In JoVE (1)

Other Publications (20)

Articles by Luisa Trombi in JoVE

 JoVE Bioengineering

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

1Dept. of Clinical and Experimental Medicine, University of Pisa, 2OtoLab, Azienda Ospedaliero-Universitaria Pisana (AOUP), 3Dept. of Civil and Industrial Engineering, University of Pisa, 4Immunohematology Operative Unit, Azienda Ospedaliero-Universitaria Pisana (AOUP), 5Dept. Of Surgical, Medical, Molecular Pathology and Emergency Medicine, University of Pisa, 6II Orthopedic and Traumatologic Clinic, Azienda Ospedaliero-Universitaria Pisana (AOUP)


JoVE 54845

Other articles by Luisa Trombi on PubMed

Carboxy-terminal Fragment of Osteogenic Growth Peptide in Vitro Increases Bone Marrow Cell Density in Idiopathic Myelofibrosis

British Journal of Haematology. Apr, 2003  |  Pubmed ID: 12670334

Idiopathic myelofibrosis (IMF) is a clonal stem cell disorder characterized by reactive fibrosis of bone marrow sustained by a complex cytokine network. At present, no efficacious therapy for this disease exists. Synthetic carboxy-terminal pentapeptide of osteogenic growth factor (sOGP10-14) can increase bone marrow cellularity and the number of haematopoietic colonies; this study evaluated the activity of sOGP10-14 in IMF. Fragments of bone marrow biopsies from patients affected by IMF were cultured with or without the addition of sOGP10-14. Cellular density was evaluated by image analysis, and transforming growth factor-beta1 (TGF-beta1) concentration was immunologically assayed in the supernatant of cultured bone marrow biopsies. The proliferation rate of the megakaryoblastic M07-e cell line, cultured in the presence of either granulocyte-macrophage colony stimulating factor or thrombopoietin (TPO), and with or without sOGP10-14, was evaluated. Megakaryocyte colony forming unit (CFU-Mk) assay was performed on bone marrow samples of IMF patients with or without sOGP10-14. After 14 d, bone marrow cellularity was significantly increased in samples cultured with the pentapeptide. Moreover, sOGP10-14 induced a significant increase of TGF-beta in culture supernatants. TPO-primed proliferation of M07-e was reduced by sOGP10-14, and the pentapeptide significantly reduced CFU-Mk on IMF bone-marrow-derived cells. sOGP10-14 increased ex vivo bone marrow cellularity in IMF. This action could be related to the megakaryocyte inhibition induced by the interference of this pentapeptide with growth factor activities. These findings suggest that a deficiency of osteoblast-related factors may play a role in bone marrow failure in IMF.

Bone and Bone Marrow Interactions: Hematological Activity of Osteoblastic Growth Peptide (OGP)-derived Carboxy-terminal Pentapeptide III. Action on Human Megakaryocytopoiesis: Focus on Essential Thrombocythemia

Leukemia Research. Oct, 2004  |  Pubmed ID: 15289024

The increase of megakaryocytes and platelets that characterizes essential thrombocythemia (ET) appears to be secondary to a deregulation of megakaryocytopoiesis. The carboxy-terminal fragment of osteogenic growth peptide (OGP10-14) promotes bone formation and hemopoiesis, while it inhibits megakaryocytopoiesis. In this paper we show that treatment with synthetic OGP10-14 (sOGP10-14) induces a significant reduction of mid and large colony-forming unit-megakaryocytes (CFU-Mk) in ET patients as well as in controls, and is associated with a significant inhibition of thrombopoietin (TPO)-primed MO-7e megakaryoblastic cells proliferation. These actions appear to be related to sOGP10-14 modulation of TGF-beta(1) synthesis and/or secretion, although a direct effect on TGF-beta receptor expression cannot be excluded.

