Human mesenchymal stem cell responses to hydrostatic pressure and shear stress.

The effects of mechanical stimuli to which cells are exposed in vivo are, at best, incompletely understood; in this respect, gene-level information regarding cell functions which are pertinent to new tissue formation is of special interest and importance in applications such as tissue engineering and tissue regeneration. Motivated by this need, the present study investigated the early responses of human mesenchymal stem cells (hMSCs) to intermittent shear stress (ISS) and to cyclic hydrostatic pressure (CHP) simulating some aspects of the biological milieu in which these cells exist in vivo. Production of nitric oxide (NO) and mRNA expression of several known mechanosensitive genes as well as ERK1/2 activation in the hMSC response to the two mechanical stimuli tested were monitored and compared. NO production depended on the type of the mechanical stimulus to which the hMSCs were exposed and was significantly higher after exposure to ISS than to CHP. At the conditions of NO peak release (i.e., at 0.7 Pa for ISS and 50,000 Pa for CHP), ISS was more effective than CHP in up-regulating mechanosensitive genes. ERK1/2 was activated by ISS but not by CHP. The present study is the first to report that PGTS2, IER3, EGR1, IGF1, IGFBP1, ITGB1, VEGFA and FGF2 are involved in the response of hMSCs to ISS. These findings establish that, of the two mechanical stimuli tested, ISS is more effective than CHP in triggering expression of genes from hMSCs which are bioactive and pertinent to several cell functions (such as cell differentiation and release of specific growth factors and cytokines) and also to tissue-related processes such as wound healing.

Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer.

Transplantation of mesenchymal stem cells (MSCs) with electrotransferred bone morphogenetic protein-2 (BMP-2) transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

Survival and function of mesenchymal stem cells (MSCs) depend on glucose to overcome exposure to long-term, severe and continuous hypoxia.

Use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for various diseases but has generated marginally successful results. A consistent finding of most studies is massive death of transplanted cells. The present study examined the respective roles of glucose and continuous severe hypoxia on MSC viability and function with respect to bone tissue engineering. We hereby demonstrate for the first time that MSCs survive exposure to long-term (12 days), severe (pO(2) < 1.5 mmHg) hypoxia, provided glucose is available. To this end, an in vitro model that mimics the hypoxic environment and cell-driven metabolic changes encountered by grafted sheep cells was established. In this model, the hallmarks of hypoxia (low pO(2) , hypoxia inducible factor-1α expression and anaerobic metabolism) were present. When conditions switched from hypoxic (low pO(2) ) to ischemic (low pO(2) and glucose depletion), MSCs exhibited shrinking, decreased cell viability and ATP content due to complete exhaustion of glucose at day 6; these results provided evidence that ischemia led to the observed massive cell death. Moreover, MSCs exposed to severe, continuous hypoxia, but without any glucose shortage, remained viable and maintained both their in vitro proliferative ability after simulation with blood reperfusion at day 12 and their in vivo osteogenic ability. These findings challenge the traditional view according to which severe hypoxia per se is responsible for the massive MSC death observed upon transplantation of these cells and provide evidence that MSCs are able to withstand exposure to severe, continuous hypoxia provided that a glucose supply is available.

Human dentin characteristics of patients with osteogenesis imperfecta: insights into collagen-based biomaterials.

Osteogenesis imperfecta (OI), also known as "brittle bone disease", is a rare genetic disorder of the skeleton, whose most benign form I corresponds to autosomal dominant mutations in the genes encoding type I collagen (COLA1, COLA2). Several associated skeletal manifestations are often observed but, surprisingly, while dentin defects often reflect genetic bone disorders, about half of OI patients have no obvious oral manifestations. Here, we investigated the collagen, mineral and mechanical properties of dentin from deciduous teeth collected from patients with mild form of OI and displaying no obvious clinical signs of dentinogenesis imperfecta. For the first time, an increase in the hardness of OI dentin associated with an increase in mineral content compared to healthy patients was reported. In addition, OI altered the tissue characteristics of the dentin-enamel junction but the interfacial gradient was preserved. The impact of changes in molecular structure due to mutations in OI was assessed by Raman microspectroscopy. Our results highlighted a change in the hydroxyproline-proline ratio in direct association with collagen mineralization. Our findings suggest that the evaluation of teeth could be an important aid for mild types of OI that are often difficult to diagnose clinically and provide experimental evidence that hydroxyproline content should be considered in future studies on collagen-based biomaterials.

A comparative study of tissue-engineered constructs from Acropora and Porites coral in a large animal bone defect model.

