RESUMEN
Orthopedic implant-related bacterial infections and resultant antibiotic-resistant biofilms hinder implant-tissue integration and failure. Biofilm quorum sensing (QS) communication determines the pathogen colonization success. However, it remains unclear how implant modifications and host cells are influenced by, or influence, QS. High aspect ratio nanotopographies have shown to reduce biofilm formation of Pseudomonas aeruginosa, a sepsis causing pathogen with well-defined QS molecules. Producing such nanotopographies in relevant orthopedic materials (i.e., titanium) allows for probing QS using mass spectrometry-based metabolomics. However, nanotopographies can reduce host cell adhesion and regeneration. Therefore, we developed a polymer (poly(ethyl acrylate), PEA) coating that organizes extracellular matrix proteins, promoting bioactivity to host cells such as human mesenchymal stromal cells (hMSCs), maintaining biofilm reduction. This allowed us to investigate how hMSCs, after winning the race for the surface against pathogenic cells, interact with the biofilm. Our approach revealed that nanotopographies reduced major virulence pathways, such as LasR. The enhanced hMSCs support provided by the coated nanotopographies was shown to suppress virulence pathways and biofilm formation. Finally, we selected bioactive metabolites and demonstrated that these could be used as adjuncts to the nanostructured surfaces to reduce biofilm formation and enhance hMSC activity. These surfaces make excellent models to study hMSC-pathogen interactions and could be envisaged for use in novel orthopedic implants.
RESUMEN
The blood-brain barrier (BBB) tightly regulates substance transport between the bloodstream and the brain. Models for the study of the physiological processes affecting the BBB, as well as predicting the permeability of therapeutic substances for neurological and neurovascular pathologies, are highly desirable. Existing models, such as Transwell utilizing-models, do not mimic the extracellular environment of the BBB with their stiff, semipermeable, non-biodegradable membranes. To help overcome this, we engineered electrospun membranes from poly L-lactic acid in combination with a nanometric coating of poly(ethyl acrylate) (PEA) that drives fibrillogenesis of fibronectin, facilitating the synergistic presentation of both growth factors and integrin binding sites. Compared to commercial semi-porous membranes, these membranes significantly improve the expression of BBB-related proteins in brain endothelial cells. PEA-coated membranes in combination with different growth factors and extracellular protein coatings reveal nerve growth factor (NGF) and fibroblast growth factor (FGF-2) caused formation of better barriers in vitro. This BBB model offers a robust platform for studying key biochemical factors influencing barrier formation that marries the simplicity of the Transwell model with the highly tunable electrospun PEA-fibronectin membranes. This enables the generation of high-throughput drug permeability models without the need of complicated co-culture conditions.
RESUMEN
Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.
Asunto(s)
Matriz Extracelular , Células Madre Hematopoyéticas , Subunidad alfa del Factor 1 Inducible por Hipoxia , Nestina , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Animales , Nestina/metabolismo , Nestina/genética , Matriz Extracelular/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Nicho de Células Madre , Hidrogeles/química , Bioingeniería/métodos , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Trasplante de Células Madre Hematopoyéticas , Antígenos CD34/metabolismo , Colágeno Tipo I/metabolismo , Diferenciación Celular , Ratones Endogámicos C57BLRESUMEN
Protein materials are versatile tools in diverse biomedical fields. Among them, artificial secretory granules (SGs), mimicking those from the endocrine system, act as mechanically stable reservoirs for the sustained release of proteins as oligomeric functional nanoparticles. Only validated in oncology, the physicochemical properties of SGs, along with their combined drug-releasing and scaffolding abilities, make them suitable as smart topographies in regenerative medicine for the prolonged delivery of growth factors (GFs). Thus, considering the need for novel, safe, and cost-effective materials to present GFs, in this study, we aimed to biofabricate a protein platform combining both endocrine-like and extracellular matrix fibronectin-derived (ECM-FN) systems. This approach is based on the sustained delivery of a nanostructured histidine-tagged version of human fibroblast growth factor 2. The GF is presented onto polymeric surfaces, interacting with FN to spontaneously generate nanonetworks that absorb and present the GF in the solid state, to modulate mesenchymal stromal cell (MSC) behavior. The results show that SGs-based topographies trigger high rates of MSCs proliferation while preventing differentiation. While this could be useful in cell therapy manufacture demanding large numbers of unspecialized MSCs, it fully validates the hybrid platform as a convenient setup for the design of biologically active hybrid surfaces and in tissue engineering for the controlled manipulation of mammalian cell growth.