PEG-Filgrastim Activity on Granulocyte Functions

Leukemia Research. Oct, 2007  |  Pubmed ID: 17197023

A Micro/nanoscale Surface Mechanical Study on Morpho-functional Changes in Multilineage-differentiated Human Mesenchymal Stem Cells

Macromolecular Bioscience. May, 2007  |  Pubmed ID: 17477443

In recent years MSCs have become a very attractive tool in tissue engineering and regenerative medicine because of their ability to be committed along several lineages through chemical or physical stimuli. Nevertheless their therapeutic potential and plasticity are not yet totally understood. This report describes the use of AFM together with conventional microscopies to obtain mechanical information on cell surfaces and deposited extra cellular matrix molecules, after inducing the differentiation of human MSCs towards three typical mesoderm phenotypes. The aim is to correlate morphological, functional, and mechanical aspects of human MSCs to obtain a deeper understanding of their great potential.

Suspension of Bone Marrow-derived Undifferentiated Mesenchymal Stromal Cells for Repair of Superficial Digital Flexor Tendon in Race Horses

Tissue Engineering. Dec, 2007  |  Pubmed ID: 17919069

It has been proven that mesenchymal stromal cells (MSCs) can differentiate into tenocytes. Attempts to repair tendon lesions have been performed, mainly using scaffold carriers in experimental settings. In this article, we describe the clinical use of undifferentiated MSCs in racehorses. Significant clinical recovery was achieved in 9 of 11 horses evaluated using ultrasound analysis and their ability to return to racing. Our results show that the suspension of a small number of undifferentiated MSCs may be sufficient to repair damaged tendons without the use of scaffold support. Ultrasound scanning showed that fibers were correctly oriented. By using undifferentiated cells, no ectopic bone deposition occurred. A sufficient number of cells was recovered for therapeutic purposes in all but 1 case. We suggest that the use of autologous MSCs is a safe therapeutic method for treating incompletely (i.e., not full-thickness) damaged tendons.

Human Autologous Plasma-derived Clot As a Biological Scaffold for Mesenchymal Stem Cells in Treatment of Orthopedic Healing

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Feb, 2008  |  Pubmed ID: 17868116

Recent advances in the isolation, expansion, and characterization of human mesenchymal stem cells (hMSCs) have raised the possibility of using them in cell therapies and tissue engineering for bone reconstruction. hMSCs, isolated from the bone marrow of eight normal adult patients, were minimally expanded ex vivo and pulsed twice toward osteogenic lineage. The cells were then included into autologous plasma-derived clots. Cytofluorimetric analysis, immunocytochemistry (osteopontin), histochemistry (alkaline phosphatase, Alcian blue, Von Kossa, and alizarin red staining), and viable/proliferation tests were performed to study both stem and differentiating cells. Although two short inductions increased osteogenic markers in hMSCs, inside the clot the cells were able to terminally differentiate into osteoblasts. Moreover, we show that the clot is able to sustain cell proliferation under appropriate cell culture conditions. Our results suggested that clot could be useful for hMSC delivery into the site of the lesion to promote bone formation. Moreover, the plasticity of this material allowed good in vitro hMSC spreading and proliferation. The advantages of using this autologous biological material are its biocompatibility and reabsorption; furthermore, using a gel as scaffold, it is possible to mold it to the shape of a bone cavity.

Gelatin/PLLA Sponge-like Scaffolds Allow Proliferation and Osteogenic Differentiation of Human Mesenchymal Stromal Cells

Macromolecular Bioscience. Sep, 2008  |  Pubmed ID: 18504804

Tissue engineering has the potential to supply constructs capable of restoring the normal function of native tissue following injury. Poly(L-lactic acid) (PLLA) scaffolds are amongst the most commonly used biodegradable polymers in tissue engineering and previous studies performed on ovine fibroblasts have showed that addition of gelatin creates a favorable hydrophilic microenvironment for the growth of these cells. The attractiveness of using mesenchymal stromal cells (MSCs) in tissue regeneration is that they are able to differentiate into several lines including osteoblasts. In this study, we investigated the ability of gelatin/PLLA sponges to support the adhesion, proliferation, and osteogenic differentiation of human MSCs isolated from the bone marrow of four donors. [Figure: see text].

