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2.
N Biotechnol ; 35: 19-29, 2017 Mar 25.
Article in English | MEDLINE | ID: mdl-27810336

ABSTRACT

Umbilical cord blood (UCB) transplantation is associated with long periods of aplastic anaemia. This undesirable situation is due to the low cell dose available per unit of UCB and the immaturity of its progenitors. To overcome this, we present a cell culture strategy aimed at the expansion of the CD34+ population and the generation of granulocyte lineage-committed progenitors. Two culture products were produced after either 6 or 14days of in vitro expansion, and their characteristics compared to non-expanded UCB CD34+ controls in terms of phenotype, colony-forming activity and multilineage repopulation potential in NOD-scid IL2Rγnull mice. Both expanded cell products maintained rapid SCID repopulation activity similar to the non-expanded control, but 14-day cultured cells showed impaired long term SCID repopulation activity. The process was successfully scaled up to clinically relevant doses of 89×106 CD34+ cells committed to the granulocytic lineage and 3.9×109 neutrophil precursors in different maturation stages. Cell yields and biological properties presented by the cell product obtained after 14days in culture were superior and therefore this is proposed as the preferred production setup in a new type of dual transplant strategy to reduce aplastic periods, producing a transient repopulation before the definitive engraftment of the non-cultured UCB unit. Importantly, human telomerase reverse transcriptase activity was undetectable, c-myc expression levels were low and no genetic abnormalities were found, as determined by G-banding karyotype, further confirming the safety of the expanded product.


Subject(s)
Cord Blood Stem Cell Transplantation/methods , Fetal Blood/cytology , Anemia, Aplastic/blood , Anemia, Aplastic/etiology , Anemia, Aplastic/prevention & control , Animals , Antigens, CD34/blood , Biotechnology , Cell Differentiation , Cell Lineage , Colony-Forming Units Assay , Cord Blood Stem Cell Transplantation/adverse effects , Female , Fetal Blood/immunology , Graft Enhancement, Immunologic/methods , Granulocytes/cytology , Humans , Interleukin Receptor Common gamma Subunit/deficiency , Interleukin Receptor Common gamma Subunit/genetics , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Neutrophils/cytology
3.
Cytotherapy ; 18(1): 25-35, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26549383

ABSTRACT

BACKGROUND AIMS: Umbilical cord (UC) has been proposed as a source of mesenchymal stromal cells (MSCs) for use in experimental cell-based therapies provided that its collection does not raise any risk to the donor, and, similar to bone marrow and lipoaspirates, UC-MSCs are multipotent cells with immuno-modulative properties. However, some of the challenges that make a broader use of UC-MSCs difficult include the limited availability of fresh starting tissue, time-consuming processing for successful derivation of cell lines, and the lack of information on identity, potency and genetic stability in extensively expanded UC-MSCs, which are necessary for banking relevant cell numbers for preclinical and clinical studies. METHODS: Factors affecting the success of the derivation process (namely, time elapsed from birth to processing and weight of fragments), and methods for establishing a two-tiered system of Master Cell Bank and Working Cell Bank of UC-MSCs were analyzed. RESULTS: Efficient derivation of UC-MSCs was achieved by using UC fragments larger than 7 g that were processed within 80 h from birth. Cells maintained their immunophenotype (being highly positive for CD105, CD90 and CD73 markers), multi-potentiality and immuno-modulative properties beyond 40 cumulative population doublings. No genetic abnormalities were found, as determined by G-banding karyotype, human telomerase reverse transcriptase activity was undetectable and no toxicity was observed in vivo after intravenous administration of UC-MSCs in athymic rats. DISCUSSION: This works demonstrates the feasibility of the derivation and large-scale expansion of UC-MSCs from small and relatively old fragments of UC typically discarded from public cord blood banking programs.


Subject(s)
Cell Culture Techniques/methods , Mesenchymal Stem Cells/cytology , Tissue Banks , Wharton Jelly/cytology , Animals , Cell Proliferation , Cells, Cultured , Humans , Immunophenotyping , Male , Mesenchymal Stem Cells/metabolism , Rats, Nude , Telomerase/metabolism , Tissue Distribution , Umbilical Cord/cytology
4.
Cytotherapy ; 17(8): 1009-14, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25769789

ABSTRACT

Along with academic and charitable organizations, transfusion centers have ventured into the stem cell field, with the aim of testing of novel cell-based therapeutics in a clinical setting for future marketing approval. The fact that quality management structures, which are required for compliance with good scientific practice regulations, were originally designed for product development in corporate environments represents a major challenge for many developers. In this Commentary, challenges that non-pharmaceutical institutions must overcome to translate cell-based products into clinical therapies will be discussed from a quality standpoint. Furthermore, our development experience for a mesenchymal stromal cell-based therapy will be shared as a case study.


Subject(s)
Cell- and Tissue-Based Therapy/methods , Mesenchymal Stem Cell Transplantation/methods , Humans , Mesenchymal Stem Cells/cytology , Quality Control
5.
N Biotechnol ; 31(5): 492-8, 2014 Sep 25.
Article in English | MEDLINE | ID: mdl-25063342

ABSTRACT

Regenerative therapies using adult stem cells have attracted great interest in the recent years and offer a promising alternative to current surgical practices. In this report, we evaluated the safety and efficacy of an autologous cell-based treatment of osteoarthritis using mesenchymal stromal cells expanded from bone marrow aspirates that were administered intra-articularly. Ten 2-year old ewes were divided in two groups (for analysis at 6 and 12 months, respectively). Full thickness articular cartilage defects of approximately 60mm(2) were created arthroscopically in the medial femorotibial condyles and a meniscal tear in the anterior horn of the medial meniscus in the 20 hind legs. Intra-articular injection of 4 mL of either treatment (a suspension of cells) or control (same as treatment, without cells) were applied one month after generating a chronic condition similar to human pathology. Animals were monitored radiographically, by MRI and ultrasound scanning; and macroscopic and histological analyses were conducted at 6 and 12 months. Furthermore a full necropsy was performed at 12 months post-treatment. The intra-articular injection of autologous MSC was safe, as judged by the lack of local or systemic adverse effects during the clinical follow-up and by a full necropsy performed at 12 months post-treatment. Evidence of regeneration of articular cartilage and meniscus was case-dependent but statistically significant improvement was found in specific macroscopic and histological parameters. Such parameters included colour, rigidity, cell distribution and hyaline quality of the refill tissue as well as the structure of subchondral bone.


Subject(s)
Cartilage, Articular/injuries , Knee Injuries/therapy , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Tibial Meniscus Injuries , Animals , Autografts , Cartilage, Articular/diagnostic imaging , Chronic Disease , Disease Models, Animal , Humans , Knee Injuries/diagnostic imaging , Magnetic Resonance Imaging , Menisci, Tibial/diagnostic imaging , Radiography , Sheep
6.
Arch Orthop Trauma Surg ; 132(11): 1611-8, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22821379

ABSTRACT

BACKGROUND: The purpose of this study was to evaluate the efficacy of core decompression associated with advanced cell therapy for the treatment of femoral head osteonecrosis in an established sheep model. METHODS: Early stage osteonecrosis of the right hip was induced cryogenically in 15 mature sheep. At 6 weeks, the sheep were divided into three groups, Group A: core decompression only; Group B: core decompression followed by implantation of an acellular bone matrix scaffold; Group C: core decompression followed by implantation of a cultured BMSC loaded bone matrix scaffold. At 12 weeks, MRI hip studies were performed and then the proximal femur was harvested for histological analysis. RESULTS: In the group of advanced cell therapy, Group C, there was a tendency to higher values of the relative surface of newly formed bone with a mean of 20.3 versus 11.27 % in Group A and 13.04 % in Group B but it was not statistically significant. However, the mean relative volume of immature osteoid was 8.6 % in Group A, 14.97 in Group B, and 53.49 % in Group C (p < 0.05), revealing a greater capacity of osteoid production in the sheep treated with BMSCs. MRI findings were not conclusive due to constant bone edema artifact in all cases. CONCLUSIONS: Our findings indicate that a BMCSs loaded bone matrix scaffold is capable of stimulating bone regeneration more effectively than isolated core decompression or in association with an acellular scaffold in a preclinical femoral head osteonecrosis model in sheep.


Subject(s)
Decompression, Surgical , Femur Head Necrosis/surgery , Mesenchymal Stem Cell Transplantation , Animals , Bone Matrix , Bone Regeneration/physiology , Disease Models, Animal , Magnetic Resonance Imaging , Sheep , Tissue Scaffolds
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