Evaluation of current genetic testing reports in German-speaking countries with regard to secondary use and future electronic implementation.

Modern diagnostic methods (next-generation sequencing) are one of the current hopes with regard to a personalised medicine. By applying detailed genetic analysis, it is possible to not only improve the prediction of potential risks (as, e.g., concerning hereditary breast cancer) but also the precision of therapy by targeting it to a specific genetic variant. However, there is no international standard for creating, structuring and/or transferring the results of a genetic test report. This type of test report often contains large amounts of complex information, and a standardised and consistent structure would offer potential benefits to all. These include reduced expenditure of time (due to the elimination of information-conversion steps), improved safety (due to a reduction in the occurrence of transmission errors, misunderstanding or misinterpretation of content) and improved clinical information gathering (by the respective linkage to scientific data and literature). Especially in regard to secondary use, a standardised (electronic) format would improve the suitability of these data in retrospective studies and basic research. In this study, we analysed the format and content of 96 genetic testing reports (germline and somatic) from Germany, Switzerland and Austria. Based on these results, we summarised and discussed potentially critical data that were demonstrated to be reported inconsistently, and propose a baseline structure for reporting that would also ease future electronic conversion.

DNA damage response in neonatal and adult stromal cells compared with induced pluripotent stem cells.

UNLABELLED: Comprehensive analyses comparing individual DNA damage response (DDR) of induced pluripotent stem cells (iPSCs) with neonatal stromal cells with respect to their developmental age are limited. The imperative necessity of providing developmental age-matched cell sources for meaningful toxicological drug safety assessments in replacement of animal-based testing strategies is evident. Here, DDR after radiation or treatment with N-methyl-N-nitrosurea (MNU) was determined in iPSCs compared with neonatal and bone marrow stromal cells. Neonatal and adult stromal cells showed no significant morphologically detectable cytotoxicity following treatment with 1 Gy or 1 mM MNU, whereas iPSCs revealed a much higher sensitivity. Foci analyses revealed an effective DNA repair in stromal cell types and iPSCs, as reflected by a rapid formation and disappearance of phosphorylated ATM and γH2AX foci. Furthermore, quantitative polymerase chain reaction analyses revealed the highest basic expression level of DDR and repair-associated genes in iPSCs, followed by neonatal stromal cells and adult stromal cells with the lowest expression levels. In addition, the influence of genotoxic stress prior to and during osteogenic differentiation of neonatal and adult stromal cells was analyzed applying common differentiation procedures. Experiments presented here suggest a developmental age-dependent basic expression level of genes involved in the processing of DNA damage. In addition a differentiation-dependent downregulation of repair genes was observed during osteogenesis. These results strongly support the requirement to provide adequate cell sources for toxicological in vitro drug testing strategies that match to the developmental age and differentiation status of the presumptive target cell of interest. SIGNIFICANCE: The results obtained in this study advance the understanding of DNA damage processing in human neonatal stromal cells as compared with adult stromal cells and induced pluripotent stem cells (iPSCs). The data suggest developmental age-dependent differences in DNA damage repair capacity. In iPSCs (closest to embryonic stem cells), the highest expression level of DNA damage response and repair genes was found, followed by neonatal stromal cells and adult stromal cells with the lowest overall expression. In addition, a differentiation-dependent downregulation of repair capacity was observed during osteogenic differentiation in neonatal stromal cells. Notably, the impact of genotoxic stress on osteogenic differentiation depended on the time the genotoxic insult took place and, moreover, was agent-specific. These results strongly support the necessity of offering and establishing adequate cell sources for informative toxicological testing matching to the developmental age and differentiation status of the respective cell of interest.

Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

Hematopoietic stem cells in neonates: any differences between very preterm and term neonates?

