Jarmo Laine

N‐Glycolylneuraminic Acid Xenoantigen Contamination of Human Embryonic and Mesenchymal Stem Cells Is Substantially Reversible

Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno‐carbohydrate N‐glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N‐glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid‐linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.

Global Gene Expression Profile of Human Cord Blood–Derived CD133+ Cells

Human cord blood (CB)–derived CD133+ cells carry characteristics of primitive hematopoietic cells and proffer an alternative for CD34+ cells in hematopoietic stem cell (HSC) transplantation. To characterize the CD133+ cell population on a genetic level, a global expression analysis of CD133+ cells was performed using oligonucleotide microarrays. CD133+ cells were purified from four fresh CB units by immunomagnetic selection. All four CD133+ samples showed significant similarity in their gene expression pattern, whereas they differed clearly from the CD133+ control samples. In all, 690 transcripts were differentially expressed between CD133+ and CD133+ cells. Of these, 393 were increased and 297 were decreased in CD133+ cells. The highest overexpression was noted in genes associated with metabolism, cellular physiological processes, cell communication, and development. A set of 257 transcripts expressed solely in the CD133+ cell population was identified. Colony‐forming unit (CFU) assay was used to detect the clonal progeny of precursors present in the studied cell populations. The results demonstrate that CD133+ cells express primitive markers and possess clonogenic progenitor capacity. This study provides a gene expression profile for human CD133+ cells. It presents a set of genes that may be used to unravel the properties of the CD133+ cell population, assumed to be highly enriched in HSCs.

Human umbilical cord blood cells do not improve sensorimotor or cognitive outcome following transient middle cerebral artery occlusion in rats

The present study investigated effects of human umbilical cord blood (HUCB) cells on sensorimotor, cognitive, and histological outcome in rats subjected to transient middle cerebral artery occlusion (MCAO). Halothane anesthetized adult male Wistar rats were subjected to transient MCAO for 2 h. HUCB cells (mononuclear 1-5x10(7) or Lin(-) cells 1-5x10(5)) were administered intravenously after 24 h recovery. The limb-placing test was performed on postoperative days 2, 4, 6, 9, 12, 16, and 20. In addition, beam-walking and cylinder tests were used to assess sensorimotor function at baseline, and on postoperative days 4, 12, and 20. Morris water-maze was used to assess cognitive performance on postoperative days 22-24. Subsequently, rats were perfused for measurement of infarct volumes and detection of HUCB cells by immunohistochemistry (MAB1281). MCAO rats showed a partial spontaneous recovery in sensorimotor function during the follow-up. However, the recovery profile was similar in MCAO controls and in MCAO rats that received HUCB cells. HUCB did not affect impaired water-maze performance of MCAO rats. Only few human nuclei-specific MAB1281-positive cells were detected in the ipsilateral hemisphere in MCAO rats that received HUCB cells. Infarct volumes did not differ between the experimental groups. A group of additional rats were used to further study biodistribution of intravenously given (111)In-oxine-labelled mononuclear HUCB cells in MCAO and sham-operated rats. SPECT imaging data indicated a high tracer uptake in the lung, liver, spleen, and kidney, but not in the brain immediately after administration or 24 h post-administration. The present study suggests that HUCB cells do not improve functional recovery or histological outcome in MCAO rats after systemic administration because of limited migration of cells in the ischemic brain.

The N-glycome of human embryonic stem cells

BackgroundComplex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.ResultsThe data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans.ConclusionThe N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

Cytokine Gene Polymorphisms and Risks of Acute Rejection and Delayed Graft Function after Kidney Transplantation

Background. Pretransplantation identification of patients at an increased risk for adverse events would allow more individualized treatment strategies possibly improving long-term outcome. We studied cytokine gene polymorphisms of kidney allograft recipients and their donors to identify factors predisposing for acute rejection (AR) and delayed graft function (DGF). Methods. A total of 291 adult cadaver kidney recipients transplanted at a single transplantation centre between 1999 and 2002 were investigated. Recipients and donors were typed for TNF-&agr;(-308G/A), TGF-&bgr;1(codon 10T/C, codon 25C/G), IL-10(-1082G/A, -819C/T, -592C/A), IL-6(-174C/G), and IFN-&ggr;(+874T/A) polymorphisms using a SSP-PCR kit. An AR episode was defined based on clinical and histological findings (Banff criteria). Results. The incidence of AR was 17%. In univariate statistical analyses recipients with TNF-&agr; -308AA-genotype were found to be at a significantly increased risk for rejection (odds ratio [OR] 5.0, 95% CI 3.0–8.3, P=0.003). The association was independent from the patient-donor HLA-mismatch status. In addition, patients with IL-10 ACCACC, ATAATA, GCCATA (-1082A/G, -819C/T, -592C/A, respectively) haplotypes were predisposed to rejection (OR 1.9, 95% CI 1.1–3.1, P=0.016). Further, the combination of recipient TGF-&bgr;1 25GG-genotype and donor IL-10 -819T-allele was associated with rejection (OR 1.8, 95% CI 1.1–3.0, P=0.027). These variables remained significant risk factors also in a multivariate logistic regression analysis. The incidence of DGF was 22%. The risk was increased by a donor TNF-&agr; -308GA-genotype (OR 1.6, 95% CI 1.1–2.6, P=0.040). Conclusions. Our results confirm that cytokine gene polymorphisms influence the outcome of kidney transplantation. Our data especially identify the TNF-&agr; -308AA-genotype as a factor predisposing for AR episodes.

