Effect of a single autologous cord blood infusion on beta-cell and immune function in children with new onset type 1 diabetes: a non-randomized, controlled trial.

BACKGROUND: The application of autologous cord blood in children with type 1 diabetes has been found to be safe, but not to preserve beta-cell function in a previous study, which, however, had not included a control group. OBJECTIVE: To compare the changes of metabolic and immune function over time between cord blood infused children and natural controls. SUBJECTS AND METHODS: Seven children with newly diagnosed type 1 diabetes underwent a single autologous cord blood infusion and 10 children were enrolled as natural controls in a non-randomized, controlled, open label intervention trial. Primary analyses were performed 1 year following cord blood infusion. Cases and controls were compared regarding metabolic [area under the curve (AUC) and peak C-peptide, insulin use, and HbA1c] and immune outcome (islet autoantibody titer and T-cell response), adjusted for age, gender, diabetes duration, and baseline levels. RESULTS: There were no significant adverse events related to the infusion. Metabolic and immune outcomes were not significantly different at 12 months follow-up between infused children and controls (e.g., adjusted p = 0.244 for AUC C-peptide, adjusted p = 0.820 for insulin use, adjusted p = 0.772 for peripheral regulatory T cells). Six-month change of AUC C-peptide correlated significantly with the number of infused CD34+ cells (r = 0.931, p = 0.002). CONCLUSIONS: An autologous cord blood infusion does not change the natural course of metabolic and immune parameters after disease onset. However, the content of CD34+ cells in the stored blood sample might offer potential for improvement of future cell therapies.

Murine and human very small embryonic-like cells: a perspective.

In 2006, very small embryonic-like (VSEL) stem cells were described as a pluripotent population of prospectively isolated stem cells in adult murine bone marrow (mBM) and human umbilical cord blood (hUCB). While rigorous proof of pluripotency is still lacking, murine VSEL cells have been shown to overlap with an independently identified population of neural crest derived mesenchymal stem cells (MSC). The presence of primitive mesenchymal precursors within the VSEL cell population may partially explain the findings that have led to the concept of an "embryonic-like" stem cell in mBM. However, our own studies on human VSEL cells revealed very little similarity between murine VSEL cells and their reportedly equivalent population in hUCB. On the contrary, our data strongly suggest that human VSEL cells are an aberrant and inactive population that cannot expand in vitro and has neither embryonic nor adult stem cell like properties. Here we critically re-examine the data supporting stemness and pluripotency of murine and human VSEL cells, respectively.

Intramyocardial delivery of human CD133+ cells in a SCID mouse cryoinjury model: Bone marrow vs. cord blood-derived cells.

OBJECTIVE: The regenerative potential of endothelial and hematopoietic progenitor cells in the heart may vary according to their origin. This study was designed to compare the functional effects of CD133+ cells from human cord blood and bone marrow in a mouse model of myocardial injury. METHODS: 5 x 10(5) CD133+ cells from bone marrow (BM(CD133)) or cord blood (UCB(CD133)) were injected in the necrosis border zone of NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice with left ventricular cryoinjury (CI+). Transplanted cells were tracked by immunostaining for hNuclear antigen and by PCR for hDNA. Echocardiography was used to measure contractility. Scar size, capillary density, and cardiomyocyte apoptosis were evaluated by histology. In addition, the myogenic and endothelial differentiation capacity of BM(CD133) and UCB(CD133) was compared in vitro. RESULTS: DNA was detected 4 weeks after cell injection by PCR, but hNuc+ cells were found by immunostaining only after 48 h. Capillary density in both BM(CD133) and UCB(CD133) cell-treated CI+ mice was higher than in control CI+ mice, but not different between BM(CD133) and UCB(CD133) cell-treated hearts. There were no differences in scar size and myocardial mass among BM(CD133), UCB(CD133) and control CI+ mice, but cardiomyocyte apoptosis was reduced by both BM(CD133) and UCB(CD133) cells. The post-injury deterioration of shortening fraction (46.2+/-1% in sham-operated mice and 41.3+/-0.8% in control CI+ mice) was prevented by BM(CD133) cells (45.4+/-0.9%), but not by UCB(CD133) cells (40.8+/-0.7%). On the other hand, both BM(CD133) and UCB(CD133) cells abolished post-injury mortality. In vitro, neither cultivated BM(CD133) or UCB(CD133) cells developed into myocytes, but both readily differentiated towards an endothelial cell phenotype. CONCLUSIONS: While both cord blood and marrow CD133+ cells have some beneficial effects on post-injury angiogenesis and survival, only marrow cells appear to improve myocardial contractility.

