Zähldatenmodelle (Count Data Models): Ansätze und Anwendungen

Die Arbeit umfaßt die Darstellung und Anwendung von Regressionsmodellen für Zähldaten, deren abhängige Variable nur nichtnegative ganzzahlige Werte annehmen kann. Die Spezifikationen des Poisson- und des NegBin 2-Modells mit den zugehörigen Schätzverfahren und Beurteilungskriterien werden für den Querschnittsdaten-Bereich vorgestellt. Als Entscheidungshilfe für das eine oder andere Modell werden Tests der Daten auf Overdispersion herangezogen. Im empirischen Tell der Arbeit bestätigt sich der m der Literatur dargestellte stark positive Zusammenhang zwischen den Patentanmeldungen und den FuE-Beschäftigten bzw. den FuE-Ausgaben. Dabei wird angesichts zu großer Streuung der Daten das NegBin 2-Modell dem Poisson-Modell vorgezogen. -- This paper deals with the description and application of regression models for count data where the dependent variable takes only nonnegative integer values. The specification of the Poisson- and the NegBin 2-Model with their estimation procedures and goodness-of-fit- statistics are presented for cross-section data. To co me to a decision between the two models tests for overdispersed data are used. The strong positive relationship between patent applications and the R&D personnel respectively R&D expenditures which is presented in the literature could be confirmed with this application. Because of overdispersion in the data the NegBin2-Model can be preferred to the Poisson-Model.

Reduced Reactivation from Dormancy but Maintained Lineage Choice of Human Mesenchymal Stem Cells with Donor Age

Mesenchymal stem cells (MSC) are promising for cell-based regeneration therapies but up to date it is still controversial whether their function is maintained throughout ageing. Aim of this study was to address whether frequency, activation in vitro, replicative function, and in vitro lineage choice of MSC is maintained throughout ageing to answer the question whether MSC-based regeneration strategies should be restricted to younger individuals. MSC from bone marrow aspirates of 28 donors (5–80 years) were characterized regarding colony-forming unit-fibroblast (CFU-F) numbers, single cell cloning efficiency (SSCE), osteogenic, adipogenic and chondrogenic differentiation capacity in vitro. Alkaline phosphatase (ALP) activity, mineralization, Oil Red O content, proteoglycan- and collagen type II deposition were quantified. While CFU-F frequency was maintained, SSCE and early proliferation rate decreased significantly with advanced donor age. MSC with higher proliferation rate before start of induction showed stronger osteogenic, adipogenic and chondrogenic differentiation. MSC with high osteogenic capacity underwent better chondrogenesis and showed a trend to better adipogenesis. Lineage choice was, however, unaltered with age. Conclusion: Ageing influenced activation from dormancy and replicative function of MSC in a way that it may be more demanding to mobilize MSC to fast cell growth at advanced age. Since fast proliferation came along with high multilineage capacity, the proliferation status of expanded MSC rather than donor age may provide an argument to restrict MSC-based therapies to certain individuals

Generation time of MSC of the three donor age groups at passage 1–4 with results show mean values ± standard deviation.

#<p>sig. different to young and middle old group, p<0.05.</p

Outcome of age-relation for distinct parameters in different studies.

<p>*No. of samples used for age correlation.</p><p>→ no effect, ↓ decrease with age, ↑ increase with age.</p><p>f = female, m = male.</p

No correlation of chondrogenic differentiation with donor age and gender.

<p>5×10⁵ MSC were subjected to six weeks of chondrogenic induction in high density culture. From 6 parallel pellets per donor, 2 pellets were used for histological stainings, 2 pellets were processed for quantification of glycosaminoglycan deposition and 2 pellets for quantification of collagen type II deposition. (A) Immunohistochemical staining for collagen type II was used to confirm chondrogenic differentiation and the chondrocyte-like morphology of the cells. Staining varied from full to negative depending on the donor with similar variability in all age groups (A, inset). (B) The collagen type II content determined after pepsin digestion of pellets by ELISA revealed a weak trend of reduced chondrogenesis at older age. (C) Glycosaminoglycan deposition determined after staining the pellets with Alcian blue dye, washing and extracting the dye revealed a trend of reduced chondrogenesis at older age. (D) Collagen type II deposition in pellets from female and male donors showed no significant differences. Correlation was calculated by Spearman-Rho Test, group comparison by Mann-Whitney-U Test.</p

Osteogenic and adipogenic differentiation potential was independent of donor age.

<p>Differentiation of MSC (passage 3) was induced with adipogenic or osteogenic induction medium for 21 days in monolayer. (A) Adipogenesis of good (donor a) and impaired MSC (donor b) was detected by Oil Red O staining of the lipid vacuoles and (B) measured by extraction of bound Oil Red O dye. (C,D) Osteogenic in vitro potential was evaluated by quantification of mineral deposition by dye extraction after Alizarin Red staining. (E) Alkaline phosphatase activity of cell lysates was determined using the pNP (para-Nitrophenylphosphate) substrate. No significant correlation of age and differentiation capacity was observed. Correlation was calculated by Spearman-Rho Test. (F) Gene expression of the osteogenic marker Integrin-binding sialoprotein (IBSP) and (G) the adipogenic marker Peroxisome proliferator-activated receptor gamma (PPARG) both normalized to the housekeeping gene GAPDH. Data are given as x-fold regulation compared to undifferentiated control MSC of the same donors. Five of the youngest and the oldest donors of the whole collective were used for analysis in RT-PCR. No difference for both markers was observed between the two age groups (Mann-Whitney-U-Test).</p

Single cell cloning efficiency at passage 0 and generation time at passage 2 and 3 correlate with donor age.

<p>(A) Clonal expandability of minimally expanded MSC was evaluated by single cell cloning for MSC from 25 donors. Fourteen days after seeding of statistically one single cell per well into two 96-well plates, wells containing >30 cells were counted as single cell clones. The clonal expandability decreased significantly with increasing donor age. (B,C) Non-clonal MSC populations were expanded and generation time, specified as the time needed for one population doubling, was recorded. Proliferation rate of MSC from passage 2 (B) and passage 3 (C) showed a significant correlation with donor age. Correlation was calculated by Spearman-Rho Test and p<0.05 was considered as statistically significant.</p

Correlation of proliferation rate and differentiation capacity.

<p>(A) Osteogenic and (B) adipogenic differentiation of MSC (passage 3) were induced for 21 days in monolayer. (A) ALP enzyme activity and (B) amount of extracted Oil Red O dye were plotted against generation time of the expanded cells at start of differentiation. (C,D) Proteoglycan and collagen type II deposition of high density pellets were plotted against generation time of the expanded cells at start of differentiation. All four differentiation parameters showed a significant negative correlation with generation time suggesting a reduced differentiation capacity of more slowly growing cells. (E) Chondrogenic differentiation capacity (collagen type II deposition) revealed a strong positive correlation with osteogenic differentiation capacity (ALP activity). (F) Additionally a trend for better adipogenesis (Oil Red O staining) at enhanced osteogenesis (ALP actitivity) almost reached significance. Correlation was calculated by Spearman-Rho Test and p<0.05 was considered as statistically significant. □ donor a in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022980#pone-0022980-g003" target="_blank">Fig. 3A</a>, ◯ donor b in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022980#pone-0022980-g003" target="_blank">Fig. 3A</a>, <b>⋄</b> donor c in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022980#pone-0022980-g004" target="_blank">Fig. 4A</a>, <b>▵</b> donor e in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022980#pone-0022980-g004" target="_blank">Fig. 4A</a>.</p