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and α2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.

Umbilical Cord Blood–derived Progenitor Cells Enhance Muscle Regeneration in Mouse Hindlimb Ischemia Model

Progenitor cell therapy is a potential new treatment option for ischemic conditions in the myocardium and skeletal muscles. However, it remains unclear whether umbilical cord blood (UCB)-derived progenitor cells can provide therapeutic effects in ischemic muscles and whether ex vivo gene transfer can be used for improving the effect. In this study, the use of a lentiviral vector led to efficient transduction of both UCB-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Our method resulted in a long-term transgene expression and did not alter the differentiation potential of either HSCs or MSCs. In addition, we studied the therapeutic potential of CD133+ and MSC progenitor cells transduced ex vivo with lentiviruses encoding the mature form of vascular endothelial growth factor D (VEGF-DΔNΔC) or the enhanced green fluorescent protein (eGFP) marker gene in a nude mouse model of skeletal muscle ischemia. Progenitor cells enhanced the regeneration of ischemic muscles without a detectable long-term engraftment of either CD133+ or MSC progenitor cells. Our results show that, rather than directly participating in angiogenesis or skeletal myogenesis, UCB-derived progenitor cells indirectly enhance the regenerative capacity of skeletal muscle after acute ischemic injury. However, VEGF-D gene transfer of progenitor cells did not improve the therapeutic effect in ischemic muscles.

Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

BackgroundThere is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood.ResultsIn this study, immunomagnetic cell sorting protocols to purify CD34+, CD133+ and Lin- cells from fresh and cryopreserved cord blood were optimized. Reproducible purities of up to 97% were reached. The selected cells were highly viable having substantial colony-forming potential.ConclusionThe optimized protocols enable rapid enrichment of highly pure hematopoietic stem cells from both fresh and cryopreserved cord blood.

Graft function 5-7 years after renal transplantation in early childhood.

BACKGROUND Low recipient age is still a risk factor for graft failure after kidney transplantation (Tx). Detailed prospective reports on long-term graft function in small children after renal Tx are still lacking. METHODS Forty-nine kidney allograft recipients who received transplants before the age of 5 years were followed prospectively. The most common disease was congenital nephrotic syndrome of the Finnish type. Twenty patients were recipients of living related donors (LRD), and 29 were cadaveric kidney (CAD) recipients. All patients received triple immunosuppression. Glomerular filtration rate (GFR), effective renal plasma flow (ERPF), sodium, urate, and potassium handling, and concentrating capacity were studied for up to 7 years after Tx. RESULTS Patient survival 7 years after Tx was 100% for LRD and 96% for CAD recipients. Graft survival was 94% for LRD and 79% for CAD recipients (P=NS) and 89% and 83% for children >2 years and <2 years of age at Tx, respectively (P=NS). Five years after Tx, GFR was 70 vs. 64 and ERPF was 380 vs. 310 ml/min/1.73 m2 for LRD and CAD recipients, respectively (P=NS). Mean absolute GFR remained stable. GFR was lower in children who received transplants at <2 years than in children who received transplants at >2 years of age, 54 vs. 75 ml/min/1.73 m2 (P=0.02). Sodium handling remained intact, but hyperuricemia was seen in 43-67%; 17-33% showed abnormal handling of potassium; and most patients had a subnormal concentrating capacity. CONCLUSIONS Excellent long-term graft survival and good graft function can be achieved with triple immunosuppression, even in young CAD kidney recipients.

Mechanisms of hyperuricemia in cyclosporine-treated renal transplanted children

Mechanisms of hyperuricemia were investigated in 19 pediatric renal transplant recipients 6 months after transplantation. 51Cr-EDTA, PAH, lithium and sodium clearances, 24-hour urinary creatinine and urate excretions were measured. Ten patients had hyperuricemia. The hyperuricemic patients had lower EDTA, PAH, and urate clearances (mean 69.5 vs. 92.5, p < 0.05, 234 vs. 421, p < 0.05 and 4.3 vs. 10.6 ml/min/1.73 m2, p < 0.001, respectively). Serum urate concentration correlated with cyclosporine dose (r = 0.46, p < 0.05) and inversely with urate (r = -0.88, p < 0.001), and lithium (r = -0.55, p < 0.05) clearances. Urate clearance showed a significant positive correlation with lithium clearance (r = 0.66, p = 0.01) and an inverse correlation with fractional proximal tubular reabsorption (r = -0.63, p = 0.02). Results were not influenced by diuretic administration. Our data support increased proximal tubular urate reabsorption rather than decreased secretion as the mechanism in cyclosporine-induced hyperuricemia.