Susanne Kern

Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue

Mesenchymal stem cells (MSCs) represent a promising tool for new clinical concepts in supporting cellular therapy. Bone marrow (BM) was the first source reported to contain MSCs. However, for clinical use, BM may be detrimental due to the highly invasive donation procedure and the decline in MSC number and differentiation potential with increasing age. More recently, umbilical cord blood (UCB), attainable by a less invasive method, was introduced as an alternative source for MSCs. Another promising source is adipose tissue (AT). We compared MSCs derived from these sources regarding morphology, the success rate of isolating MSCs, colony frequency, expansion potential, multiple differentiation capacity, and immune phenotype. No significant differences concerning the morphology and immune phenotype of the MSCs derived from these sources were obvious. Differences could be observed concerning the success rate of isolating MSCs, which was 100% for BM and AT, but only 63% for UCB. The colony frequency was lowest in UCB, whereas it was highest in AT. However, UCB‐MSCs could be cultured longest and showed the highest proliferation capacity, whereas BM‐MSCs possessed the shortest culture period and the lowest proliferation capacity. Most strikingly, UCB‐MSCs showed no adipogenic differentiation capacity, in contrast to BM‐ and AT‐MSCs. Both UCB and AT are attractive alternatives to BM in isolating MSC: AT as it contains MSCs at the highest frequency and UCB as it seems to be expandable to higher numbers.

Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood.

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However, attempts to isolate MSCs from umbilical cord blood (UCB) of full‐term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi‐lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria.

Human AB Serum and Thrombin‐Activated Platelet‐Rich Plasma Are Suitable Alternatives to Fetal Calf Serum for the Expansion of Mesenchymal Stem Cells from Adipose Tissue

MSCs are currently in focus regarding their clinical potential in cell therapy and tissue engineering. However, most isolation and expansion protocols for clinical‐scale production of MSCs use fetal calf serum (FCS) as a supplement, which poses a potential risk for infections as well as immunological reactions. To find a suitable FCS substitute, we investigated the effects of pooled human AB serum (AB‐HS) and thrombin‐activated platelet‐rich plasma (tPRP) on adipose tissue MSCs (AT‐MSCs) with FCS as the standard control medium. AT‐MSCs of 10 donors were cultured under three different conditions: (a) 10% FCS, (b) 10% AB‐HS, and (c) 10% tPRP. Colony‐forming units, cumulative population doubling rates, and differentiation capacity toward the adipogenic and osteogenic lineages were assessed, along with immunophenotype. We demonstrated that AB‐HS and tPRP provide a significantly higher proliferative effect on AT‐MSCs than does FCS. In the first six passages, AB‐HS and tPRP MSCs exhibited a fold expansion of 66.6 ± 15.7 and 68.1 ± 6.7, respectively, compared with 24.4 ± 0.7 for FCS. Differentiation capacity was preserved throughout long‐term culture. Immunophenotype was characteristic for MSCs and comparable for all culture conditions with the exception of a distinct CD45‐/CD14‐positive side population for AB‐HS and tPRP that tended to diminish with prolonged culture. We showed that pooled human AB serum and thrombin‐activated platelet‐rich plasma are alternatives to FCS for AT‐MSCs. These human sources are better characterized regarding potential infectious threats, while providing a higher proliferation rate and retaining differentiation capacity and mesenchymal stem cell marker expression throughout long‐term culture.

Engraftment capacity of umbilical cord blood cells processed by either whole blood preparation or filtration.

Umbilical cord blood (UCB) preparation needs to be optimized in order to develop more simplified procedures for volume reduction, as well as to reduce the amount of contaminating cells within the final stem cell transplant. We evaluated a novel filter device (StemQuick™E) and compared it with our routine buffy coat (BC) preparation procedure for the enrichment of hematopoietic progenitor cells (HPCs). Two groups of single or pooled UCB units were filtered (each n = 6), or equally divided in two halves and processed by filtration and BC preparation in parallel (n = 10). The engraftment capacity of UCB samples processed by whole blood (WB) preparation was compared with paired samples processed by filtration in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse animal model. Filtration of UCB units in the two groups with a mean volume of 87.8 and 120.7 ml, respectively, and nucleated cell (NC) content of 9.7 and 23.8 × 108 resulted in a sufficient mean cell recovery for mononucleated cells ([MNCs] 74.2%‐77.5%), CD34+ cells (76.3%‐79.0%), and colony‐forming cells (64.1%‐86.3%). Moreover, we detected a relevant depletion of the transplants for RBCs (89.2%‐90.0%) and platelets ([PLTs] 77.5%‐86.1%). In contrast, the mean depletion rate using BC processing proved to be significantly different for PLTs (10%, p = 0.03) and RBCs (39.6%, p < 0.01). The NC composition showed a highly significant increase in MNCs and a decrease in granulocytes after filtration (p < 0.01), compared with a less significant MNC increase in the BC group (p < 0.05). For mice transplanted with WB‐derived progenitors, we observed a mean of 15.3% ± 15.5% of human CD45+ cells within the BM compared with 19.9% ± 16.8% for mice transplanted with filter samples (p = 0.03). The mean percentage of human CD34+ cells was 4.2% ± 3.1% for WB samples and 4.5% ± 3.2% for filter samples (p = 0.68). As the data of NOD/SCID mice transplantation demonstrated a significant engraftment capacity of HPCs processed by filtration, no negative effect on the engraftment potential of filtered UCB cells versus non‐volume‐reduced cells from WB transplants was found. The StemQuick™E filter devices proved to be a useful tool for Good Manufacturing Practices conform enrichment of HPCs and MNCs out of UCB. Filtration enables a quick and standardized preparation of a volume‐reduced UCB transplant, including a partial depletion of granulocytes, RBCs, and PLTs without the need for centrifugation. Therefore, it seems very probable that filter‐processed UCB transplants will also result in sufficient hematopoietic reconstitution in humans.