Human cord blood cells induce angiogenesis following myocardial infarction in NOD/scid-mice.

OBJECTIVE: We tested the hypothesis that intravenously administered human umbilical cord blood (hUCB) cells contribute to repair processes following myocardial infarction. METHODS: hUCB mononuclear cells containing 0.11% to 1.1% CD34(+) cells were injected in the tail vein of NOD/scid mice that had (MI+) or had not (MI-) previously undergone ligation of the left anterior coronary artery (LAD). Homing to bone marrow and solid organs was determined by polymerase chain reaction (PCR) for human DNA (hDNA) using human-specific primers of Locus D7Z1. Immunostaining was used for phenotypic analysis, and capillary density as well as myocardial scar formation was assessed. Moreover, expression of stromal cell-derived factor-1 (SDF-1) was studied in infarcted and in normal hearts. RESULTS: hDNA was detected in marrow, spleen, and liver of both MI+ and MI- mice 24 h, 1 week, and 3 weeks after cell injection. In the heart, however, hDNA was detected in 10 of 19 MI+ mice but in none of the MI- mice (p=0.002). Infarct size was smaller in cell-treated MI+ mice than in untreated MI+ hearts (38.7 versus 47.8%, P<0.05), and there was also less collagen deposition. In cell-treated MI+ mice, capillary density in the infarct border zone was approximately 20% higher (p=0.03), and clusters of hUCB-derived cells were detected in the perivascular interstitium. Occasionally, chimeric capillaries composed of human and mouse endothelial cells were found, but the vast majority of neo-vessels appeared to consist of mouse cells only. Up to 70% of the cord blood-derived cells in the heart were CD45(+). There was no evidence of cardiomyocyte differentiation as determined by co-localization of HNA or HLA-I with GATA-4 or Connexin 43. In infarcted myocardium, expression of SDF-1 mRNA was approximately 7-fold higher than in normal hearts. CONCLUSIONS: hUCB cells migrate to infarcted, not to normal myocardium, where they engraft, participate in neoangiogenesis, and beneficially influence remodelling processes. Cord blood cells may hence be useful for cell therapy of ischemic heart disease.

Very small embryonic-like stem cells purified from umbilical cord blood lack stem cell characteristics.

Very small embryonic-like (VSEL) cells have been described as putatively pluripotent stem cells present in murine bone marrow and human umbilical cord blood (hUCB) and as such are of high potential interest for regenerative medicine. However, there remain some questions concerning the precise identity and properties of VSEL cells, particularly those derived from hUCB. For this reason, we have carried out an extensive characterisation of purified populations of VSEL cells from a large number of UCB samples. Consistent with a previous report, we find that VSEL cells are CXCR4(+), have a high density, are indeed significantly smaller than HSC and have an extremely high nuclear/cytoplasmic ratio. Their nucleoplasm is unstructured and stains strongly with Hoechst 33342. A comprehensive FACS screen for surface markers characteristic of embryonic, mesenchymal, neuronal or hematopoietic stem cells revealed negligible expression on VSEL cells. These cells failed to expand in vitro under a wide range of culture conditions known to support embryonic or adult stem cell types and a microarray analysis revealed the transcriptional profile of VSEL cells to be clearly distinct both from well-defined populations of pluripotent and adult stem cells and from the mature hematopoietic lineages. Finally, we detected an aneuploid karyotype in the majority of purified VSEL cells by fluorescence in situ hybridisation. These data support neither an embryonic nor an adult stem cell like phenotype, suggesting rather that hUCB VSEL cells are an aberrant and inactive population that is not comparable to murine VSEL cells.

Comparison of hematopoietic stem cells derived from fresh and cryopreserved whole cord blood in the generation of humanized mice.