Asunto(s)
Matriz Extracelular , Fibronectinas , Células Madre Mesenquimatosas , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibronectinas/química , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proliferación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Nanoestructuras/químicaRESUMEN
Conventional osteogenic platforms utilize active growth factors to repair bone defects that are extensive in size, but they can adversely affect patient health. Here, an unconventional osteogenic platform is reported that functions by promoting capture of inactive osteogenic growth factor molecules to the site of cell growth for subsequent integrin-mediated activation, using a recombinant fragment of latent transforming growth factor beta-binding protein-1 (rLTBP1). It is shown that rLTBP1 binds to the growth-factor- and integrin-binding domains of fibronectin on poly(ethyl acrylate) surfaces, which immobilizes rLTBP1 and promotes the binding of latency associated peptide (LAP), within which inactive transforming growth factor beta 1 (TGF-ß1) is bound. rLTBP1 facilitates the interaction of LAP with integrin ß1 and the subsequent mechanically driven release of TGF-ß1 to stimulate canonical TGF-ß1 signaling, activating osteogenic marker expression in vitro and complete regeneration of a critical-sized bone defect in vivo.
Asunto(s)
Osteogénesis , Factor de Crecimiento Transformador beta1 , Animales , Humanos , Factor de Crecimiento Transformador beta1/metabolismo , Fibronectinas/metabolismo , Fibronectinas/química , Proteínas de Unión a TGF-beta Latente/metabolismo , Proteínas de Unión a TGF-beta Latente/química , Regeneración Ósea , Propiedades de Superficie , Integrinas/metabolismo , Unión Proteica , Integrina beta1/metabolismo , Transducción de SeñalRESUMEN
There is an unmet clinical need to provide viable bone grafts for clinical use. Autologous bone, one of the most commonly transplanted tissues, is often used but is associated with donor site morbidity. Tissue engineering strategies to differentiate an autologous cell source, such as mesenchymal stromal cells (MSCs), into a potential bone-graft material could help to fulfill clinical demand. However, osteogenesis of MSCs can typically require long culture periods that are impractical in a clinical setting and can lead to significant cost. Investigation into strategies that optimize cell production is essential. Here, we use the piezoelectric copolymer poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE), functionalized with a poly(ethyl acrylate) (PEA) coating that drives fibronectin network formation, to enhance MSC adhesion and to present growth factors in the solid phase. Dynamic electrical cues are then incorporated, via a nanovibrational bioreactor, and the MSC response to electromechanical stimulation is investigated.
RESUMEN
During bone regeneration, the periosteum acts as a carrier for key regenerative cues, delivering osteochondroprogenitor cells and crucial growth factors to the injured bone. We developed a biocompatible, 3D polycaprolactone (PCL) melt electro-written membrane to act as a mimetic periosteum. Poly (ethyl acrylate) coating of the PCL membrane allowed functionalization, mediated by fibronectin and low dose recombinant human BMP-2 (rhBMP-2) (10-25 µg/ml), resulting in efficient, sustained osteoinduction in vitro. In vivo, rhBMP-2 functionalized mimetic periosteum demonstrated regenerative potential in the treatment of rat critical-size femoral defects with highly efficient healing and functional recovery (80%-93%). Mimetic periosteum has also proven to be efficient for cell delivery, as observed through the migration of transplanted periosteum-derived mesenchymal cells to the bone defect and their survival. Ultimately, mimetic periosteum demonstrated its ability to deliver key stem cells and morphogens to an injured site, exposing a therapeutic and translational potential in vivo when combined with unprecedentedly low rhBMP-2 doses.
RESUMEN
Cell-based liver therapies based on retrieving and steadying failed metabolic function(s) for acute and chronic diseases could be a valuable substitute for liver transplants, even though they are limited by the low engraftment capability and reduced functional quality of primary human hepatocytes (PHH). In this paper we propose the use of gelatin-hyaluronic acid (Gel-HA) scaffolds seeded with PHH for the treatment of liver failure. We first optimized the composition using Gel-HA hydrogels, looking for the mechanical properties closer to the human liver and determining HepG2 cells functionality. Gel-HA scaffolds with interconnected porosity (pore size 102 µm) were prepared and used for PHH culture and evaluation of key hepatic functions. PHH cultured in Gel-HA scaffolds exhibited increased albumin and urea secretion and metabolic capacity (CYP and UGT activity levels) compared to standard monolayer cultures. The transplant of the scaffold containing PHH led to an improvement in liver function (transaminase levels, necrosis) and ameliorated damage in a mouse model of acetaminophen (APAP)-induced liver failure. The study provided a mechanistic understanding of APAP-induced liver injury and the impact of transplantation by analyzing cytokine production and oxidative stress induction to find suitable biomarkers of cell therapy effectiveness.