BACKGROUND: In the last decades, human full-term cord blood was extensively investigated as a potential source of hematopoietic stem and progenitor cells (HSPCs). Despite the growing interest of regenerative therapies in preterm neonates, only little is known about the biological function of HSPCs from early preterm neonates under different perinatal conditions. Therefore, we investigated the concentration, the clonogenic capacity and the influence of obstetric/perinatal complications and maternal history on HSPC subsets in preterm and term cord blood. METHODS: CD34+ HSPC subsets in UCB of 30 preterm and 30 term infants were evaluated by flow cytometry. Clonogenic assays suitable for detection of the proliferative potential of HSPCs were conducted. Furthermore, we analyzed the clonogenic potential of isolated HSPCs according to the stem cell marker CD133 and aldehyde dehydrogenase (ALDH) activity. RESULTS: Preterm cord blood contained a significantly higher concentration of circulating CD34+ HSPCs, especially primitive progenitors, than term cord blood. The clonogenic capacity of HSPCs was enhanced in preterm cord blood. Using univariate analysis, the number and clonogenic potential of circulating UCB HSPCs was influenced by gestational age, birth weight and maternal age. Multivariate analysis showed that main factors that significantly influenced the HSPC count were maternal age, gestational age and white blood cell count. Further, only gestational age significantly influenced the clonogenic potential of UCB HSPCs. Finally, isolated CD34+/CD133+, CD34+/CD133- and ALDH(high) HSPC obtained from preterm cord blood showed a significantly higher clonogenic potential compared to term cord blood. CONCLUSION: We demonstrate that preterm cord blood exhibits a higher HSPC concentration and increased clonogenic capacity compared to term neonates. These data may imply an emerging use of HSPCs in autologous stem cell therapy in preterm neonates.

Neonatal mesenchymal-like cells adapt to surrounding cells.

Hematopoietic cord blood (CB) transplantations are performed to treat patients with life-threatening diseases. Besides endothelial cells, the neonatal multipotent stromal cell subpopulations CDSCs (CB-derived stromal cells) and USSCs (unrestricted somatic stromal cells) are like bone marrow (BM) SCs interesting candidates for clinical applications if detailed knowledge is available. Clonal USSC compared to CDSC and BMSC lines differ in their developmental origin reflected by a distinct HOX expression. About 20 (out of 39) HOX genes are expressed in CDSCs (HOX+), whereas native USSCs reveal no HOX gene expression (HOX-). Moreover, USSCs display a lineage-specific absence of the adipogenic differentiation potential. As the specific HOX code can be ascribed to topographic bodysites it may be important to match the HOX code of transplanted cells to the tissue of interest. Herein co-culture experiments were performed, presenting a novel approach to modulate the differentiation potency of USSCs towards HOX positive stromal cells. After co-culturing native USSCs with CDSCs and BMSCs, USSCs adapt a positive HOX code and gain the adipogenic differentiation capacity. These results present for the first time modulation of a lineage-specific differentiation potential by co-culture. Finally, USSCs can be claimed as potential candidates to substitute unique progenitor cell populations in clinical approaches.

Increased Haematopoietic Supportive Function of USSC from Umbilical Cord Blood Compared to CB MSC and Possible Role of DLK-1.

Multipotent stromal cells can be isolated from a variety of different tissues in the body. In contrast to stromal cells from the adult bone marrow (BM) or adipose tissue, cord blood (CB) multipotent stromal cells (MSC) are biologically younger. Since first being described by our group, delta like 1 homologue (DLK-1) was determined as a discriminating factor between the distinct cord blood-derived subpopulations: the unrestricted somatic stromal cells (USSC), which lack adipogenic differentiation capacity, and the BM MSC-like CB MSC. In this study, experiments assessing the haematopoiesis-supporting capacity and molecular biological analyses were conducted and clearly confirmed different properties. Compared to CB MSC, USSC lead to a higher expansion of haematopoietic cells and in addition express significantly higher levels of insulin-like growth factor binding protein 1 (IGFBP1), but lower levels of IGF2. The data presented here also indicate that DLK-1 might not be the sole factor responsible for the inhibition of adipogenic differentiation potential in USSC but nevertheless indicates a biological diversity among cord blood-derived stromal cells.

Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood.

Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells' properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF), thrombopoietin (THPO), interleukin 6 (IL-6), and fms-related tyrosine kinase 3 ligand (FLT3lg). At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

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.

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.

Potential application of cord blood-derived stromal cells in cellular therapy and regenerative medicine.

Neonatal stromal cells from umbilical cord blood (CB) are promising alternatives to bone marrow- (BM-) derived multipotent stromal cells (MSCs). In comparison to BM-MSC, the less mature CB-derived stromal cells have been described as a cell population with higher differentiation and proliferation potential that might be of potential interest for clinical application in regenerative medicine. Recently, it has become clear that cord blood contains different stromal cell populations, and as of today, a clear distinction between unrestricted somatic stromal cells (USSCs) and CB-MSC has been established. This classification is based on the expression of DLK-1, HOX, and CD146, as well as functional examination of the adipogenic differentiation potential and the capacity to support haematopoiesis in vitro and in vivo. However, a marker enabling a prospective isolation of the rare cell populations directly out of cord blood is yet to be found. Further analysis may help to reveal even more subpopulations with different properties, which could be useful for the directed application of these cells in preclinical models.