To study the function and maturation of the human hematopoietic and immune system without endangering individuals, translational human-like animal models are needed. We compare the efficiency of CD34(+) stem cells isolated from cryopreserved cord blood from a blood bank (CCB) and fresh cord blood (FCB) in generating highly engrafted humanized mice in NOD-SCID IL2Rγ(null) (NSG) rodents. Interestingly, the isolation of CD34(+) cells from CCB results in a lower yield and purity compared to FCB. The purity of CD34(+) isolation from CCB decreases with an increasing number of mononuclear cells that is not evident in FCB. Despite the lower yield and purity of CD34(+) stem cell isolation from CCB compared to FCB, the overall reconstitution with human immune cells (CD45) and the differentiation of its subpopulations e.g., B cells, T cells or monocytes is comparable between both sources. In addition, independent of the cord blood origin, human B cells are able to produce high amounts of human IgM antibodies and human T cells are able to proliferate after stimulation with anti-CD3 antibodies. Nevertheless, T cells generated from FCB showed increased response to restimulation with anti-CD3. Our study reveals that the application of CCB samples for the engraftment of humanized mice does not result in less engraftment or a loss of differentiation and function of its subpopulations. Therefore, CCB is a reasonable alternative to FCB and allows the selection of specific genotypes (or any other criteria), which allows scientists to be independent from the daily changing birth rate.

ABCG2 expression is correlated neither to side population nor to hematopoietic progenitor function in human umbilical cord blood.

OBJECTIVE: The ABCG2 transporter has been identified as a determinant of the side population (SP) in murine bone marrow and a potential marker for primitive stem cells. To assess the potential of the SP phenotype and ABCG2 expression to identify stem cells in human umbilical cord blood (hUCB), we have examined directly the relationship between SP; expression of ABCG2, CD133, and CD34; and hematopoietic potential in UCB samples. MATERIALS AND METHODS: Multicolor fluorescence activated cell sorting analysis was combined with the Hoechst SP procedure to allow the simultaneous detection of the SP phenotype together with surface markers in cells from fresh and cryopreserved UCB. Sorted populations were analyzed for cobblestone area-forming cell (CAFC) activity and by quantitative reverse transcriptase polymerase chain reaction for expression of mRNA from the ABC transporters ABCG2, MDR1, and MRP1. ABCG2(+) cells were enriched by magnetic-activated cell sorting for stringent analysis. RESULTS: hUCB-derived SP cells were negative for ABCG2, but comprise approximately 20% CD133(+)/CD34(+) cells. Sorted SP cells from UCB were enriched 20-fold for week 13 CAFC activity, while magnetic-activated cell sorting-enriched ABCG2(+) cells retained no hematopoietic activity either in CAFC or liquid cultures. There was no significant difference in the SP frequency, immunophenotype, or CAFC potential of fresh and cryopreserved UCB. ABCG2 mRNA was not enriched in the SP and was specifically diminished ninefold in CD133 cells, which were eightfold enriched for MDR1 mRNA. CONCLUSION: We find no evidence for an association of ABCG2 with SP activity or hematopoietic progenitor function in hUCB.

Autologous umbilical cord blood mononuclear cell transplantation preserves right ventricular function in a novel model of chronic right ventricular volume overload.

We aimed to evaluate the feasibility and efficacy of autologous umbilical cord blood mononuclear cell (UCMNC) transplantation on right ventricular (RV) function in a novel model of chronic RV volume overload. Four-month-old sheep (n = 20) were randomized into cell (n = 10) and control groups (n = 10). After assessment of baseline RV function by the conductance catheter method, a transannular patch (TAP) was sutured to the right ventricular outflow tract (RVOT). Following infundibulotomy the ring of the pulmonary valve was transected without cardiopulmonary bypass. UCMNC implantation (8.22 +/- 6.28 x 10(7)) in the cell group and medium injection in the control group were performed into the RV myocardium around the TAP. UCMNCs were cultured for 2 weeks after fluorescence-activated cell sorting (FACS) analysis for CD34 antigen. Transthoracic echocardiography (TTE) and computed tomography were performed after 6 weeks and 3 months, respectively. RV function was assessed 3 months postoperatively before the hearts were excised for immunohistological examinations. FACS analysis revealed 1.2 +/- 0.22% CD34(+) cells within the isolated UCMNCs from which AcLDL(+) endothelial cells were cultured in vitro. All animals survived surgery. TTE revealed grade II-III pulmonary regurgitation in both groups. Pressure-volume loops under dobutamine stress showed significantly improved RV diastolic function in the cell group (dP/dt(min): p = 0.043; E(ed): p = 0.009). CD31 staining indicated a significantly enhanced number of microvessels in the region of UCMNC implantation in the cell group (p < 0.001). No adverse tissue changes were observed. TAP augmentation and pulmonary annulus distortion without cardiopulmonary bypass constitutes a valid large animal model mimicking the surgical repair of tetralogy of Fallot. Our results indicate that the chronically volume-overloaded RV profits from autologous UCMNC implantation by enhanced diastolic properties with a probable underlying mechanism of increased angiogenesis.

