Survival, integration, and differentiation of unrestricted somatic stem cells in the heart.

Unrestricted somatic stem cells (USSCs) derived from human umbilical cord blood represent an attractive cell source to reconstitute the damaged heart. We have analyzed the cardiomyogenic potential and investigated the fate of USSCs after transplantation into rat heart in vivo. USSCs demonstrated cardiomyogenic differentiation properties characterized by the spontaneously beating activity and the robust expression of cardiac α-actinin and troponin T (cTnT) at protein and mRNA level after cocultivation with neonatal rat cardiomyocytes. To study the fate in vivo, eGFP⁺ USSCs were injected transcoronarily into immunosuppressed rats via a catheter-based technique. Nearly 80% USSCs were retained within the myocardium without altering cardiac hemodynamics. After 7 days, 20% of the transplanted cells survived in the host myocardium and showed elongated morphology with weak expression of cardiac-specific markers, while some eGFP⁺ USSCs were found to integrate into the vascular wall. After 21 days, only a small fraction of USSCs were found in the myocardium (0.13%); however, the remaining cells clearly exhibited a sarcomeric structure similar to mature cardiomyocytes. Identical results were also obtained in nude rats. In addition, we found some cells stained positively for activated caspase 3 paralleled by the massive infiltration of CD11b⁺ cells into the myocardium. In summary, USSCs can differentiate into beating cardiomyocytes by cocultivation in vitro. After coronary transplantation in vivo, however, long-term survival of differentiated USSCs was rather low despite a high initial fraction of trapped cells.

Oxygen tension modifies the 'stemness' of human cord blood-derived stem cells.

BACKGROUND AIMS: Amongst different stem cell populations derived from human cord blood (CB), unrestricted somatic stem cells (USSC) are distinguished from CB mesenchymal stromal cells (CB MSC) by expression patterns of homeobox (HOX) genes, delta-like1 homolog (DLK1) expression and adipogenic differentiation potential. In this study we investigated the effects of oxygen tension on the generation, proliferation and expression of stem cell marker genes, which could be critical during large-scale cell culture for clinical applications. METHODS: We cultured CB-derived stem cells at 5% and 20% O(2). Telomere length shortening was analyzed and we investigated gene expression using reverse-transcription (RT)-polymerase chain reaction (PCR) and real-time PCR. Additionally we performed adipogenic and osteogenic in vitro differentiation. Results. Altering the cultivation conditions of USSC or CB MSC from 20% to 5% O(2) had no significant impact. In contrast, cell populations derived from primary cultures prepared at 5% O(2) qualified as neither USSC nor as CB MSC. When converted to 20%, their proliferation was diminished, telomere shortening was accelerated, and two of six cell lines ceased expression of HOX genes. The HOX code of the other cell populations was not been affected by culture conditions. CONCLUSIONS: Altering culture conditions during generation can impact cell characteristics such as the HOX code. These effects need to be considered when dealing with cell cultures for clinical applications.

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.

Inactivation of CD73 promotes atherogenesis in apolipoprotein E-deficient mice.

AIMS: CD73 (ecto-5'-nucleotidase) is expressed by a broad range of immune cells and attenuates inflammation in several acute disease models. This study therefore explored the role of CD73-derived adenosine in a model of chronic vascular inflammation such as atherogenesis. METHODS AND RESULTS: CD73(-/-) mice were backcrossed into the apolipoprotein E (ApoE(-/-)) background. In CD73(-/-)/ApoE(-/-) double mutants, atherosclerotic lesion formation was increased by ∼50% compared with ApoE(-/-). However, the cellular composition and extracellular matrix of the plaques did not differ. Surprisingly, we found significant activity and expression of CD73 in the plaque of ApoE(-/-) mice which increased over time. CD73 co-localized with macrophages, Tregs, and cells of mesenchymal origin. Genome-wide microarray analysis of the aorta lacking CD73 revealed upregulation of endothelin-1 (Edn1) mRNA together with changes of genes in lipid metabolism and the Wnt and nuclear factor kappa B pathways. Measurement of plasma levels verified the upregulation of Edn1 in CD73(-/-) and double mutants. Plasma triglycerides (TG) were also found to be significantly elevated in the CD73(-/-)/ApoE(-/-) mice compared with ApoE(-/-) controls. CONCLUSION: Lack of CD73 promotes atherogenesis most likely by de-inhibition of resident macrophages and T cells. Elevated Edn1 and TG levels may have contributed. This establishes CD73-derived adenosine as a direct or indirect regulator of atherogenesis.

Induction of pluripotency in human cord blood unrestricted somatic stem cells.