Good Manufacturing Practice-grade Fibrin Gel is Useful As a Scaffold for Human Mesenchymal Stromal Cells and Supports in Vitro Osteogenic Differentiation

Transfusion. Oct, 2008  |  Pubmed ID: 18657082

Recently, there has been an increased interest in using mesenchymal stromal cells (MSCs) in bone tissue engineering coupled with a suitable scaffold of both biological and synthetic origin. The cells and these constructs can be combined in vitro or directly in vivo to enhance tissue repair. MSCs are spindle-shaped cells capable of self-renewal and can be induced to differentiate mainly into osteo-, chondro-, and adipogenic-progeny types. Several biomaterials are currently available and, among them, fibrin-based constructs seem to be suitable for guiding the cells during tissue repair or regeneration due to their biocompatibility and biodegradability.

Morpho-functional Characterization of Human Mesenchymal Stem Cells from Umbilical Cord Blood for Potential Uses in Regenerative Medicine

Stem Cells and Development. Mar, 2009  |  Pubmed ID: 18444788

Mesenchymal stem cells (MSCs) represent a promising source of progenitor cells having the potential to repair and to regenerate diseased or damaged skeletal tissues. Bone marrow (BM) has been the first source reported to contain MSCs. However, BM-derived cells are not always acceptable, due to the highly invasive drawing and the decline in MSC number and differentiative capability with increasing age. Human umbilical cord blood (UCB), obtainable by donation with a noninvasive method, has been introduced as an alternative source of MSCs. Here human UCB-derived MSCs isolation and morpho-functional characterization are reported. Human UCB-derived mononuclear cells, obtained by negative immunoselection, exhibited either an osteoclast-like or a mesenchymal-like phenotype. However, we were able to obtain homogeneous populations of MSCs that displayed a fibroblast-like morphology, expressed mesenchym-related antigens and showed differentiative capacities along osteoblastic and early chondroblastic lineages. Furthermore, this study is one among a few papers investigating human UCB-derived MSC growth and differentiation on three-dimensional scaffolds focusing on their potential applications in regenerative medicine and tissue engineering. UCB-derived MSCs were proved to grow on biodegradable microfiber meshes; additionally, they were able to differentiate toward mature osteoblasts when cultured inside human plasma clots, suggesting their potential application in orthopedic surgery.

Identification and Purification of Mesodermal Progenitor Cells from Human Adult Bone Marrow

Stem Cells and Development. Jul-Aug, 2009  |  Pubmed ID: 18991503

Bone marrow-derived mesodermal stem cells may differentiate toward several lines and are easily cultured in vitro. Some putative progenitors of these cells have been described in both humans and mice. Here, we describe a new mesodermal progenitor population [mesodermal progenitors cells (MPCs)] able to differentiate into mesenchymal cells upon appropriate culture conditions. When cultured in presence of autologous serum, these cells are strongly adherent to plastic, resistant to trypsin detachment, and resting. Mesodermal progenitor cells may be pulsed to proliferate and differentiate by substituting autologous serum for human cord blood serum or fetal calf serum. By these methods cells proliferate and differentiate toward mesenchymal cells and thus may further differentiate into osteoblats, chondrocytes, or adipocytes. Moreover MPCs are capable to differentiate in endothelial cells (ECs) showing characteristics similar to microvessel endothelium cells. Mesodermal progenitors cells have a defined phenotype and carry embryonic markers not present in mesenchymal cells. Moreover MPCs strongly express aldehyde dehydrogenase activity, usually present in hematopoietic precursors but absent in mesenchymal cells. When these progenitors are pulsed to differentiate, they lose these markers and acquire the mesenchymal ones. Interestingly, mesenchymal cells may not be induced to back differentiate into MPCs. Our results demonstrate the adult serum role in maintaining pluripotent mesodermal precursors and allow isolation of these cells. After purification, MPCs may be pulsed to proliferate in a very large scale and then induced to differentiate, thus possibly allowing their use in regenerative medicine.

