Treg sensitivity to FasL and relative IL-2 deprivation drive idiopathic aplastic anemia immune dysfunction.

Idiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell-mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Tissue-Specific Decellularization Methods: Rationale and Strategies to Achieve Regenerative Compounds.

The extracellular matrix (ECM) is a complex network with multiple functions, including specific functions during tissue regeneration. Precisely, the properties of the ECM have been thoroughly used in tissue engineering and regenerative medicine research, aiming to restore the function of damaged or dysfunctional tissues. Tissue decellularization is gaining momentum as a technique to obtain potentially implantable decellularized extracellular matrix (dECM) with well-preserved key components. Interestingly, the tissue-specific dECM is becoming a feasible option to carry out regenerative medicine research, with multiple advantages compared to other approaches. This review provides an overview of the most common methods used to obtain the dECM and summarizes the strategies adopted to decellularize specific tissues, aiming to provide a helpful guide for future research development.

Role of Activator Protein-1 Complex on the Phenotype of Human Osteosarcomas Generated from Mesenchymal Stem Cells.

Osteosarcoma (OS) is a highly aggressive bone tumor that usually arises intramedullary at the extremities of long bones. Due to the fact that the peak of incidence is in the growth spurt of adolescence, the specific anatomical location, and the heterogeneity of cells, it is believed that osteosarcomagenesis is a process associated with bone development. Different studies in murine models showed that the tumor-initiating cell in OS could be an uncommitted mesenchymal stem cell (MSC) developing in a specific bone microenvironment. However, only a few studies have reported transgene-induced human MSCs transformation and mostly obtained undifferentiated sarcomas. In our study, we demonstrate that activator protein 1 family members induce osteosarcomagenesis in immortalized hMSC. c-JUN or c-JUN/c-FOS overexpression act as tumorigenic factors generating OS with fibroblastic or pleomorphic osteoblastic phenotypes, respectively. Stem Cells 2018;36:1487-1500.

Bone microenvironment signals in osteosarcoma development.

The bone is a complex connective tissue composed of many different cell types such as osteoblasts, osteoclasts, chondrocytes, mesenchymal stem/progenitor cells, hematopoietic cells and endothelial cells, among others. The interaction between them is finely balanced through the processes of bone formation and bone remodeling, which regulates the production and biological activity of many soluble factors and extracellular matrix components needed to maintain the bone homeostasis in terms of cell proliferation, differentiation and apoptosis. Osteosarcoma (OS) emerges in this complex environment as a result of poorly defined oncogenic events arising in osteogenic lineage precursors. Increasing evidence supports that similar to normal development, the bone microenvironment (BME) underlies OS initiation and progression. Here, we recapitulate the physiological processes that regulate bone homeostasis and review the current knowledge about how OS cells and BME communicate and interact, describing how these interactions affect OS cell growth, metastasis, cancer stem cell fate and therapy outcome.

Bone environment is essential for osteosarcoma development from transformed mesenchymal stem cells.

The cellular microenvironment plays a relevant role in cancer development. We have reported that mesenchymal stromal/stem cells (MSCs) deficient for p53 alone or together with RB (p53(-/-)RB(-/-)) originate leiomyosarcoma after subcutaneous (s.c.) inoculation. Here, we show that intrabone or periosteal inoculation of p53(-/-) or p53(-/-)RB(-/-) bone marrow- or adipose tissue-derived MSCs originated metastatic osteoblastic osteosarcoma (OS). To assess the contribution of bone environment factors to OS development, we analyzed the effect of the osteoinductive factor bone morphogenetic protein-2 (BMP-2) and calcified substrates on p53(-/-)RB(-/-) MSCs. We show that BMP-2 upregulates the expression of osteogenic markers in a WNT signaling-dependent manner. In addition, the s.c. coinfusion of p53(-/-)RB(-/-) MSCs together with BMP-2 resulted in appearance of tumoral osteoid areas. Likewise, when p53(-/-)RB(-/-) MSCs were inoculated embedded in a calcified ceramic scaffold composed of hydroxyapatite and tricalciumphosphate (HA/TCP), tumoral bone formation was observed in the surroundings of the HA/TCP scaffold. Moreover, the addition of BMP-2 to the ceramic/MSC implants further increased the tumoral osteoid matrix. Together, these data indicate that bone microenvironment signals are essential to drive OS development.

