Gesine Kögler

Functional differences between mesenchymal stem cell populations are reflected by their transcriptome.

Stem cells are widely studied to enable their use in tissue repair. However, differences in function and differentiation potential exist between distinct stem cell populations. Whether those differences are due to donor variation, cell culture, or intrinsic properties remains elusive. Therefore, we compared 3 cell lines isolated from 3 different niches using the Affymetrix Exon Array platform: the cord blood-derived neonatal unrestricted somatic stem cell (USSC), adult bone marrow-derived mesenchymal stem cells (BM-MSC), and adult adipose tissue-derived stem cells (AdAS). While donor variation was minimal, large differences between stem cells of different origin were detected. BM-MSC and AdAS, outwardly similar, are more closely related to each other than to USSC. Interestingly, USSC expressed genes involved in the cell cycle and in neurogenesis, consistent with their reported neuronal differentiation capacity. The BM-MSC signature indicates that they are primed toward developmental processes of tissues and organs derived from the mesoderm and endoderm. Remarkably, AdAS appear to be highly enriched in immune-related genes. Together, the data suggest that the different mesenchymal stem cell types have distinct gene expression profiles, reflecting their origin and differentiation potential. Furthermore, these differences indicate a demand for effective differentiation protocols tailored to each stem cell type.

Unrestricted somatic stem cells from human umbilical cord blood can be differentiated into neurons with a dopaminergic phenotype.

Recently, it has been shown that human unrestricted somatic stem cells (USSCs) from umbilical cord blood represent pluripotent, neonatal, nonhematopoietic stem cells with the potential to differentiate into the neural lineage. However, molecular and functional characterization of the neural phenotype and evaluation of the degree of maturity of the resulting cells are still lacking. In this study, we addressed the question of neuronal differentiation and maturation induced by a defined composition of growth and differentiation factors (XXL medium). We demonstrated the expression of different neuronal markers and their enrichment in USSC cultures during XXL medium incubation. Furthermore, we showed enrichment of USSCs expressing tyrosine hydroxylase (TH), an enzyme specific for dopaminergic neurons and other catecholamine-producing neurons, accompanied by induction of Nurr1, a factor regulating dopaminergic neurogenesis. The functionality of USSCs has been analyzed by patch-clamp recordings and high-performance liquid chromatography (HPLC). Voltage-gated sodium-channels could be identified in laminin-predifferentiated USSCs. In addition, HPLC analysis revealed synthesis and release of the neurotransmitter dopamine by USSC-derived cells, thus correlating well with the detection of TH transcripts and protein. This study provides novel insight into the potential of unrestricted somatic stem cells from human umbilical cord blood to acquire a neuronal phenotype and function.

MicroRNA hsa-miR-135b regulates mineralization in osteogenic differentiation of human unrestricted somatic stem cells.

Unrestricted somatic stem cells (USSCs) have been recently identified in human umbilical cord blood and have been shown to differentiate into lineages representing all 3 germ layers. To characterize microRNAs that may regulate osteogenic differentiation of USSCs, we carried out expression analysis for 157 microRNAs using quantitative RT-PCR before and after osteogenic induction (t = 0.5, 24, 72, 168, 216 h). Three microRNAs, hsa-miR-135b, hsa-miR-224, and hsa-miR-31, were consistently down-regulated during osteogenesis of USSC line 1. Hsa-miR-135b was shown to be the most profoundly down-regulated in osteogenesis of USSC line 1 and further confirmed to be down-regulated in the osteogenic differentiation of 2 additional USSC lines. Function of hsa-miR-135b in osteogenesis of USSCs was examined by retroviral overexpression, which resulted in an evident decreased mineralization, indicating that hsa-miR-135b down-regulation is functionally important for full osteogenic differentiation of USSCs. MicroRNAs have been shown to regulate negatively expression of their target gene(s). To identify putative targets of hsa-miR-135b, we performed cDNA microarray expression analysis. We selected in total 10 transcripts that were down-regulated (>or=2-fold) in response to hsa-miR-135b overexpression at day 7 and day 9 of osteogenic differentiation. The function of most of these targets in human osteogenesis is unknown and requires further investigation. Markedly, quantitative RT-PCR data showed decreased expression of osteogenic markers IBSP and Osterix, both known to be involved in bone mineralization, in osteogenesis of USSCs that overexpress hsa-miR-135b. This finding suggests that hsa-miR-135b may control osteoblastic differentiation of USSCs by regulating expression of bone-related genes.