Selective Culture of Mesodermal Progenitor Cells

Stem Cells and Development. Oct, 2009  |  Pubmed ID: 19331526

We have recently identified mesodermal progenitor cells (MPCs) isolated from adult human bone marrow. These cells show unusual phenotypes, having putative embryonic markers and aldehyde dehydrogenase (ALDH) activity. Interestingly, these resting cells, which have been selected by culturing them in the presence of adult human serum, can easily be induced to differentiate into mature mesenchymal stromal cells (MSCs) after substituting the adult human serum for fetal bovine serum (FBS) or human cord serum. MPC-derived MSCs are, in turn, able to differentiate toward osteoblasts, chondrocytes, and adipocytes. Furthermore, MPCs are able to differentiate into endothelial cells. MPCs have been proven to be strongly adherent to plastic culture bottles and to be trypsin-resistant. In the present article, we show a simple and inexpensive method to isolate highly selected mesodermal progenitors from bone marrow or cord blood. The optimization of standard culture conditions (using commercial human AB sera and appropriate concentrations for cell seeding in plastics) allows a pure population of MPCs to be obtained even after a short culture period. We believe that this simple, repeatable, and standardized method will facilitate studies on MPCs.

Constitutive Expression of Pluripotency-associated Genes in Mesodermal Progenitor Cells (MPCs)

PloS One. 2010  |  Pubmed ID: 20360837

We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation.

Mesodermal Progenitor Cells (MPCs) Differentiate into Mesenchymal Stromal Cells (MSCs) by Activation of Wnt5/calmodulin Signalling Pathway

PloS One. 2011  |  Pubmed ID: 21980498

Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90(neg) phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4(neg)CD105+CD90(bright) and MSCA-1+), in the primary cultures, resulted lower than 2%.

HOX and TALE Signatures Specify Human Stromal Stem Cell Populations from Different Sources

Journal of Cellular Physiology. Apr, 2013  |  Pubmed ID: 23018864

Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues.

Growing Bone Tissue-engineered Niches with Graded Osteogenicity: an in Vitro Method for Biomimetic Construct Assembly

Tissue Engineering. Part C, Methods. Dec, 2013  |  Pubmed ID: 23537352

The traditional bone tissue-engineering approach exploits mesenchymal stem cells (MSCs) to be seeded once only on three-dimensional (3D) scaffolds, hence, differentiated for a certain period of time and resulting in a homogeneous osteoblast population at the endpoint. However, after achieving terminal osteodifferentiation, cell viability is usually markedly compromised. On the other hand, naturally occurring osteogenesis results from the coexistence of MSC progenies at distinct differentiative stages in the same microenvironment. This diversification also enables long-term viability of the mature tissue. We report an easy and tunable in vitro method to engineer simple osteogenic cell niches in a biomimetic fashion. The niches were grown via periodic reseeding of undifferentiated MSCs on MSC/scaffold constructs, the latter undergoing osteogenic commitment. Time-fractioning of the seeded cell number during differentiation time of the constructs allowed graded osteogenic cell populations to be grown together on the same scaffolds (i.e., not only terminally differentiated osteoblasts). In such cell-dynamic systems, the overall differentiative stage of the constructs could also be tuned by varying the cell density seeded at each inoculation. In this way, we generated two different biomimetic niche models able to host good reservoirs of preosteoblasts and other osteoprogenitors after 21 culture days. At that time, the niche type resulting in 40.8% of immature osteogenic progenies and only 59.2% of mature osteoblasts showed a calcium content comparable to the constructs obtained with the traditional culture method (i.e., 100.03 ± 29.30 vs. 78.51 ± 28.50 pg/cell, respectively; p=not significant), the latter colonized only by fully differentiated osteoblasts showing exhausted viability. This assembly method for tissue-engineered constructs enabled a set of important parameters, such as viability, colonization, and osteogenic yield of the MSCs to be balanced on 3D scaffolds, thus achieving biomimetic in vitro models with graded osteogenicity, which are more complex and reliable than those currently used by tissue engineers.

Use of Autologous Human Mesenchymal Stromal Cell/fibrin Clot Constructs in Upper Limb Non-unions: Long-term Assessment

PloS One. 2013  |  Pubmed ID: 24023694

Tissue engineering appears to be an attractive alternative to the traditional approach in the treatment of fracture non-unions. Mesenchymal stromal cells (MSCs) are considered an appealing cell source for clinical intervention. However, ex vivo cell expansion and differentiation towards the osteogenic lineage, together with the design of a suitable scaffold have yet to be optimized. Major concerns exist about the safety of MSC-based therapies, including possible abnormal overgrowth and potential cancer evolution.

