Regenerative medicine – Adult stem cells study

Objective:
One of our fundamental aims is to increase the knowledge about the biology of stem cells and to harness their potential for differentiation and the body’s own regenerative capacity to restore the function and structure of damaged tissues and organs. We intend to develop standardized GMP methods of culturing adult mesenchymal stem cells (MSCs) for clinical applications for repairing human tissues. MSCs derived from different sources will be used: bone marrow (BM), umbilical cord blood (CB), adipose tissue (AT), or amniotic membrane (AM). The therapeutic potential and the function of cultured MSCs will be evaluated extensively in vitro and in vivo, resulting in clinical application protocols.

a) Mesenchymal stem cells for tissue reconstruction

1. GENOSTEM – Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side, FP6, 2004-2008

Short description:
The objective of this Integrated Project Genostem was to establish a European international scientific leadership for stem cell regenerative medicine in the field of connective tissue disorders. Genostem brings together a unique combination of expertises in Cellular Biology, Molecular Biology, Biomechanics, Genomics, Proteomics, Bioinformatics and Molecular Medicine. The critical mass achieved by this Consortium that comprises 30 partners, enabled breakthroughs in MSC engineering directly amenable for clinical applications.

Autologous adult Mesenchymal Stem Cells (MSCs) are optimal candidates to serve as the building blocks for the engineering of connective tissues being multipotential stem cells that give rise to osteoblasts, chondrocytes and tenocytes, muscle cells and adipocytes. Genostem compared different tissue sources of MSCs and isolated subsets in order to obtain undifferentiated MSCs, committed MSCs at early stage of differentiation, progeny blocked at specific differentiation stage and fully differentiated progeny. Genostem studied the complete MSC gene product repertoire using genomic and proteomic analysis providing the understanding of the molecules and pathways potentially operative for the maintenance and differentiation of stem cells. Genostem developed innovative technologies to generate biodegradable matrices, scaffolds and microcarriers that bind pharmacologically active proteins and allow their delivery in a controlled way. Genostem developed transplantation models of MSCs mimicking human pathological processes operative in bone cartilage and tendon diseases. The final goal was to create the premises for developing clinical trials using MSCs, in bone, cartilage and tendon disorders, in partnership with SMEs and regulatory bodies for the scale up of safe procedures. This project aimed to place the EU at the forefront of tissue engineering and to ensure strict safety standards for the connective tissues produced.

Link: http://cordis.europa.eu/project/rcn/75276_en.html

2. CASCADE – Cultivated Adult Stem Cells as Alternative for Damaged tissuE, FP7-223236, 2008-2011

Short description:
The main objective of the proposed work is to develop standardized GMP methods of culturing adult mesenchymal stem cells (MSCs) for clinical applications for repairing human tissue. MSCs derived from different sources will be used: bone marrow (BM), umbilical cord blood (CB), adipose tissue (AT), or amniotic membrane (AM). The therapeutic potential and the function of cultured MSCs will be evaluated extensively in vitro and in vivo. The consortium will develop clinical protocols for MSC application in defined diseases related to chronic wound healing (chronic venous leg ulcer and corneal ulcer).

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3. REBORNE – Regenerating Bone defects using New biomedical Engineering approaches, FP7-241879, 2010-2015

Short description:
The objective of REBORNE is to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. In order to reach this goal, five phase II clinical studies with 20 patients are proposed in 12 clinical centres spread in 8 European countries. Three orthopaedic trials concerning the treatment of long bone fractures and osteonecrosis of the femoral head in adults or children will be conducted using bioceramics, hydrogel for percutaneous injection and stem cells from autologous or allogeneic sources. Clinical research will also concern maxillofacial surgery with bone augmentation prior to dental implant and the reconstruction of cleft palates in children. The safety and efficacy of the new therapies will be assessed clinically using X-rays, CT scans and MRI as well as histology of biopsies.

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b) Characterization of pluripotent and adult stem cells (HU-RO)

In a trans-border collaboration with the group of Dr. Elen Gocza from ABC Institute in Godollo, Hungary, we compared the multipotential capacities of human mesenchymal stem cells against the pluripotentiality of embryonic stem cells and evaluated their ability to differentiate into cell lineages from all three germ layers: mesodermal, neuroectodermal, or endodermal lineages. The differences, as well as the similarities between the two cell types suggested possible solutions for improving the differentiation and renewing potential of adult stem cells.

a) Biology of mesenchymal stem cell-derived adipocytes – Studies regarding in vitro differentiation of mesenchymal stem cells towards adipogenic lineage and validation of molecular factors involved in adipogenesis (PNII-Idei, 1748/2008, 2008-2011)

Short description:
From the perspective of increasing prevalence of obesity around the world, even among children, this project aims a systemic aproach of molecular factors involved in mesenchymal stem cells differentiation towards the adipocytic lineage. After harvesting human bone marrow and isolation of mesenchymal stem cells (MSC) we will try to develop optimal new protocols for differentiation, under the action of inductors and maintenance reagents. Adipocytic differentiation will be validated by specific immunohystochemical and molecular biology methods, and the genes involved in the adipogenesis process will be further investigated using microarray technique. Identification of molecular markers involved in adipogenesis, mainly in the early stages of human MSC differentiation towards pre-adipocytes, opens important practical perspectives for future therapeutic approaches in obesity treatment. Once these markers and the genic expression involved in adipogenesis are identified, new modern methods of inhibition of adipose tissue formation process can emerge, thus aiming at the molecular level. Until now, the genes regulating adipogenesis are not clearly identified, mainly in the early stages of this process. For this purpose we will use a modern transfection technique, which uses microRNA (miRNA). We will identify by microarray techniques the genes for mRNA which is coding the adipogenesis process during different stages. After establishing the interest mRNA, this will be interfered with miRNA by gene-silencing process, followed by identification of key genes that will be used in further stages. Our results will be checked and compared with similar results obtained by the German research team, having agood collavoration plan for the entire duration of the project. At the end of our project, we will be able to draw a gene map and will elaborate a theoretical concept regarding the complex mechanisms involved in development of obesity.