Asunto(s)
Acetaminofén , Fallo Hepático Agudo , Ratones , Animales , Humanos , Acetaminofén/toxicidad , Acetaminofén/metabolismo , Hepatocitos/metabolismo , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/terapia , Fallo Hepático Agudo/metabolismo , Células Hep G2 , Ácido Hialurónico/metabolismoRESUMEN
In this work, Engineered Living Materials (ELMs), based on the combination of genetically-modified bacteria and mineral-reinforced organic matrices, and endowed with self-healing or regenerative properties and adaptation to specific biological environments were developed. Concretely, we produced ELMs combining human mesenchymal stem cells (hMSCs) and Lactococcus lactis (L. lactis), which was specifically programmed to deliver bone morphogenetic protein (BMP-2) upon external stimulation using nisin, into mineralized alginate matrices. The hybrid organic/inorganic matrix was built through a protocol, inspired by bone mineralization, in which alginate (Alg) assembly and apatite (HA) mineralization occurred simultaneously driven by calcium ions. Chemical composition, structure and reologhical properties of the hybrid 3D matrices were dedicately optimized prior the incorportation of the living entities. Then, the same protocol was reproduced in the presence of hMSC and engineered L. lactis that secrete BMP-2 resulting in 3D hybrid living hydrogels. hMSC viability and osteogenic differentiation in the absence and presence of the bacteria were evaluated by live/dead and quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence assays, respectively. Results demonstrate that these 3D engineered living material support osteogenic differentiation of hMSCs due to the synergistic effect between HA and the growth factors BMP-2 delivered by L. lactis.
Asunto(s)
Calcinosis , Células Madre Mesenquimatosas , Humanos , Osteogénesis/genética , Andamios del Tejido/química , Células Madre Mesenquimatosas/metabolismo , Alginatos , Diferenciación Celular , Calcinosis/metabolismoRESUMEN
Mimicking bone extracellular matrix (ECM) is paramount to develop novel biomaterials for bone tissue engineering. In this regard, the combination of integrin-binding ligands together with osteogenic peptides represents a powerful approach to recapitulate the healing microenvironment of bone. In the present work, we designed polyethylene glycol (PEG)-based hydrogels functionalized with cell instructive multifunctional biomimetic peptides (either with cyclic RGD-DWIVA or cyclic RGD-cyclic DWIVA) and cross-linked with matrix metalloproteinases (MMPs)-degradable sequences to enable dynamic enzymatic biodegradation and cell spreading and differentiation. The analysis of the intrinsic properties of the hydrogel revealed relevant mechanical properties, porosity, swelling and degradability to engineer hydrogels for bone tissue engineering. Moreover, the engineered hydrogels were able to promote human mesenchymal stem cells (MSCs) spreading and significantly improve their osteogenic differentiation. Thus, these novel hydrogels could be a promising candidate for applications in bone tissue engineering, such as acellular systems to be implanted and regenerate bone or in stem cells therapy.
RESUMEN
Collagen type I lacks affinity for growth factors (GFs) and yet it is clinically used to deliver bone morphogenic protein 2 (BMP-2), a potent osteogenic growth factor. To mitigate this lack of affinity, supra-physiological concentrations of BMP-2 are loaded in collagen sponges leading to uncontrolled BMP-2 leakage out of the material. This has led to important adverse side effects such as carcinogenesis. Here, we design recombinant dual affinity protein fragments, produced in E. Coli, which contain two regions, one that spontaneously binds to collagen and a second one that binds BMP-2. By adding the fragment to collagen sponges, BMP-2 is sequestered enabling solid phase presentation of BMP-2. We demonstrate osteogenesis in vivo with ultra-low doses of BMP-2. Our protein technology enhances the biological activity of collagen without using complex chemistries or changing the manufacturing of the base material and so opens a pathway to clinical translation.
RESUMEN
Fibronectin (FN) mediates cell-material interactions during events such as tissue repair, and therefore the biomimetic modeling of this protein in vitro benefits regeneration. The nature of the interface is crucial in determining cell adhesion, morphology, and differentiation. Poly(ethyl acrylate) (PEA) spontaneously organizes FN into biological nanonetworks, resulting in exceptional bone regeneration in animal models. Spontaneous network organization of FN is also observed in poly(buthyl acrylate) (PBA) substrates that have higher surface mobility than PEA. C2C12 myoblasts differentiate efficiently on PEA and PBA substrates. In this study, we investigate if intermediate surface mobilities between PEA and PBA induce cell differentiation more efficiently than PEA. A family of P(EA-co-BA) copolymers were synthesized in the entire range of compositions to finely tune surface mobility between PEA and PBA. Surface characterization demonstrates that FN mobility steadily increased with the PBA content. All compositions allowed the biological organization of FN with similar exposure of cell binding domains. C2C12 myoblasts adhered well in all the materials, with higher focal adhesions in PEA and PBA. The increase of the interfacial mobility had an impact in cell adhesion by increasing the number of FAs per cell. In addition, cell differentiation decreased proportionally with surface mobility, from PEA to PBA.