In-Hydrogel Cell-Free Protein Expression System as Biocompatible and Implantable Biomaterial.

Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

Development of bioactive solid-foam scaffolds from decellularized cartilage with chondrogenic and osteogenic properties.

Full osteochondral regeneration remains a major clinical challenge. Among other experimental cartilage regenerative approaches, decellularized cartilage (DCC) is considered a promising material for generating potentially implantable scaffolds useful as cartilage repair strategy. In this work, we focus on screening and comparing different decellularization methods, aiming to generate DCC potentially useful in biomedical context, and therefore, with biological activity and functional properties in terms of induction of differentiation and regeneration. Data indicates that enzymatic and detergents-based decellularization methods differentially affect ECM components, and that it has consequences in further biological behavior. SDS-treated DCC powder is not useful to be further processed in 2D or 3D structures, because these structures tend to rapidly solubilize, or disaggregate, in physiologic media conditions. Conversely, Trypsin-treated DCC powders can be processed to mechanically stable 2D films and 3D solid-foam scaffolds, presumably due to partial digestion of collagens during decellularization, which would ease crosslinking at DCC during solubilization and processing. In vitro cell culture studies indicate that these structures are biocompatible and induce and potentiate chondrogenic differentiation. In vivo implantation of DCC derived 3D porous scaffolds in rabbit osteochondral defects induce subchondral bone regeneration and fibrocartilage tissue formation after implantation. Therefore, this work defines an optimal cartilage tissue decellularization protocol able to generate DCC powders processable to biocompatible and bioactive 2D and 3D structures. These structures are useful for in vitro cartilage research and in vivo subchondral bone regeneration, while hyaline cartilage regeneration with DCC alone as implantable material remains elusive.

rhBMP-2 induces terminal differentiation of human bone marrow mesenchymal stromal cells only by synergizing with other signals.

BACKGROUND: Recombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro. METHODS: rhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes. RESULTS: The data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo. CONCLUSIONS: Altogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment.

Low molecular weight poly((D,L)-lactide-co-caprolactone) liquid inks for diluent-free DLP printing of cell culture platforms.

Digital light processing (DLP) printing offers the possibility of fabricating complex objects in a fast and reproducible manner. A main requirement for DLP printing is the use of inks with low viscosities that can flow under the printing platform in a short period of time. Its exploitation in tissue engineering applications has been centered on the use of hydrogel forming materials diluted in aqueous solutions or the use of polyesters in combination with diluents and heating platforms that aid in the reduction of their viscosity. The use of diluents, however, modifies the mechanical properties and reduces the shape fidelity of the printed objects and, the use of heating platforms results in vats with heterogeneous temperatures and ink viscosities. Here, we report on the synthesis of a library of methacrylated low molecular weight (<3000 g mol-1) homopolymers ((P(D,L)LA and PCL) and copolymers (P((D,L)LA-co-CL)) of 2- and 3-arms based on (D,L)-lactide and ε-caprolactone. The resulting inks possessed low viscosity that made them printable in the absence of diluents and heating elements. DLP printing of cubical and cylindrical patterns resulted in objects with a higher shape fidelity than their counterparts fabricated using diluents and with printed features on the order of 300 µm. The printed materials were biocompatible and supported the growth of human mesenchymal stem cells (hMSCs). Moreover, the variations in the composition resulted in polymers that enabled the attachment of hMSCs to different extents, leading to the formation of well-adhered cell monolayers or loosely adhered cell aggregates.

Polycaprolactone with multiscale porosity and patterned surface topography prepared using sacrificial 3D printed moulds: Towards tailor-made scaffolds.