Unrestricted somatic stem cells: interaction with CD34+ cells in vitro and in vivo, expression of homing genes and exclusion of tumorigenic potential.

BACKGROUND AIMS: Transplantation of allogeneic hematopoietic stem cells (HSC) within the framework of hematologic oncology or inherited diseases may be associated with complications such as engraftment failure and long-term pancytopenia. HSC engraftment can be improved, for example by co-transplantation with mesenchymal stem cells (MSC). Recently, a new multipotent MSC line from umbilical cord blood, unrestricted somatic stem cells (USSC), has been described. It was demonstrated that USSC significantly support proliferation of HSC in an in vitro feeder layer assay. METHODS: A NOD/SCID mouse model was used to assess the effect of USSC on co-transplanted CD34(+) cells and look for the fate of transplanted USSC. The migration potential of USSC was studied in a Boyden chamber migration assay and in vivo. Quantitative real-time polymerase chain reaction (qRT-PCR) for CXCR4, CD44, LFA1, CD62L, VLA4, RAC2, VLA5A and RAC1 were performed. NMR1 nu/nu mice were used for a tumorigenicity test. RESULTS: After 4 weeks, homing of human cells (CD45(+)) to the bone marrow of NOD/SCID mice was significantly increased in mice co-transplanted with CD34(+) cells and USSC (median 30.9%, range 7-50%) compared with the CD34(+) cell-only control group (median 5.9%, range 3-10%; P = 0.004). Homing of USSC could not be shown in the bone marrow. A cell-cell contact was not required for the graft enhancing effect of USSC. An in vivo tumorigenicity assay showed no tumorigenic potential of USSC. CONCLUSIONS: This pre-clinical study clearly shows that USSC have an enhancing effect on engraftment of human CD34(+) cells. USSC are a safe graft adjunct.

In vitro differentiation of unrestricted somatic stem cells into functional hepatic-like cells displaying a hepatocyte-like glucose metabolism.

The hepatic-like phenotype resulting from in vitro differentiation of unrestricted somatic stem cells (USSC) derived from human umbilical cord blood (CB) was analyzed with regard to functional and metabolic aspects. USSC can be differentiated into cells of all three germ layers in vitro and in vivo and, although they share many features with mesenchymal stroma cells (MSC), can be distinguished from these by their expression of DLK1 as well as a restricted adipogenic differentiation potential. For the differentiation procedure described herein, a novel three-stage differentiation protocol resembling embryonic developmental processes of hepatic endoderm was applied. Hepatic pre-induction was performed by activinA and FGF4 resulting in enhanced SOX17 and FOXA2 expression. Further differentiation was achieved sequentially by retinoic acid, FGF4, HGF, EGF, and OSM resulting in a hepatic endodermal identity, characterized by the expression of AFP and HNF1alpha. Thereafter, expression of G6PC, ARG1, FBP1, and HNF4alpha was observed, thus indicating progressive differentiation. Functional studies concerning albumin secretion, urea formation, and cytochrome-p450-3A4 (CYP3A4) enzyme activity confirmed the hepatic-like phenotype. In order to characterize the differentiated cells at a metabolic level, USSC were incubated with [1-(13)C]glucose. By tracing the fate of the molecule's label via isotopomer analysis using (13)C NMR spectroscopy, formation of both glycogen and some gluconeogenetic activity could be observed providing evidence of a hepatocyte-like glucose metabolism in differentiated USSC. In conclusion, the results of the present study indicate that USSC represent a stem cell source with a substantial hepatic differentiation capacity which hold the potential for clinical applications.

Good manufacturing practice-grade production of unrestricted somatic stem cell from fresh cord blood.

