Biomedical subjects

Norbert Stute

Publications and source records attributed to Norbert Stute.

6 recordsLinked to original sources

PubMed2005

Reduction of myocardial scar size after implantation of mesenchymal stem cells in rats: what is the mechanism?

The use of a cellular therapy offers a promising approach for the treatment of heart disease. Besides other precursor cells, bone marrow (BM)-derived stem cells were discovered that migrate into ischemic myocardium and participate in myogenesis as well as angiogenesis. A subpopulation of those are the mesenchymal stem cells (MSC), which may be potential candidates for repairing ischemic heart tissue. MSC are easy to prepare and can be used in an autologous strategy. Here we demonstrate the effect of transplanted MSC in our autologous rat model of myocardial injury. BM was isolated from tibiae and femurs of Wistar rats. After 24 h, the adhering MSC were separated, expanded, retrovirally transduced using green fluorescent protein (GFP), and cloned. A cryo-infarct was generated in the rat hearts, and immediately after this the cells were injected into the border zone of the lesion. After a 10-week follow up, the hearts were excised and the myocardial scar areas were measured using computer-guided morphometry. When comparing transplanted rats (n = 8) with control animals (n = 5) treated rats demonstrated a significant reduction in the width (p < 0.05) of the myocardial scar area. The depth of the scars of the cell therapy rats was less extended (p > 0.05) and the myocardium of these animals was thicker than in the controls (p > 0.05). Immunohistochemical analyses revealed neither evidence of MSC transdifferentiation into cardiomyocytes, nor could an increased neovascularization be found. In conclusion, MSC are responsible for a remarkable reduction of the myocardial scar size in the treated animals. But, whether this strategy is directly transferable to the patient suffering from heart disease has to be determined. In addition, the mechanism by which MSC act in the ischemic heart remains to be determined.

Animals↗

PubMed2005

High-potential human mesenchymal stem cells.

Bone marrow-derived stromal mesenchymal stem cells (MSCs) have been characterized in vitro by their growth characteristics, the expression of a panel of surface antigens, and their potential to differentiate into mesenchymal lineages. They can be separated by physical methods as well as by immunological or chemical separation or cultivation. Different protocols are used in different laboratories, making the comparison of various reported MSC populations difficult. Here we describe a population of bone marrow-derived adult stem cells that has been separated on a Percoll gradient with low density. It is characterized by an extraordinary high proliferative potential and a conserved phenotype characteristic of MSCs that retain their plutipotentiality in culture, as evidenced by their ability to differentiate into osteo-, chondro-, and adipogenic lineages. Separation of these cells provide an effective and convenient method for rapid expansion of pluripotential human MSCs for clinical use where large amounts of stem cells are needed.

Adipocytes↗

PubMed2005

Pilot study of reduced-intensity conditioning followed by allogeneic stem cell transplantation from related and unrelated donors in patients with myelofibrosis.

A prospective pilot study was performed to evaluate the effect of reduced-intensity conditioning with busulphan (10 mg/kg), fludarabine (180 mg/qm) and anti-thymocyte globulin followed by allogeneic stem cell transplantation from related (n = 8) and unrelated donors (n = 13) in 21 patients with myelofibrosis. The median age of the patients was 53 years (range, 32-63). No primary graft failure occurred. The median time until leucocyte (>1.0 x 10(9)/l) and platelet (>20 x 10(9)/l) engraftment was 16 (range, 11-26) and 23 d (range, 9-139) respectively. Complete donor chimaerism on day 100 was seen in 20 patients (95%). Acute graft-versus-host disease (GvHD) grades II-IV and III/IV occurred in 48% and 19% of cases and 55% of the patients had chronic GvHD. Treatment-related mortality was 0% at day 100 and 16% [95% confidence interval (CI): 0-32%] at 1 year. Haematological response was seen in 100% and complete histopathological remission was observed in 75% of the patients and 25% of the patients showed partial histopathological remission with a continuing decline in the grade of fibrosis. After a median follow-up of 22 months (range, 4-59), the 3-year estimated overall and disease-free survival was 84% (95% CI: 67-100%).

Adult↗

PubMed2004

Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use.

OBJECTIVE: Mesenchymal stem cells (MSC) are promising candidates for cell-based therapies. One major obstacle for their clinical use is the biosafety of fetal calf serum (FCS), which is a crucial part of all media currently used for the culture of MSC. METHODS: Nine donors each contributed 5 mL of bone marrow aspirate. Isolation of MSC was conducted according to Caplan et al., although for expansion we used low-density seeding with 20 MSC/cm2. Four different media A, B, C, and D were tested, containing 1%, 3%, or 10% autologous serum (AS), or 10% selected FCS, respectively. MSC were cultured on 24-well plates until passage 2 and counted under the microscope at regular intervals. Osteogenic and adipogenic differentiation were induced in vitro by using a modified standard cocktail and were evaluated semi-quantitatively through a microscope. RESULTS: Isolation of MSC after 3 days appeared best in media C with almost always C>D congruent with B>A. Proliferation was exponential with generally C>D>B>A. Morphologically, MSC isolated and expanded in medium C were indistinguishable from those in medium D. Phenotypic markers of MSC grown in medium C were: CD34-, CD45-, CD90+, CD105+, MHC class I+, MHC class II-, similar to MSC isolated and grown in medium D. Moreover, MSC grown in medium C showed more osteogenic potential than those from medium D in all cases: C+++, D++, B+, A 0. Cells retained their immaturity as shown by adipogenic differentiation and it always was: D+++, C++, B+, A 0. CONCLUSIONS: Growth of MSC in a FCS-free medium is feasible without addition of growth factors. Ten percent AS appears at least as good as 10% FCS with regard to both isolation and expansion of human MSC, while 1% and 3% AS appear inferior. With respect to osteogenic differentiation, 10% AS proved superior to the other serum conditions.

Adipocytes↗

PubMed2002

Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels.

Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. (c)2002 Elsevier Science (USA).

Cell Culture Techniques↗

PubMed2002

Human mesenchymal stem cells are not of donor origin in patients with severe aplastic anemia who underwent sex-mismatched allogeneic bone marrow transplant.

Stromal defects are part of the etiology of severe aplastic anemia (SAA), and hematopoietic engraftment is poor in unrelated and mismatched transplant. Therefore, we wanted to find out whether human mesenchymal stem cells (MSC) are partly of donor origin in patients with SAA years after successful bone marrow transplant (BMT). Three SAA patients 3, 5, and 8 years after BMT (cyclophosphamide, ATG) with bone marrow from an HLA-identical sibling donor of the opposite sex were investigated. MSC were grown from patients' bone marrow aspirates according to Caplan et al. The number of MSC that were isolated from SAA bone marrow post transplant was about 10 times lower than in normal controls. Primary cultures of adherent MSC and passage-one cells were analyzed by dual-color interphase fluorescence in situ hybridization (FISH) analysis using centromere-specific DNA probes for X and Y chromosome. FISH did not show any clear evidence of donor cells in the adherent MSC: In all cases, less than 0.5% of nuclei showed a donor-type signal pattern that is well within assay limits. In a female patient, the absence of male donor cells was confirmed by sensitive and quantitative, Y chromosome-specific TaqMan PCR (QYCS-PCR). In contrast, Ficoll-separated hematopoietic cells from the same aspirates were greater than 90% of donor origin, as expected. In SAA, as previously found in patients with lysosomal and peroxisomal storage disease, bone marrow MSC remain host-derived despite successful hematopoietic engraftment years after allogeneic BMT.

Adult↗