Mesenchymal Stem Cells Derived from Vertebrae (vMSCs) Show Best Biological Properties

European Spine Journal : Official Publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society. Nov, 2013  |  Pubmed ID: 24061975

Due to their properties and characteristics human mesenchymal stem cells (MSCs) appear to have great therapeutic potential. Many different populations of MSCs have been described and to understand whether they have equivalent biological properties is a critical issue for their therapeutic application.

Plasticity of Human Dental Pulp Stromal Cells with Bioengineering Platforms: a Versatile Tool for Regenerative Medicine

Micron (Oxford, England : 1993). Dec, 2014  |  Pubmed ID: 25180486

In recent years, human dental pulp stromal cells (DPSCs) have received growing attention due to their characteristics in common with other mesenchymal stem cells, in addition to the ease with which they can be harvested. In this study, we demonstrated that the isolation of DPSCs from third molar teeth of healthy individuals allowed the recovery of dental mesenchymal stem cells that showed self-renewal and multipotent differentiation capability. DPSCs resulted positive for CD73, CD90, CD105, STRO-1, negative for CD34, CD45, CD14 and were able to differentiate into osteogenic and chondrogenic cells. We also assayed the angiogenic potential of DPSCs, their capillary tube-like formation was assessed using an in vitro angiogenesis assay and the uptake of acetylated low-density lipoprotein was measured as a marker of endothelial function. Based on these results, DPSCs were capable of differentiating into cells with phenotypic and functional features of endothelial cells. Furthermore, this study investigated the growth and differentiation of human DPSCs under a variety of bioengineering platforms, such as low frequency ultrasounds, tissue engineering and nanomaterials. DPSCs showed an enhanced chondrogenic differentiation under ultrasound application. Moreover, DPSCs were tested on different scaffolds, poly(vinyl alcohol)/gelatin (PVA/G) sponges and human plasma clots. We showed that both PVA/G and human plasma clot are suitable scaffolds for adhesion, growth and differentiation of DPSCs toward osteoblastic lineages. Finally, we evaluated the interactions of DPSCs with a novel class of nanomaterials, namely boron nitride nanotubes (BNNTs). From our investigation, DPSCs have appeared as a highly versatile cellular tool to be employed in regenerative medicine.

Multiscale Fabrication of Biomimetic Scaffolds for Tympanic Membrane Tissue Engineering

Biofabrication. May, 2015  |  Pubmed ID: 25947357

The tympanic membrane (TM) is a thin tissue able to efficiently collect and transmit sound vibrations across the middle ear thanks to the particular orientation of its collagen fibers, radiate on one side and circular on the opposite side. Through the combination of advanced scaffolds and autologous cells, tissue engineering (TE) could offer valuable alternatives to autografting in major TM lesions. In this study, a multiscale approach based on electrospinning (ES) and additive manufacturing (AM) was investigated to fabricate scaffolds, based on FDA approved copolymers, resembling the anatomic features and collagen fiber arrangement of the human TM. A single scale TM scaffold was manufactured using a custom-made collector designed to confer a radial macro-arrangement to poly(lactic-co-glycolic acid) electrospun fibers during their deposition. Dual and triple scale scaffolds were fabricated combining conventional ES with AM to produce poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer scaffolds with anatomic-like architecture. The processing parameters were optimized for each manufacturing method and copolymer. TM scaffolds were cultured in vitro with human mesenchymal stromal cells, which were viable, metabolically active and organized following the anisotropic character of the scaffolds. The highest viability, cell density and protein content were detected in dual and triple scale scaffolds. Our findings showed that these biomimetic micro-patterned substrates enabled cell disposal along architectural directions, thus appearing as promising substrates for developing functional TM replacements via TE.

Grafting of Expanded Mesenchymal Stem Cells Without Associated Procedure in a Healed Case of Ulna Pseudarthrosis: A Case Report

Surgical Technology International. Apr, 2016  |  Pubmed ID: 27121410

The surgical management of pseudoarthrosis is often a challenge. The use of mesenchymal multipotent cells expanded and manipulated in the laboratory is an interesting treatment of pseudoarthrosis, because they can lead to differentiation into osteocytes and thus the formation of bone tissue.

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