Biocompatible three-dimensional porous scaffolds are widely used in multiple biomedical applications. However, the fabrication of tailor-made 3D structures with controlled and combined multiscale macroscopic-microscopic, surface and inner porosities in a straightforward manner is still a current challenge. Herein, we use multimaterial fused deposition modeling (FDM) to generate poly (vinyl alcohol) (PVA) sacrificial moulds filled with poly (Ɛ-caprolactone) (PCL) to generate well defined PCL 3D objects. Further on, the supercritical CO2 (SCCO2) technique, as well as the breath figures mechanism (BFs), were additionally employed to fabricate specific porous structures at the core and surfaces of the 3D PCL object, respectively. The biocompatibility of the resulting multiporous 3D structures was tested in vitro and in vivo, and the versatility of the approach was assessed by generating a vertebra model fully tunable at multiple pore size levels. In sum, the combinatorial strategy to generate porous scaffolds offers unique possibilities to fabricate intricate structures by combining the advantages of additive manufacturing (AM), which provides flexibility and versatility to generate large sized 3D structures, with advantages of the SCCO2 and BFs techniques, which allow to finely tune the macro and micro porosity at material surface and material core levels.

The Effect of Ca2+ and Mg2+ Ions Loaded at Degradable PLA Membranes on the Proliferation and Osteoinduction of MSCs.

Biodegradable membranes, including Polylactic acid (PLA)-based membranes, are commonly used in bone-tissue-related clinical procedures as biointerface to promote bone tissue regeneration. Calcium (Ca2+) and Magnesium (Mg2+) ions have been related to the promotion of osteogenesis, where the PLA membranes could be used as carrier and delivery substrate for them to provide osteogenic properties to this material. For this aim, a new ion delivery system based on biodegradable PLA membranes loaded with Mg and hydroxyapatite (HA) particles has been processed by the combination of tape casting and colloidal route. Materials characterization shows that the incorporation of Mg and HA particles changes the surface and hydrophobicity of the PLA membrane, and the in vitro degradation test shows Mg2+ and Ca2+ ion release and occasionally the precipitation of different ion species onto the membrane surface. Mouse and human Mesenchymal Stem Cells (MSC) were used to define the biocompatibility and bioactivity of these PLA membrane composites, and data indicated Mg2+ promotes cell proliferation and potentiates osteoinductive signals, while Ca2+ induces the expression of ALP osteogenic marker in human MSCs. Biodegradable PLA membranes loaded with Mg and HA particles is a promising new ion delivery system of Mg2+ and Ca2+ ions that provides osteogenic signals and works as functional biointerface interfaces with bone tissues.

Decellularization of xenografted tumors provides cell-specific in vitro 3D environment.

In vitro cell culture studies are common in the cancer research field, and reliable biomimetic 3D models are needed to ensure physiological relevance. In this manuscript, we hypothesized that decellularized xenograft tumors can serve as an optimal 3D substrate to generate a top-down approach for in vitro tumor modeling. Multiple tumor cell lines were xenografted and the formed solid tumors were recovered for their decellularization by several techniques and further characterization by histology and proteomics techniques. Selected decellularized tumor xenograft samples were seeded with the HCC1806 human triple-negative breast cancer (TNBC) basal-like subtype cell line, and cell behavior was compared among them and with other control 2D and 3D cell culture methods. A soft treatment using Freeze-EDTA-DNAse allows proper decellularization of xenografted tumor samples. Interestingly, proteomic data show that samples decellularized from TNBC basal-like subtype xenograft models had different extracellular matrix (ECM) compositions compared to the rest of the xenograft tumors tested. The in vitro recellularization of decellularized ECM (dECM) yields tumor-type-specific cell behavior in the TNBC context. Data show that dECM derived from xenograft tumors is a feasible substrate for reseeding purposes, thereby promoting tumor-type-specific cell behavior. These data serve as a proof-of-concept for further potential generation of patient-specific in vitro research models.

The transcription factor DDIT3 is a potential driver of dyserythropoiesis in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 (DDIT3). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients.

Ectopic humanized mesenchymal niche in mice enables robust engraftment of myelodysplastic stem cells.