The Formation of Extracellular Matrix During Chondrogenic Differentiation of Mesenchymal Stem Cells Correlates with Increased Levels of Xylosyltransferase I

In vitro differentiation of mesenchymal stem cells (MSCs) into chondrogenic cells and their transplantation is promising as a technique for the treatment of cartilaginous defects. But the regulation of extracellular matrix (ECM) formation remains elusive. Therefore, the objective of this study was to analyze the regulation of proteoglycan (PG) biosynthesis during the chondrogenic differentiation of MSCs. In different stages of chondrogenic differentiation, we analyzed mRNA and protein expression of key enzymes and PG core proteins involved in ECM development. For xylosyltransferase I (XT‐I), we found maximum mRNA levels 48 hours after chondrogenic induction with a 5.04 ± 0.58 (mean ± SD)‐fold increase. This result correlates with significantly elevated levels of enzymatic XT‐I activity (0.49 ± 0.03 μU/1 × 106 cells) at this time point. Immunohistochemical staining of XT‐I revealed a predominant upregulation in early chondrogenic stages. The highly homologous protein XT‐II showed 4.7‐fold (SD 0.6) increased mRNA levels on day 7. To determine the differential expression of heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) chains, we analyzed the mRNA expression of EXTL2 (α‐4‐N‐acetylhexosaminyltransferase), GalNAcT (β‐1,4‐N‐acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5 epimerase). All key enzymes showed a similar regulation with temporarily downregulated mRNA levels (up to −87‐fold) after chondrogenic induction. In accordance to previous studies, we observed a similar increase in the expression of PG core proteins. In conclusion, we could show that key enzymes for CS, DS, and HS synthesis, especially XT‐I, are useful markers for the developmental stages of chondrogenic differentiation.

Lipoaspirate-derived adult mesenchymal stem cells improve functional outcome during intracerebral hemorrhage by proliferation of endogenous progenitor cells stem cells in intracerebral hemorrhages.

Stem cell therapy seems promising in reducing deficits after focal cerebral ischemia. As stroke may result from intracerebral hemorrhage (ICH) in up to 20% we investigated whether human processed lipoaspirate mesenchymal stem cells (PLA-MSC) influence the functional outcome, migration behavior and the activation of endogenous progenitor cells. Experimental ICH was induced by stereotactic administration of collagenase in rats randomly assigned to the control or treatment group. The latter received 3 x 10(6) PLA-MSC by intravenous (i.v.) injection 24h after ICH induction. The outcome was continuously monitored using the RotaRod test over a period of 4 weeks. Morphometric analysis of ICH was performed consecutively by magnetic resonance imaging (MRI) studies and immunohistochemical analysis. The RotaRod test revealed a significant 1.5-fold improvement (p<0.005) in functional outcome for the PLA-MSC treated group after 4 weeks compared to controls. Histological and MRI assessment of lesion size showed no difference between the two groups. Although i.v. injected human cells could not be detected in the post mortem brain, evaluation of the number of endogenous progenitor cells revealed a twofold increase in the treated animals compared to controls. Treatment with PLA-MSC improved the functional outcome significantly in an experimental ICH model. This effect was achieved by stimulation of endogenous progenitor cells rather than integration and differentiation of the infused PLA-MSC.

Induction of retinal pigment epithelium properties in ciliary margin progenitor cells

Purpose:  Degenerative processes in the retinal pigment epithelium (RPE) are known to play a pivotal role in the development of age‐related maculopathy. Substitute RPE analogue cells could be used to preserve visual function. In this paper we investigate methods for the isolation, cultivation and RPE differentiation of undifferentiated cells from the ciliary marginal zone (CMZ) of rat eyes.

Influence of S9 mix composition on the SOS response in Escherichia coli PQ37 by polycyclic aromatic hydrocarbons

To investigate the variability in test results obtained with the SOS chromotest (Escherichia coli PQ37 genotoxicity assay) when varying the composition of the exogenous metabolizing system (S9 mix), we examined the influence of different S9 and NADP concentrations, of buffer pH value, of SDS concentrations, the effects of E. coli PQ37 density and centrifugation steps on the expression of beta-galactosidase (beta g) and alkaline phosphatase (ap) activity, the calculated induction factors (IFs) and SOS-inducing potencies (SOSIPs). Additionally we examined the metabolic potency (stability) of S9 mix when stored at 37 degrees C before use. Initially, we used 0-5000 ng (= 0-20 nmole) benzo[a]pyrene (B[a]P) as a reference compound for the test procedure in the presence of standard S9 mix. Subsequently, to evaluate the results of S9 mix variations we examined several polycyclic aromatic hydrocarbons (PAHs) using both the standard and a modified S9 mix composition and test protocol. We observed the highest beta g and ap activities and/or IFs using only 11-27 microliters 9000 x g liver supernatant (S9) from Aroclor 1254-induced rats per assay (20-50% of standard amount) and calibrating the S9 mix Tris buffer to pH 7.8-8.0. 60-300 micrograms NADP/assay (10-50% of standard) was sufficient for optimum activation of PAHs. In contrast to previous investigations about the variability of the SOS chromotest in the absence of a metabolizing system, higher induction factors were obtained when using higher bacterial densities (12-18 x 10(6) cfu/assay). Centrifugation steps as recommended by other investigators were not necessary when using optimum S9 amounts. The metabolic activity of S9 mix remained nearly constant approximately 20 min after preparation, but decreased to 80% of its activity in about 1 h.