Congenital Infections with Human Herpesvirus 6

Cord blood DNA was tested for the presence of human herpesvirus 6 (HHV-6) DNA by the polymerase chain reaction. Specific DNA could be detected in the specimens of 5 (1.6%) of 305 babies born to ostensibly healthy mothers, indicating that intrauterine infection had occurred. These transmissions would not have been detected by serologic methods, because no specific IgM antibody could be found in the fetal sera. These results indicate that, in addition to infections acquired in early childhood, congenital infections may account for the HHV-6 seropositivity in children.

Distinct differentiation potential of "MSC" derived from cord blood and umbilical cord: are cord-derived cells true mesenchymal stromal cells?

Mesenchymal stromal cells (MSC) with distinct differentiation properties have been reported in many adult [eg, bone marrow (BM)] or fetal tissues [eg, cord blood (CB); umbilical cord (UC)] and are defined by their specific surface antigen expression and multipotent differentiation potential. The MSC identity of these cells should be validated by applying well-defined readout systems if a clinical application is considered. In order to determine whether cells isolated from human UC fulfill the criteria defined for MSC, the immunophenotype and differentiation potential including gene expression analysis of the most relevant lineage-specific markers were analyzed in the presented report in combination with the HOX-gene expression. Cells from the UC do not differentiate into osteoblasts demonstrated by Alizarin Red and Von Kossa staining in addition to real-time polymerase chain reaction (PCR)-analysis of runt-related transcription factor 2, bone sialoprotein, osteocalcin, osterix, bone morphogenetic proteins 2 and 4. Oil Red O staining as well as PCR analysis of peroxisome proliferator-activated receptor-gamma, fatty acid-binding protein 4, and perilipin revealed an absent adipogenic differentiation. The lack of potential to differentiate into chondrocytes was documented by Alcian-Blue periodic acid-Schiff, Safranin O staining, and real-time PCR analysis of SOX9. Furthermore, neither endothelial nor myogenic differentiation was documented after induction of UC-MSC. In comparison to CB- and BM-derived cells, UC cells revealed an absent trilineage differentiation capacity in vitro. Therefore, these cells should not be termed mesenchymal stromal cells. The UC cells can be distinguished from CB- and BM-derived cells as well as from pericytes and foreskin fibroblasts by the expression of HOX-genes and the cell surface antigens CD56 and CD146.

Hepatocyte Growth Factor/c-MET Axis-mediated Tropism of Cord Blood-derived Unrestricted Somatic Stem Cells for Neuronal Injury

An under-agarose chemotaxis assay was used to investigate whether unrestricted somatic stem cells (USSC) that were recently characterized in human cord blood are attracted by neuronal injury in vitro. USSC migrated toward extracts of post-ischemic brain tissue of mice in which stroke had been induced. Moreover, apoptotic neurons secrete factors that strongly attracted USSC, whereas necrotic and healthy neurons did not. Investigating the expression of growth factors and chemokines in lesioned brain tissue and neurons and of their respective receptors in USSC revealed expression of hepatocyte growth factor (HGF) in post-ischemic brain and in apoptotic but not in necrotic neurons and of the HGF receptor c-MET in USSC. Neuronal lesion-triggered migration was observed in vitro and in vivo only when c-MET was expressed at a high level in USSC. Neutralization of the bioactivity of HGF with an antibody inhibited migration of USSC toward neuronal injury. This, together with the finding that human recombinant HGF attracts USSC, document that HGF signaling is necessary for the tropism of USSC for neuronal injury. Our data demonstrate that USSC have the capacity to migrate toward apoptotic neurons and injured brain. Together with their neural differentiation potential, this suggests a neuroregenerative potential of USSC. Moreover, we provide evidence for a hitherto unrecognized pivotal role of the HGF/c-MET axis in guiding stem cells toward brain injury, which may partly account for the capability of HGF to improve function in the diseased central nervous system.