Myelodysplastic syndrome (MDS) are clonal stem cell diseases characterized mainly by ineffective hematopoiesis. Here, we present an approach that enables robust long-term engraftment of primary MDS stem cells (MDS-SCs) in mice by implantation of human mesenchymal cell-seeded scaffolds. Critically for modelling MDS, where patient sample material is limiting, mononuclear bone marrow cells containing as few as 104 CD34+ cells can be engrafted and expanded by this approach with the maintenance of the genetic make-up seen in the patients. Non-invasive high-resolution ultrasound imaging shows that these scaffolds are fully perfused. Our data shows that human microenvironment but not mouse is essential to MDS-SCs homing and engraftment. Notably, the alternative niche provided by healthy donor MSCs enhanced engraftment of MDS-SCs. This study characterizes a new tool to model MDS human disease with the level of engraftment previously unattainable in mice, and offers insights into human-specific determinants of MDS-SC microenvironment.

Integrated OMICs unveil the bone-marrow microenvironment in human leukemia.

The bone-marrow (BM) niche is the spatial environment composed by a network of multiple stromal components regulating adult hematopoiesis. We use multi-omics and computational tools to analyze multiple BM environmental compartments and decipher their mutual interactions in the context of acute myeloid leukemia (AML) xenografts. Under homeostatic conditions, we find a considerable overlap between niche populations identified using current markers. Our analysis defines eight functional clusters of genes informing on the cellular identity and function of the different subpopulations and pointing at specific stromal interrelationships. We describe how these transcriptomic profiles change during human AML development and, by using a proximity-based molecular approach, we identify early disease onset deregulated genes in the mesenchymal compartment. Finally, we analyze the BM proteomic secretome in the presence of AML and integrate it with the transcriptome to predict signaling nodes involved in niche alteration in AML.

In vivo comparison of the effects of rhBMP-2 and rhBMP-4 in osteochondral tissue regeneration.

The aim of this work is to investigate the use of bone morphogenetic proteins (rhBMP-2, rhBMP-4) alone or in combination with cells delivered in a calcium alginate gel for the treatment of osteochondral defects. For this purpose, alginate gels were prepared mixing a 2% sodium alginate solution and a 200 mM calcium chloride solution (1:1). Osteochondral defects were created (4 mm wide, 5 mm deep) in the internal femoral condyle of rabbit knee and gels were directly formed into the defects. 3 months after surgery samples were harvested, gross morphology was documented and histological appearance was evaluated. The performed histological observations revealed subchondral bone regeneration in rhBMP-2 samples and moderate hyaline cartilage regeneration in rhBMP-4 samples. Thus, results indicate that alginate gel may serve as an appropriate delivery vehicle for rhBMP-2, rhBMP-4 and stromal cells. With this carrier material, differential behaviour between the evaluated proteins was observed. rhBMP-2 shows better restoration of subchondral bone in contrast to the superior efficiency of rhBMP-4 for hyaline cartilage repair.

Mesenchymal niche remodeling impairs hematopoiesis via stanniocalcin 1 in acute myeloid leukemia.

Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.

Correction: Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment.

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches.

A Humanized Bone Niche Model Reveals Bone Tissue Preservation Upon Targeting Mitochondrial Complex I in Pseudo-Orthotopic Osteosarcoma.

A cogent issue in cancer research is how to account for the effects of tumor microenvironment (TME) on the response to therapy, warranting the need to adopt adequate in vitro and in vivo models. This is particularly relevant in the development of strategies targeting cancer metabolism, as they will inevitably have systemic effects. For example, inhibition of mitochondrial complex I (CI), despite showing promising results as an anticancer approach, triggers TME-mediated survival mechanisms in subcutaneous osteosarcoma xenografts, a response that may vary according to whether the tumors are induced via subcutaneous injection or by intrabone orthotopic transplantation. Thus, with the aim to characterize the TME of CI-deficient tumors in a model that more faithfully represents osteosarcoma development, we set up a humanized bone niche ectopic graft. A prominent involvement of TME was revealed in CI-deficient tumors, characterized by the abundance of cancer associated fibroblasts, tumor associated macrophages and preservation of osteocytes and osteoblasts in the mineralized bone matrix. The pseudo-orthotopic approach allowed investigation of osteosarcoma progression in a bone-like microenvironment setting, without being invasive as the intrabone cell transplantation. Additionally, establishing osteosarcomas in a humanized bone niche model identified a peculiar association between targeting CI and bone tissue preservation.