OBJECTIVES: To compare the therapeutic potential of tissue-engineered constructs (TECs) combining mesenchymal stem cells (MSCs) and coral granules from either Acropora or Porites to repair large bone defects. MATERIALS AND METHODS: Bone marrow-derived, autologous MSCs were seeded on Acropora or Porites coral granules in a perfusion bioreactor. Acropora-TECs (n = 7), Porites-TECs (n = 6) and bone autografts (n = 2) were then implanted into 25 mm long metatarsal diaphyseal defects in sheep. Bimonthly radiographic follow-up was completed until killing four months post-operatively. Explants were subsequently processed for microCT and histology to assess bone formation and coral bioresorption. Statistical analyses comprised Mann-Whitney, t-test and Kruskal-Wallis tests. Data were expressed as mean and standard deviation. RESULTS: A two-fold increaseof newly formed bone volume was observed for Acropora-TECs when compared with Porites-TECs (14 sd 1089 mm3versus 782 sd 507 mm3; p = 0.09). Bone union was consistent with autograft (1960 sd 518 mm3). The kinetics of bioresorption and bioresorption rates at four months were different for Acropora-TECs and Porites-TECs (81% sd 5% versus 94% sd 6%; p = 0.04). In comparing the defects that healed with those that did not, we observed that, when major bioresorption of coral at two months occurs and a scaffold material bioresorption rate superior to 90% at four months is achieved, bone nonunion consistently occurred using coral-based TECs. DISCUSSION: Bone regeneration in critical-size defects could be obtained with full bioresorption of the scaffold using coral-based TECs in a large animal model. The superior performance of Acropora-TECs brings us closer to a clinical application, probably because of more suitable bioresorption kinetics. However, nonunion still occurred in nearly half of the bone defects.Cite this article: A. Decambron, M. Manassero, M. Bensidhoum, B. Lecuelle, D. Logeart-Avramoglou, H. Petite, V. Viateau. A comparative study of tissue-engineered constructs from Acropora and Porites coral in a large animal bone defect model. Bone Joint Res 2017;6:208-215. DOI: 10.1302/2046-3758.64.BJR-2016-0236.R1.

Correction of the enzyme defect in cultured congenital erythropoietic porphyria disease cells by retrovirus-mediated gene transfer.

Congenital erythropoietic porphyria (CEP) is a genetic disease characterized by an overproduction and accumulation of porphyrins in bone marrow. The enzyme defect concerns uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the heme biosynthetic pathway. It is the most severe porphyria and the treatment is largely symptomatic: gene therapy would represent a great therapeutic improvement. As a step toward the development of an effective gene therapy, we have constructed two retroviral vectors, LUSN and pMFG-US (with and without the selectable marker Neo), containing a full-length human cDNA for UROIIIS. Recombinant retroviruses were obtained by transfection of the LUSN or pMFG-US plasmid into the amphotropic packaging cell line psi CRIP. For each construct, three different producing clones were selected for their high titer (LUSN) or for their ability to express the message at a high level (pMFG-US). In vitro amplification of genomic DNA from target tissue demonstrated the presence of vector sequences. Murine fibroblasts infected in vitro expressed the human enzyme efficiently, as indicated by RNA and enzymatic studies. Retroviral-mediated gene transfer was then used to introduce the UROIIIS cDNA into human deficient cells. Enzyme activity was increased from 2% (deficient fibroblasts) to 121-274% of the normal value for the different clones. Transduced cells selected with G418 presented an 18-fold increase in enzyme activity compared to the normal cells. Furthermore, high gene transfer rate into peripheral blood progenitor cells (PBPB) was documented by in vitro amplification (PCR). These results demonstrate the potential usefulness of somatic gene therapy for the treatment of CEP.

A systematic analysis of the mutations of the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) or Günther's disease is an inborn error of heme biosynthesis, transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase activity (UROIIIS). The molecular defects observed in CEP are mainly heterogeneous, except for one missense mutation, C73R (Cys to Arg substitution at codon 73) which represents nearly 40% of the disease alleles. A convenient strategy was designed to establish a rapid diagnosis at the genetic level in samples from patients with CEP. As a first step, the most frequent mutation is searched for by restriction analysis from genomic. DNA amplified by PCR. Next, the nine coding exons and intron-exon boundaries are sequenced from genomic DNA. As an alternative, the mutation can be determined by sequencing the UROIIIS cDNA of the patient, using the RT-PCR technique on RNAs when a lymphoblastoid cell line can be established. Finally, for each new mutation in UROIIIS coding sequence, the corresponding mutant protein is expressed in Escherichia coli, in order to demonstrate the pathological significance of the mutation. This work describes the analysis of UROIIIS gene mutations in 10 new families with CEP and summarizes the data from 20 unrelated families studied in our laboratory. Three new missense mutations of UROIIIS coding sequence (H173Y, Q187P and P248Q) have been observed together with 8 known mutations. The significance of three intronic base changes (476 -31 T-->C; 562 -4 A-->T; 562 -23 A-->G) is discussed. In 6 alleles out of 40 (15%), the mutation remains undetermined.

Identification of two new mutations in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) or Günther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity. Six missense mutations in the UROIIIS gene, a deletion and an insertion have already been described in CEP. This work brings further evidence for the heterogeneity in the genetic defect found in CEP. Two new mutations are described, a point mutation (V99A) and a frame-shift mutation (633insA) in the same patient who had a mild to moderate form of Günther's disease. The mutation (V99A) had a detectable residual activity when expressed in Escherichia coli while the insertion (633insA), which introduced a premature stop, had no activity. In the patients studied in our laboratory, the mutation C73R, associated with a severe phenotype, remains the most frequently seen.