The stairway: a novel behavioral test detecting sensomotoric stroke deficits in rats.

New therapeutic concepts of cell and tissue replacement therapies have been introduced in treatment of neurological disorders which require robust and sensitive sensomotoric behavioral diagnostic test systems for experimental research. However, most test arrays available are either expensive and/or sensitive to environmental disturbances. Moreover, an extensive training phase for experimental animals is often required by conventional tests to get reproducible data. In this study, we describe a new test array, the Stairway, that is robust, cost-effective, and needs only a minimum training phase. Its efficiency is evaluated in comparison to the well-established RotaRod and modified neurological severity score. Stroke was induced by middle cerebral artery occlusion. Human umbilical cord blood transplantation (n = 18) was performed 24 h after stroke by intravenous injection of suspended cells in comparison to control injections (n = 18), while behavioral assessment was carried out continuously for 29 days. Furthermore, development of lesions was monitored by magnetic resonance imaging. The results reveal the Stairway test as a simple, highly reproducible, and easy manageable test system thus reflecting therapeutic benefits precisely.

Experimental treatment of stroke in spontaneously hypertensive rats by CD34+ and CD34- cord blood cells.

Human umbilical cord blood as a source of stem cells has recently been reported in experimental treatment of cerebral disorders. However, little is known about the nature of cells and cellular mechanisms leading to neurofunctional improvement. Here we investigated the potential of separated CD34(+) versus CD34(-) human umbilical cord blood cells (HUCBC) to promote functional recovery following stroke. The experiments were performed in spontaneously hypertensive (SH) rats, known for a risk profile comparable to stroke patients. After three weeks of behavioral training in the RotaRod and Beamwalk test arrays, stroke was induced by permanent middle cerebral artery occlusion (MCAO). For cell therapy, 1 x 10(6) cryopreserved cells were administered systemically between 8 and 10 hours after MCAO. The behavioral tests were performed together with a neurological severity score (mNSS) until day 29 to assess neurofunctional disabilities. Nearly complete functional remission was observed with both subpopulations CD34(+) as well as CD34(-) cells. To localize cells histologically, they were labeled with a fluorescence dye (CFSE) before injection. Again, after administration of CD34(+) as well as CD34(-) cells, CFSE labelled cells were found that accumulated in the border zone between the central necrosis of the ischemic lesion and functional brain tissue, thus indicating active attraction towards the lesion for both cell populations. Immunohistology with anti-CD68 and antibodies to human neuronal markers (NF-L, chromogranin) indicated an accumulation of human and rat monocytes in the border zone of the lesion while neuronal cells of human origin could not be detected in host brains.

Duplex polymerase chain reaction quantification of human cells in a murine background.

Studies of the regenerative potential of human stem cells commonly involve their transplantation into immune-deficient mice or in vitro coculture with mouse cells. The optimal use of such models requires the detection and quantification of relatively low numbers of human cells in a murine background. We report here a duplex polymerase chain reaction (PCR) approach involving the coamplification of human-and mouse-specific repetitive sequences. The determination of product ratios compensates against variations in sample quality and enables quantitation from >50% down to 0.01% human-in-mouse from a single reaction. Product ratios are determined by standard electrophoresis of end-stage PCR reactions followed by image analysis techniques using freely available software, with no requirement for real-time PCR. The approach has been used to analyze tissue from mice transplanted with human cells and cocultures between differentiating mouse embryonal stem cells and human umbilical cord blood cells.