Asunto(s)
Acrilatos , Animales , Adhesión Celular , Diferenciación Celular , Acrilatos/farmacologíaRESUMEN
Tissue engineered cartilage for external ear reconstruction of congenital deformities, such as microtia or resulting from trauma, remains a significant challenge for plastic and reconstructive surgeons. Current strategies involve harvesting autologous costal cartilage or expanding autologous chondrocytes ex vivo. However, these procedures often lead to donor site morbidity and a cell source with limited expansion capacity. Stromal stem cells such as perivascular stem cells (pericytes) offer an attractive alternative cell source, as they can be isolated from many human tissues, readily expanded in vitro and possess chondrogenic differentiation potential. Here, we successfully isolate CD146+ pericytes from the microtia remnant from patients undergoing reconstructive surgery (Microtia pericytes; MPs). Then we investigate their chondrogenic potential using the polymer poly(ethyl acrylate) (PEA) to unfold the extracellular matrix protein fibronectin (FN). FN unfolding exposes key growth factor (GF) and integrin binding sites on the molecule, allowing tethering of the chondrogenic GF transforming growth factor beta 1 (TGFß1). This system leads to solid-phase, matrix-bound, GF presentation in a more physiological-like manner than that of typical chondrogenic induction media (CM) formulations that tend to lead to off-target effects. This simple and controlled material-based approach demonstrates similar chondrogenic potential to CM, while minimising proclivity toward hypertrophy, without the need for complex induction media formulations.
Asunto(s)
Microtia Congénita , Humanos , Microtia Congénita/cirugía , Pericitos , Condrogénesis , Fibronectinas , CartílagoRESUMEN
Tissue engineering aims at replicating tissues and organs to develop applications in vivo and in vitro. In vivo, by engineering artificial constructs using functional materials and cells to provide both physiological form and function. In vitro, by engineering three-dimensional (3D) models to support drug discovery and enable understanding of fundamental biology. 3D culture constructs mimic cell-cell and cell-matrix interactions and use biomaterials seeking to increase the resemblance of engineered tissues with its in vivo homologues. Native tissues, however, include complex architectures, with compartmentalized regions of different properties containing different types of cells that can be captured by multicompartment constructs. Recent advances in fabrication technologies, such as micropatterning, microfluidics or 3D bioprinting, have enabled compartmentalized structures with defined compositions and properties that are essential in creating 3D cell-laden multiphasic complex architectures. This review focuses on advances in engineered multicompartment constructs that mimic tissue heterogeneity. It includes multiphasic 3D implantable scaffolds and in vitro models, including systems that incorporate different regions emulating in vivo tissues, highlighting the emergence and relevance of 3D bioprinting in the future of biological research and medicine.
Asunto(s)
Bioimpresión , Impresión Tridimensional , Hidrogeles/química , Ingeniería de Tejidos/métodos , Materiales Biocompatibles/química , Andamios del Tejido/químicaRESUMEN
Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are of considerable clinical potential in transplantation and anti-inflammatory therapies due to their capacity for tissue repair and immunomodulation. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunomodulatory therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can influence paracrine activities of MSCs. Here, we show that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension and increasing oxidative glycolysis. We use nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture and during larger scale cell manufacture. Our findings demonstrate an effective route to support large-scale expansion of functional MSCs for therapeutic purposes.
Asunto(s)
Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Células Madre Multipotentes/metabolismo , Diferenciación Celular , Inmunomodulación , FenotipoRESUMEN
Pancreatic cancer is a deadly and highly metastatic disease, although how metastatic lesions establish is not fully understood. A key feature of pancreatic tumours is extensive fibrosis and deposition of extracellular matrix (ECM). While pancreatic cancer cells are programmed by stimuli derived from a stiff ECM, metastasis requires loss of attachment and adaptation to a softer microenvironment at distant sites. Growing evidence suggests that stiff ECM influences pancreatic cancer cell behaviour. Here, we argue that this influence is reversible and that pancreatic cancer cells can be reprogrammed upon sensing soft substrates. Using engineered polyacrylamide hydrogels with tuneable mechanical properties, we show that collagen VI is specifically upregulated in pancreatic cancer cells on soft substrates, due to a lack of integrin engagement. Furthermore, the expression of collagen VI is inversely correlated with mechanosensing and activity of YAP (also known as YAP1), which might be due to a direct or indirect effect on transcription of genes encoding collagen VI. Collagen VI supports migration in vitro and metastasis formation in vivo. Metastatic nodules formed by pancreatic cancer cells lacking Col6a1 display stromal cell-derived collagen VI deposition, suggesting that collagen VI derived from either cancer cells or the stroma is an essential component of the metastatic niche. This article has an associated First Person interview with Vasileios Papalazarou, joint first author of the paper.
Asunto(s)
Colágeno , Neoplasias Pancreáticas , Humanos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Integrinas/metabolismo , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
The tumor microenvironment plays an important role in cancer development and the use of 3D in vitro systems that decouple different elements of this microenvironment is critical for the study of cancer progression. In neuroblastoma (NB), vitronectin (VN), an extracellular matrix protein, has been linked to poor prognosis and appears as a promising therapeutic target. Here, we developed hydrogels that incorporate VN into 3D polyethylene glycol (PEG) hydrogel networks to recapitulate the native NB microenvironment. The stiffness of the VN/PEG hydrogels was modulated to be comparable to the in vivo values reported for NB tissue samples. We used SK-N-BE (2) NB cells to demonstrate that PEGylated VN promotes cell adhesion as the native protein does. Furthermore, the PEGylation of VN allows its crosslinking into the hydrogel network, providing VN retention within the hydrogels that support viable cells in 3D. Confocal imaging and ELISA assays indicate that cells secrete VN also in the hydrogels and continue to reorganize their 3D environment. Overall, the 3D VN-based PEG hydrogels recapitulate the complexity of the native tumor extracellular matrix, showing that VN-cell interaction plays a key role in NB aggressiveness, and that VN could potentially be targeted in preclinical drug studies performed on the presented hydrogels.
RESUMEN
Mesenchymal stem cell (MSC)-based tissue engineering strategies are of interest in the field of bone tissue regenerative medicine. MSCs are commonly investigated in combination with growth factors (GFs) and biomaterials to provide a regenerative environment for the cells. However, optimizing how biomaterials interact with MSCs and efficiently deliver GFs, remains a challenge. Here, via plasma polymerization, tissue culture plates are coated with a layer of poly (ethyl acrylate) (PEA), which is able to spontaneously permit fibronectin (FN) to form fibrillar nanonetworks. However, vitronectin (VN), another important extracellular matrix (ECM) protein forms multimeric globules on the polymer, thus not displaying functional groups to cells. Interestingly, when FN and VN are co-absorbed onto PEA surfaces, VN can be entrapped within the FN fibrillar nanonetwork in the monomeric form providing a heterogeneous, open ECM network. The combination of FN and VN promote MSC adhesion and leads to enhanced GF binding; here we demonstrate this with bone morphogenetic protein-2 (BMP2). Moreover, MSC differentiation into osteoblasts is enhanced, with elevated expression of osteopontin (OPN) and osteocalcin (OCN) quantified by immunostaining, and increased mineralization observed by von Kossa staining. Osteogenic intracellular signalling is also induced, with increased activity in the SMAD pathway. The study emphasizes the need of recapitulating the complexity of native ECM to achieve optimal cell-material interactions.
RESUMEN
Living biointerfaces are a new class of biomaterials combining living cells and polymeric matrices that can act as biologically active and instructive materials that host and provide signals to surrounding cells. Here, living biomaterials based on Lactococcus lactis to control hematopoietic stem cells in 2D surfaces and 3D hydrogels are introduced. L. lactis is modified to express C-X-C motif chemokine ligand 12 (CXCL12), thrombopoietin (TPO), vascular cell adhesion protein 1 (VCAM1), and the 7th-10th type III domains of human plasma fibronectin (FN III7-10 ), in an attempt to mimic ex vivo the conditions of the human bone marrow. These results suggest that living biomaterials that incorporate bacteria expressing recombinant CXCL12, TPO, VCAM1, and FN in both 2D systems direct hematopoietic stem and progenitor cells (HSPCs)-bacteria interaction, and in 3D using hydrogels functionalized with full-length human plasma fibronectin allow for a notable expansion of the CD34+ /CD38- /CD90+ HSPC population compared to the initial population. These results provide a strong evidence based on data that suggest the possibility of using living materials based on genetically engineered bacteria for the ex-vivo expansion of HSPC with eventual practical clinical applications in HSPCs transplantation for hematological disorders.