (AML) yet the precise biological impact of GATA2 expression on human AML cell fate decisions remains ambiguous. Herein, we performed large-scale bioinformatics that demonstrated relatively frequent GATA2 overexpression in AML patients as well as select human AML (or AML-like) cell lines. By using shRNAi to target GATA2 in these AML cell lines, and an AML cell line expressing normal levels of GATA2, we found that inhibition of GATA2 caused attenuated cell proliferation and enhanced apoptosis exclusively in AML cell lines that overexpress GATA2. We proceeded to pharmacologically inhibit GATA2 in concert with AML chemotherapeutics and found this augmented cell killing in AML cell lines that overexpress GATA2, but not in an AML cell line expressing normal levels of GATA2. These data indicate that inhibition of GATA2 enhances chemotherapy-mediated apoptosis in human AML cells overexpressing GATA2. Thus, we define novel insights into the oncogenic role of GATA2 in human AML cells and suggest the potential utilization of transient GATA2 therapeutic targeting in AML.

Immune surveillance is an established regulatory mechanism that spares tissues from malignant transformation. Agudo et al. (2018) find that the chief cell type to generate tissues in the body-somatic stem cells-is subject to immune surveillance only during proliferation.

The prospects for using autologous induced pluripotent stem cells (iPSCs) in cell replacement therapy have been tempered by evidence that undifferentiated, syngeneic mouse iPSCs are immunogenic upon transplantation. However, the immunogenicity of more therapeutically relevant differentiated cells remains unexplored. Here, we differentiated mouse iPSCs into embryoid bodies (EBs) or representative cell types spanning the three embryonic germ layers and assessed their immunogenicity in vitro and after their transplantation into syngeneic recipients. We found no evidence of increased T cell proliferation in vitro, rejection of syngeneic iPSC-derived EBs/tissue-specific cells (TSCs) after transplantation, or an antigen-specific secondary immune response. Thus, differentiated cells derived from syngeneic iPSCs do not appear to be rejected after transplantation. We also found little evidence of an immune response to undifferentiated, syngeneic iPSCs. Our data support the idea that differentiated cells generated from autologous iPSCs could be applied for cell replacement therapy without eliciting immune rejection.

The adaptive immune system protects organisms from harmful environmental insults. In parallel, regulatory mechanisms control immune responses in order to assure preservation of organ integrity. Yet, molecules involved in the control of T-cell responses in peripheral tissues are poorly characterized. Here, we investigated the function of Dickkopf-3 in the modulation of local T-cell reactivity. Dkk3 is a secreted, mainly tissue-derived protein with highest expression in organs considered as immune-privileged such as the eye, embryo, placenta, and brain. While T-cell development and activation status in naïve Dkk3-deficient mice was comparable to littermate controls, we found that Dkk3 contributes to the immunosuppressive microenvironment that protects transplanted, class-I mismatched embryoid bodies from T-cell-mediated rejection. Moreover, genetic deletion or antibody-mediated neutralization of Dkk3 led to an exacerbated experimental autoimmune encephalomyelitis (EAE). This phenotype was accompanied by a change of T-cell polarization displayed by an increase of IFNγ-producing T cells within the central nervous system. In the wild-type situation, Dkk3 expression in the brain was up-regulated during the course of EAE in an IFNγ-dependent manner. In turn, Dkk3 decreased IFNγ activity and served as part of a negative feedback mechanism. Thus, our findings suggest that Dkk3 functions as a tissue-derived modulator of local CD4+ and CD8+ T-cell responses.