OBJECTIVE: Generation of induced pluripotent stem (iPS) cells from human cord blood (CB)-derived unrestricted somatic stem cells and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells. MATERIALS AND METHODS: Unrestricted somatic stem cells isolated from human CB were reprogrammed to iPS cells using retroviral expression of the transcription factors OCT4, SOX2, KLF4, and C-MYC. The reprogrammed cells were analyzed morphologically, by quantitative reverse transcription polymerase chain reaction, genome-wide microRNA and methylation profiling, and gene expression microarrays, as well as in their pluripotency potential by in vivo teratoma formation in severe combined immunodeficient mice and in vitro differentiation. RESULTS: CB iPS cells are very similar to human embryonic stem cells morphologically, at their molecular signature, and in their differentiation potential. CONCLUSIONS: Human CB-derived unrestricted somatic stem cells offer an attractive source of cells for generation of iPS cells. Our findings open novel perspectives to generate human leukocyte antigen-matched pluripotent stem cell banks based on existing CB banks. Besides the obvious relevance of a second-generation CB iPS cell bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible.

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.

The HOX Code as a "biological fingerprint" to distinguish functionally distinct stem cell populations derived from cord blood.

Mesenchymal stem cells (MSC) have been isolated from almost every adult tissue. In cord blood (CB), different non-hematopoietic CD45-, CD34- adherent cell populations can be generated: the cord blood derived MSC (CB-MSC), that behave almost like MSC from bone marrow (BM-MSC), and unrestricted somatic stem cells (USSC) which show a distinct differentiation potential into all three germ layers. However, distinguishing these populations easily by molecular markers is still a concern. In this study we were able to present the HOX expression pattern of USSC, CB-MSC and BM-MSC, which in fact allows a discrimination of these populations. Briefly, RT-PCR analysis of the HOX code revealed a high similarity between BM-MSC and CB-MSC, which are both HOX-positive, whereas USSC resembled H9 embryonic stem cells HOX-negative.Especially HOXA9, HOXB7, HOXC10 and HOXD8 are good candidate markers to discriminate MSC from USSC. Thus, our data suggest that the "biological fingerprint" based on the HOX code can be used to distinguish functionally distinct MSC populations derived from bone marrow and cord blood.

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.

Cord blood for tissue regeneration.

Umbilical cord blood (CB) has become a commonly accepted source of hematopoietic stem cells for transplantation in children and adults. It is readily available and outperforms bone marrow (BM) as well as peripheral blood stem cells in terms of tolerance for HLA-mismatches between donor and recipient and its decreased graft-versus-host disease. Clinical use has been expanded from hematological malignancies to various areas such as treatment of metabolic genetic disorders or to induce angiogenesis. For the last years CB has been under intense experimental investigation in in vitro differentiation models as well as in preclinical animal models. Since CB-derived stem cells offer multiple advantages over adult stem cells from other sources like BM, CB may provide a future source of stem cells for tissue repair and regeneration. To facilitate the use of CB-derived stem cells in clinical scenarios, the biology of these cells needs to be further explored in detail particularly with regard to the fact that different non-hematopoietic stem cell populations occur within CB. Here we explore the most consistent and the most contradictory data referring to the differentiation potential of CB-derived stem cells and give an outlook on their potential clinical value including and possible reprogramming into IPS cells.

Adenosine produced via the CD73/ecto-5'-nucleotidase pathway has no impact on erythropoietin production but is associated with reduced kidney weight.

CD73/ecto-5'-nucleotidase, which catalyzes the conversion of adenosine monophosphate to adenosine, has been implicated in vascular homeostasis. The aim of the present study was to evaluate the role of CD73 in erythropoietin (EPO) production and to determine its influence on basal kidney perfusion using a CD73 knockout mutant recently generated by us. Of all organs investigated, kidneys showed the most prominent CD73 activity, preferentially located in peritubular fibroblasts of the renal cortex and the glomerular mesangium. In the absence of CD73, alkaline phosphatase remained unchanged, but tissue adenosine was reduced under control conditions (by 76%) and during normobaric hypoxia (by 72%). Despite the loss of CD73 activity, EPO mRNA and plasma protein concentrations were not different under basal conditions as well as after normobaric hypoxia (8% O2) and carbon monoxide (0.1% CO) inhalation (both for 4 h). Although there were no differences in blood pressure and urine flow volume, average weight of both kidneys was reduced by 21% in the knockout (wild type 7.17+/-1.18 mg g-1 body wt, CD73-/- 5.70+/-1.91 mg g-1 body wt). Measurement of renal plasma flow and glomerular filtration revealed no significant differences when related to respective kidney weights. We conclude that adenosine derived by the extracellular CD73 pathway has no impact on EPO production under basal conditions and after hypoxic challenge but may determine kidney weight.

Targeted disruption of cd73/ecto-5'-nucleotidase alters thromboregulation and augments vascular inflammatory response.

To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5'-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (-14%; P<0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl3) was significantly reduced by 20% in cd73-/- mice (P<0.05). Bleeding time after tail tip resection tended to be shorter in cd73-/- mice (-35%). In vivo platelet cAMP levels were 0.96+/-0.46 in WT versus 0.68+/-0.27 pmol/106 cells in cd73-/- mice (P<0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 micromol/L) was undistinguishable between the two strains. In the cremaster model of ischemia-reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73-/- compared with WT littermates (WT 98% versus cd73-/- 245%; P<0.005). The constitutive adhesion of monocytes in ex vivo-perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73-/- mice (P<0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo.