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Life Extension Magazine June 2013

Stem cells

Adult and umbilical cord blood-derived platelet-rich plasma for mesenchymal stem cell proliferation, chemotaxis, and cryo-preservation.

Platelet-rich plasma (PRP) was prepared from human adult peripheral blood and from human umbilical cord (uc) blood and the properties were compared in a series of in vitro bioassays. Quantification of growth factors in PRP and platelet-poor plasma (PPP) fractions revealed increased levels of mitogenic growth factors PDGF-AB, PDGF-BB, and FGF-2, the angiogenic agent VEGF and the chemokine RANTES in ucPRP compared to adult PRP (aPRP) and PPP. To compare the ability of the various PRP products to stimulate proliferation of human bone marrow (BM), rat BM and compact bone (CB)-derived mesenchymal stem cells (MSC), cells were cultured in serum-free media for 4 and 7 days with varying concentrations of PRP, PPP, or combinations of recombinant mitogens. It was found that while all forms of PRP and PPP were more mitogenic than fetal bovine serum, ucPRP resulted in significantly higher proliferation by 7 days than adult PRP and PPP. We observed that addition of as little as 0.1% ucPRP caused greater proliferation of MSC effects than the most potent combination of recombinant growth factors tested, namely PDGF-AB + PDGF-BB + FGF-2, each at 10 ng/mL. Similarly, in chemotaxis assays, ucPRP showed greater potency than adult PRP, PPP from either source, or indeed than combinations of either recombinant growth factors (PDGF, FGF, and TGF-β1) or chemokines previously shown to stimulate chemotactic migration of MSC. Lastly, we successfully demonstrated that PRP and PPP represented a viable alternative to FBS containing media for the cryo-preservation of MSC from human and rat BM.

Biomaterials.2012 Jul;33(21):5308-16

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.

Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.

Cell.2006 Aug 25;126(4):663-76

Clinical transplantation of a tissue-engineered airway.

BACKGROUND: The loss of a normal airway is devastating. Attempts to replace large airways have met with serious problems. Prerequisites for a tissue-engineered replacement are a suitable matrix, cells, ideal mechanical properties, and the absence of antigenicity. We aimed to bioengineer tubular tracheal matrices, using a tissue-engineering protocol, and to assess the application of this technology in a patient with end-stage airway disease. METHODS: We removed cells and MHC antigens from a human donor trachea, which was then readily colonised by epithelial cells and mesenchymal stem-cell-derived chondrocytes that had been cultured from cells taken from the recipient (a 30-year old woman with end-stage bronchomalacia). This graft was then used to replace the recipient’s left main bronchus. FINDINGS: The graft immediately provided the recipient with a functional airway, improved her quality of life, and had a normal appearance and mechanical properties at 4 months. The patient had no anti-donor antibodies and was not on immunosuppressive drugs. INTERPRETATION: The results show that we can produce a cellular, tissue-engineered airway with mechanical properties that allow normal functioning, and which is free from the risks of rejection. The findings suggest that autologous cells combined with appropriate biomaterials might provide successful treatment for patients with serious clinical disorders.

Lancet.2008 Dec 13;372(9655):2023-30

Bioartificial tracheobronchial transplantation. Interview with Paolo Macchiarini.

We caught up with Paolo Macchiarini to find out more about the international collaboration responsible for creating and implanting the world’s first artificial trachea in June 2011. Paolo Macchiarini, Professor at the Karolinska University Hospital and Karolinska Institutet (Stockholm, Sweden), has dedicated his career to improving the outcomes of tracheal transplant patients. He became well known in the regenerative medicine field in 2008 when he performed the first adult stem cell-grown trachea transplant and has since carried out several transplants using decellularized deceased donor trachea reseeded with the patient’s own cells. In June 2011, Macchiarini led an international team to successfully implant an artificial trachea, seeded with autologous stem cells, in a human patient, a world first. The patient, a 36-year-old man with late-stage tracheal cancer, has since made a good recovery.

Regen Med. 2011 Nov;6(6 Suppl):14-5

Tissue-engineered autologous bladders for patients needing cystoplasty.