BACKGROUND AIMS: The discovery of unrestricted somatic stem cells (USSC), a non-hematopoietic stem cell population, brought cord blood (CB) to the attention of regenerative medicine for defining more protocols for non-hematopoietic indications. We demonstrate that a reliable and reproducible method for good manufacturing practice (GMP)-conforming generation of USSC is possible that fulfils safety requirements as well as criteria for clinical applications, such as adherence of strict regulations on cell isolation and expansion. METHODS: In order to maintain GMP conformity, the automated cell processing system Sepax (Biosafe) was implemented for mononucleated cell (MNC) separation from fresh CB. After USSC generation, clinical-scale expansion was achieved by multi-layered CellSTACKs (Costar/Corning). Infectious disease markers, pyrogen and endotoxin levels, immunophenotype, potency, genetic stability and sterility of the cell product were evaluated. RESULTS: The MNC isolation and cell cultivation methods used led to safe and reproducible GMP-conforming USSC production while maintaining somatic stem cell character. CONCLUSIONS: Together with implemented in-process controls guaranteeing contamination-free products with adult stem cell character, USSC produced as suggested here may serve as a universal allogeneic stem cell source for future cell treatment and clinical settings.

DLK-1 as a marker to distinguish unrestricted somatic stem cells and mesenchymal stromal cells in cord blood.

In addition to hematopoietic stem cells, cord blood (CB) also contains different nonhematopoietic CD45-, CD34- adherent cell populations: cord blood mesenchymal stromal cells (CB MSC) that behave almost like MSC from bone marrow (BM MSC) and unrestricted somatic stem cells (USSC) that differentiate into cells of all 3 germ layers. Distinguishing between these populations is difficult due to overlapping features such as the immunophenotype or the osteogenic and chondrogenic differentiation pathway. Functional differences in the differentiation potential suggest different developmental stages or different cell populations. Here we demonstrate that the expression of genes and the differentiation toward the adipogenic lineage can discriminate between these 2 populations. USSC, including clonal-derived cells lacking adipogenic differentiation, strongly expressed δ-like 1/preadipocyte factor 1 (DLK-1/PREF1) correlating with high proliferative potential, while CB MSC were characterized by a strong differentiation toward adipocytes correlating with a weak or negative DLK-1/PREF1 expression. Constitutive overexpression of DLK-1/PREF1 in CB MSC resulted in a reduced adipogenic differentiation, whereas silencing of DLK-1 in USSC resulted in adipogenic differentiation.

Cytokine production and hematopoiesis supporting activity of cord blood-derived unrestricted somatic stem cells.

OBJECTIVE: Cytokine production and hematopoiesis-supporting stromal activity of cord blood (CB)-derived unrestricted somatic stem cells (USSC) in comparison to bone marrow mesenchymal stem cells (BMMSC) and hematopoietic progenitor expansion solely driven by recombinant cytokines were assessed. METHODS: USSC generation was initiated from fresh and cryopreserved CB. Cytokine production by USSC and BMMSC was determined qualitatively by cytokine mRNA expression array analyses or quantitatively by Multiplex or ELISA analyses. To evaluate hematopoiesis-supporting activity, CB CD34+ cells were expanded in cocultures with USSC and BMMSC or in the presence of Flt3-L, SCF, and TPO. Expansion of CD34+ cells, total cells, colony-forming cells (CFC), and LTC-IC were determined after 1, 2, 3, and 4 weeks of culture. RESULTS: USSC constitutively produced SCF, LIF, TGF-1beta, M-CSF, GM-CSF, VEGF, IL-1beta, IL-6, IL-8, IL-11, IL-12, IL-15, SDF-1alpha, and HGF. When USSC were stimulated with IL-1beta, G-CSF was released. Production of SCF and LIF were significantly higher in USSC compared to BMMSC. At 1, 2, 3, and 4 weeks, cocultivation of CD34+ cells on the USSC layer resulted in a 14.6-fold +/- 1.1-fold, 110.1-fold +/- 17.9-fold, 151.8-fold +/- 39.7-fold, and 183.6-fold +/- 40.4-fold amplification of total cells and in a 30.6-fold +/- 4.4-fold, 101.4-fold +/- 27.5-fold, 64.7-fold +/- 15.8-fold, and 29.4-fold +/- 3.1-fold amplification of CFC, respectively. LTC-IC expansion at 1 and 2 weeks was, with 2.0-fold +/- 0.1-fold and 2.5-fold +/- 0.3-fold, significantly higher for USSC than BMMSC (1.1-fold +/- 0.03-fold and 1.1-fold +/- 0.1-fold), but declined after day 21. Transwell cocultures of USSC did not significantly alter total cell or CFC expansion. CONCLUSIONS: USSC produce functionally significant amounts of hematopoiesis-supporting cytokines and are superior to BMMSC in expansion of CD34+ cells from CB. USSC is therefore a suitable candidate for stroma-driven ex vivo expansion of hematopoietic CB cells for short-term reconstitution.