Clonal culture of fetal cells from maternal blood

Successful isolation and genetic testing of fetal cells obtained from maternal blood could eliminate the risks associated with invasive prenatal testing. We used clonal in-vitro expansion of fetal haemopoietic cells and micromanipulation and fluorescent PCR of single colonies to obtain pure fetal colonies from peripheral blood of 12 healthy pregnant women. Of 2966 randomly selected colonies, 42 contained fetal and other cells and, for four women, two to four colonies each contained purely fetal cells. Detection of fetal cells has been hampered by rarity in maternal blood, but with our approach many cells are available for analysis.

HLA-DRB1,3,4,5 and -DQB1 allele frequencies and HLA-DR/DQ linkage disequilibrium of 231 German Caucasoid patients and their corresponding 821 potential unrelated stem cell transplants

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.

Phenotypic and functional comparison of monocytes from cord blood and granulocyte colony-stimulating factor–mobilized apheresis products

OBJECTIVEnIt is well established that T cells are effector cells in graft-vs-host disease (GVHD), yet the contribution of graft monocytes is less well characterized. Therefore, monocytes in cord blood (CB) and granulocyte colony-stimulating factor-mobilized apheresis products (G-AP), two stem cell grafts associated with reduction of acute and chronic GVHD and relative reduction of acute GVHD, respectively, were compared phenotypically and functionally.nnnMATERIALS AND METHODSnThe frequencies, phenotypes, and pinocytosis activities of monocytes from CB and G-AP were determined by flow cytometry and their allostimulatory potential in a primary mixed leukocyte reaction.nnnRESULTSnG-AP contained significantly more monocytes than CB (24.9% +/- 7.1% vs 8.8% +/- 1.5% CD14+ and 62.4 +/- 27.5 x 10(6) vs 0.9 +/- 0.2 x 10(6) CD14+ cells/mL). Monocytes from both sources revealed similar phenotypes. They expressed CD4, CD11a, CD11b, CD11c, CD18, CD32, CD33, CD45R0, CD48, CD50, CD54, CD58, CD64, CD86, CD102, CD116, CD123, and HLA-DR; showed no expression of CD1a and CD83; and weak expression of CD16, CD45RA, and CD80. The levels of CD80 and CD86 expression were comparable; however, in contrast to G-AP monocytes, CB monocytes lacked CD40. There was no difference in pinocytosis activity and allostimulatory capacity of CB and G-AP monocytes.nnnCONCLUSIONSnMonocytes in CB and G-AP are phenotypically and functionally comparable. The only difference observed is the lack of CD40 on CB monocytes.

The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays.

The assessment of nonviable haematopoietic cells by Annexin V staining method in flow cytometry has recently been published by Duggleby et al. Resulting in a better correlation with the observed colony formation in methylcellulose assays than the standard ISHAGE protocol, it presents a promising method to predict cord blood potency. Herein, we applied this method for examining the parameters during processing which potentially could affect cord blood viability. We could verify that the current standards regarding time and temperature are sufficient, since no significant difference was observed within 48 hours or in storage at 4°C up to 26°C. However, the addition of DMSO for cryopreservation alone leads to an inevitable increase in nonviable haematopoietic stem cells from initially 14.8% ± 4.3% to at least 30.6% ± 5.5%. Furthermore, CFU-assays with varied seeding density were performed in order to evaluate the applicability as a quantitative method. The results revealed that only in a narrow range reproducible clonogenic efficiency (ClonE) could be assessed, giving at least a semiquantitative estimation. We conclude that both Annexin V staining method and CFU-assays with defined seeding density are reliable means leading to a better prediction of the final potency. Especially Annexin V, due to its fast readout, is a practical tool for examining and optimising specific steps in processing, while CFU-assays add a functional confirmation.