BACKGROUND: Patients with end-stage bladder disease can be treated with cystoplasty using gastrointestinal segments. The presence of such segments in the urinary tract has been associated with many complications. We explored an alternative approach using autologous engineered bladder tissues for reconstruction. METHODS: Seven patients with myelomeningocele, aged 4-19 years, with high-pressure or poorly compliant bladders, were identified as candidates for cystoplasty. A bladder biopsy was obtained from each patient. Urothelial and muscle cells were grown in culture, and seeded on a biodegradable bladder-shaped scaffold made of collagen, or a composite of collagen and polyglycolic acid. About 7 weeks after the biopsy, the autologous engineered bladder constructs were used for reconstruction and implanted either with or without an omental wrap. Serial urodynamics, cystograms, ultrasounds, bladder biopsies, and serum analyses were done. RESULTS: Follow-up range was 22-61 months (mean 46 months). Post-operatively, the mean bladder leak point pressure decrease at capacity, and the volume and compliance increase was greatest in the composite engineered bladders with an omental wrap (56%, 1.58-fold, and 2.79-fold, respectively). Bowel function returned promptly after surgery. No metabolic consequences were noted, urinary calculi did not form, mucus production was normal, and renal function was preserved. The engineered bladder biopsies showed an adequate structural architecture and phenotype. CONCLUSIONS: Engineered bladder tissues, created with autologous cells seeded on collagen-polyglycolic acid scaffolds, and wrapped in omentum after implantation, can be used in patients who need cystoplasty.

Lancet.2006 Apr 15;367(9518):1241-6

Paths to stemness: building the ultimate antitumour T cell.

Stem cells are defined by the ability to self-renew and to generate differentiated progeny, qualities that are maintained by evolutionarily conserved pathways that can lead to cancer when deregulated. There is now evidence that these stem cell-like attributes and signalling pathways are also shared among subsets of mature memory T lymphocytes. We discuss how using stem cell-like T cells can overcome the limitations of current adoptive T cell therapies, including inefficient T cell engraftment, persistence and ability to mediate prolonged immune attack. Conferring stemness to antitumour T cells might unleash the full potential of cellular therapies.

Nat Rev Cancer.2012 Oct;12(10):671-84

Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction.

BACKGROUND: Pilot trials suggest that the intracoronary administration of autologous progenitor cells may improve left ventricular function after acute myocardial infarction. METHODS: In a multicenter trial, we randomly assigned 204 patients with acute myocardial infarction to receive an intracoronary infusion of progenitor cells derived from bone marrow (BMC) or placebo medium into the infarct artery 3 to 7 days after successful reperfusion therapy. RESULTS: At 4 months, the absolute improvement in the global left ventricular ejection fraction (LVEF) was significantly greater in the BMC group than in the placebo group (mean [+/-SD] increase, 5.5+/-7.3% vs. 3.0+/-6.5%; P=0.01). Patients with a baseline LVEF at or below the median value of 48.9% derived the most benefit (absolute improvement in LVEF, 5.0%; 95% confidence interval, 2.0 to 8.1). At 1 year, intracoronary infusion of BMC was associated with a reduction in the prespecified combined clinical end point of death, recurrence of myocardial infarction, and any revascularization procedure (P=0.01). CONCLUSIONS: Intracoronary administration of BMC is associated with improved recovery of left ventricular contractile function in patients with acute myocardial infarction. Large-scale studies are warranted to examine the potential effects of progenitor-cell administration on morbidity and mortality.

N Engl J Med. 2006 Sep 21;355(12):1210-21

A degradable, bioactive, gelatinized alginate hydrogel to improve stem cell/growth factor delivery and facilitate healing after myocardial infarction.

Despite remarkable effectiveness of reperfusion and drug therapies to reduce morbidity and mortality following myocardial infarction (MI), many patients have debilitating symptoms and impaired left ventricular (LV) function highlighting the need for improved post-MI therapies. A promising concept currently under investigation is intramyocardial injection of high-water content, polymeric biomaterial gels (e.g., hydrogels) to modulate myocardial scar formation and LV adverse remodeling. We propose a degradable, bioactive hydrogel that forms a unique microstructure of continuous, parallel capillary-like channels (Capgel). We hypothesize that the innovative architecture and composition of Capgel can serve as a platform for endogenous cell recruitment and drug/cell delivery, therefore facilitating myocardial repair after MI.

Med Hypotheses.2012 Nov;79(5):673-7

Adipose-derived mesenchymal stromal cells for chronic myocardial ischemia (MyStromalCell Trial): study design.

Adipose tissue represents an abundant, accessible source of multipotent adipose-derived stromal cells (ADSCs). Animal studies have suggested that ADSCs have the potential to differentiate in vivo into endothelial cells and cardiomyocytes. This makes ADSCs a promising new cell source for regenerative therapy to replace injured tissue by creating new blood vessels and cardiomyocytes in patients with chronic ischemic heart disease. The aim of this special report is to review the present preclinical data leading to clinical stem cell therapy using ADSCs in patients with ischemic heart disease. In addition, we give an introduction to the first-in-man clinical trial, MyStromalCell Trial, which is a prospective, randomized, double-blind, placebo-controlled study using culture-expanded ADSCs obtained from adipose-derived cells from abdominal adipose tissue and stimulated with VEGF-A(165) the week before treatment.

Regen Med. 2012 May;7(3):421-8

Preclinical derivation and imaging of autologously transplanted canine induced pluripotent stem cells.

Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.

J Biol Chem. 2011 Sep 16;286(37):32697-704