Teja Falk Radke

Separation of adult bone marrow mononuclear cells using the automated closed separation system Sepax.

BACKGROUND The Düsseldorf-based cardiologist Professor Strauer was the first to present a therapeutic concept for the repair of acute infarcted myocardium in 2001: the autologous intracoronary transplantation of unfractionated human bone marrow (BM) mononuclear cells (MNC). The Division of Cardiology, Pneumology and Angiology, University of Duesseldorf Medical School, Duesseldorf, Germany, was also able to show the regenerative potential of BM stem cell transplantation in patients with chronic heart disease (CHD) and peripheral arterial disease (PAD). In the mean time, several clinical trials have been set up worldwide, predominantly by using MNC isolated manually from BM aspirates via density-gradient centrifugation; 374 patients have been treated here with unselected BM MNC since 2001. Altogether 217 BM aspirates have been processed manually. In order to maintain the high standards required for cellular therapeutics, the Sepax cell-separation system was implemented into routine BM processing in 2006. The closed Sepax system provides a reproducible MNC isolation method, and 157 BM samples have been processed with the Sepax device. The results of manual MNC isolation were compared with the Sepax-mediated MNC isolation. METHODS The manual Ficoll separation method was compared with the Sepax density gradient-based separation (DGBS) protocol using Ficoll with the kit CS-900 and the Sepax S-100 main processing unit from Biosafe. RESULTS Nucleated cell and MNC recovery were significantly higher after Sepax processing (P<0.0001) whereas no significance was found for red blood cell depletion. DISCUSSION The Sepax cell-separation system is a time-saving method providing clinical-grade MNC isolated automatically from human BM by Ficoll density centrifugation.

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.

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.

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 HNF1α. Thereafter, expression of G6PC, ARG1, FBP1, and HNF4α 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 [1‐13C]glucose. By tracing the fate of the molecules label via isotopomer analysis using 13C 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. J. Cell. Physiol. 225: 545–554, 2010.

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.

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.

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.

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 ALDHhigh 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.

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.