Relevant publications:

  1. Tatu CS, Bojin FM, Gruia AT, Ordodi VI, Mic FA, Iman V, Cean A, Gavriliuc OI, Paunescu V. Features of Lipid Metabolism along Differentiation Pathway of Human Mesenchymal Stem Cells towards Mature Adipocytes. Romanian Biotechnological Letters, 2014; 19(2): 9257-71.
  2. Bojin FM, Gruita AT, Cristea MI, Ordodi VL, Paunescu V, Mic FA. Adipocytes differentiated in vitro from rat mesenchymal stem cells lack essential free fatty acids compared to adult adipocytes. Stem Cells and Development. 2012; 21(4): 507-12.

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Fig.1. MSC-differentiated adipocytes stained for Oil Red O and FABP4

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Fig.2. Gene map – Signaling and regulatory pathways during the adipogenesis process

b) Genetic engineering of stem cells for therapy of diabetes (SInPro)

Short description:
We addressed both the cause and the consequences of Type 1 Diabetes in a combined therapy pairing β-cell replacement by genetic engineering of insulin secreting cells with protection of the newly transplanted β-cell surrogates from the recurrence of islet-specific autoimmune attack. We hypothesized that this could be accomplished by using the immunosuppressive mesenchymal stem cells as cell substitutes in which to reconstruct the insulin secretory machinery by means of genetic transfer vectors.

Link: www.simp.ro
c) Pluripotent stem cells for cardiac reconstruction (ReCell)
Short description:
The main objective of ReCell is to establish the first hIPSCs based in vitro model system of chemotherapy induced cardiotoxicity. The project will offer an alternative option for repairing injured or dysfunctional heart and vessels involving in vitro engineering of cardiac tissue based on cutting-edge induced pluripotent stem cell methods. Fundamental research-derived knowledge in this field will subsequently serve to establish working and reproducible procedures which will guide GMP production of clinical-grade cells and tissue. In adddition, the project aims to create the first human induced pluripotent stem cell (hIPSC)-based in vitro model system of chemotherapy-induced cardiotoxicity, which can be developed and applied for diagnosis or even pre-emptive therapeutic reasons in support of personalised medicine in Romania.

By completing the goals of ReCell it will be possible to establish human cardiac tissue models from samples (biopsies or small amount of blood) provided by the principal hospitals in Romania. Hence, patient-specific hiPSC-CMCs can be further used to identify and evaluate drugs such as in chemotherapy induced cardiotoxicity. Development of quick screening kits for diagnosis and preemptive purposes in order to predict chemotherapy induced cardiotoxicity is also a possible outcome of the research efforts of ReCell.

The ReCell project will include the following activities:

  1. Generation and characterization of hIPSCs by episomal mediated gene transfer
  2. Generation and characterization of hIPSCs by the CRISPR/Cas9 system mediated gene transfer
  3. Generation and characterization of cardiomyocytes (CMs) from hIPSCs
  4. Generation and characterization of vascular derivatives from hIPSCs
  5. Engineering of cardiac tissue
  6. Engineering of vessels
  7. iPS –based cardiopathy model
  8. Optimization of the reprogramming and differentiation protocol in GMP conditions
  9. Optimization of the decellularisation and recellularisation protocol in GMP conditions
  10. Generation of cardiac patches

ReCell is aimed to have an impact on both the Romanian and European Biotechnology sector. The main impact of the project is expected to be in integrating efforts in stem cell biology, biotechnology, tissue engineering and personalized medicine. Furthermore, directly applicable outcomes of the project are expected from better stem cell differentiation protocols in human and a new model system for chemotherapy induced cardiotoxycity.

By establishing the first human iPSCs in the country, further applications such as toxicology and drug screening can be also subsequently developed and applied for newer type of diagnostic techniques or even preemptive therapeutic approaches. Development of quick screening kits might further partnerships of the research institutes with private pharmaceutical companies in order to offer as quickly as possible the new options to the main beneficiaries, the patients.

The target groups of the project activities are ultimately patients as beneficiaries of healthcare services which include the scientific results of the project. Patients with cardiovascular and coronary disorders will benefit form the project results regarding recellularized cardiac tissues (especially blood vessels), as well as patients with cancer under chemotherapy, which are the direct beneficiaries of the project results in terms of cardiotoxicity associated with widely prescribed chemotherapeutic drugs.

The achievement of the project is also important in terms of socio-economic reasons:

  • It will ensure the creation of new jobs in medicine
  • It will decrease the tendency of migration of skilled labor towards centers from other regions/ abroad, better equipped through the development of modern research facilities
  • It will provide training of qualified personnel and exchange of experience capable of sustaining the continuity in aproaching issues of modern research
  • It will stimulate the production of sustainable research – industry partnerships.

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