[Congenital erythropoietic porphyria. Apropos of a fatal case in the neonatal period due to acute hemolysis with hepatic failure]. / Porphyrie érythropoïétique congénitale. A propos d'un cas fatal en période néonatale dû à une hémolyse aiguë avec insuffisance hépatique.

BACKGROUND: Congenital erythropoietic porphyria, an autosomal recessive disease, is characterized by deficiency of uroporphyrinogen III synthase. Clinical variability of the disease is related to the different mutations found in the patients. CASE REPORT: A newborn suffered one hour after birth from jaundice and polypnea with acute hemolysis. Severe cutaneous photosensitivity occurred after phototherapy. Congenital erythropoietic porphyria was suspected because of reddish-colored urine and confirmed by porphyrin analyses. The baby died one month later due to severe hemolytic anemia with hepatic failure. Uroporphyrinogen III synthase activity was decreased by 99% in bone marrow cells and established lymphoblastoid cells from the patient. Molecular biology studies demonstrated the presence of the Cys 73-->Arg substitution at the homozygous state in the patient. CONCLUSION: This mutation, the most frequently found in this disease, is responsible for a severe phenotype. Molecular characterization provides genotype/phenotype correlations in this porphyria and allows to clarify unusual cases of porphyrias.

Selected Stro-1-enriched bone marrow stromal cells display a major suppressive effect on lymphocyte proliferation.

Mesenchymal stem cells (MSCs) have an immunosuppressive effect and can inhibit the proliferation of alloreactive T cells in vitro and in vivo. Cotransplantation of MSCs and hematopoietic stem cells (HSCs) from HLA-identical siblings has been shown to reduce the incidence of acute graft-vs.-host disease. MSCs are heterogeneous and data on the inhibitory effects of different MSC subsets are lacking. The antigen Stro1 is a marker for a pure primitive MSC subset. We investigated whether Stro-1-enriched induce a more significant suppressive effect on lymphocytes in a mixed lymphocyte reaction (MLR), and whether this action is related to a specific gene expression profile in Stro-1-enriched compared to other MSCs. We demonstrated that the Stro-1-enriched population elicits a significantly more profound dose-dependent inhibition of lymphocyte proliferation in a MLR than MSCs. One thousand expanded Stro-1-enriched induced an inhibitory effect comparable to that of 10 times as many MSCs. Inhibition by Stro-1-enriched was more significant in contact-dependent cultures than in noncontact-dependant cultures at higher ratio. The Stro-1-enriched inhibitory effect in both culture types was linked to increased gene expression for soluble inhibitory factors such as interleukin-8 (IL-8), leukemia inhibitory factor (LIF), indoleamine oxidase (IDO), human leukocyte antigen-G (HLA-G), and vascular cell adhesion molecule (VCAM1). However, tumor growth factor-beta1 (TGF-beta) and IL-10 were only up-regulated in contact-dependant cultures. These results may support using a purified Stro-1-enriched population to augment the suppressive effect in allogeneic transplantation.

Metabolic correction of congenital erythropoietic porphyria by retrovirus-mediated gene transfer into Epstein-Barr virus-transformed B-cell lines.

Congenital erythropoietic porphyria (CEP) is an inherited metabolic disorder resulting from the accumulation of porphyrins because of defective uroporphyrinogen III synthase (UROIIIS). This autosomal recessive disorder is phenotypically heterogeneous with respect to the age of onset and the severity of the symptoms. Different exonic point mutations in the UROIIIS gene have been identified, providing phenotype-genotype correlations in this disease. Severe cases may be treated by bone marrow transplantation and are potential candidates for somatic gene therapy. Epstein-Barr virus-transformed B-cell lines from patients with CEP provide a model system for the disease. We have used retrovirus-mediated expression of UROIIIS to restore enzymatic activity in a B-cell line from a patient. We have also demonstrated the metabolic correction of the disease, ie, porphyrin accumulation into the deficient transduced cells was reduced to the normal level. These data show the potential of gene therapy for this disease.

Immunological, enzymatic and biochemical studies of uroporphyrinogen III-synthase deficiency in 20 patients with congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP), a rare autosomal recessive inborn error of heme biosynthesis, results from reduced activity of uroporphyrinogen III synthase (URO-III-S) leading to an excessive production and accumulation of porphyrins. Various clinical and biochemical observations point to a relationship between degree of disease expression and metabolic disturbance. We investigated 20 patients with early onset of clinical symptoms of CEP and, additionally, the relatives of six patients. CEP was confirmed by porphyrinemia and porphyrinuria with dominance of uroporphyrin and its isomer I. The investigation of the immunological nature of the defective URO-III-S gene from unrelated patients with unknown mutations was possible thanks to an antibody against the human enzyme. URO-III-S concentration in erythrocytes was determined by ELISA. No signal was achieved when assaying nonimmune serum by ELISA, whereas there was a positive reaction with the serum after immunisation. Furthermore, specificity of immune sera is demonstrated by immunoprecipitation of URO-III-S activity which caused a 33% reduction of enzyme activity. Normal levels of immunoreactive enzyme protein 100+/-10% of control (x +/- SD, n = 12) with a reduced specific activity 15+/-8.5% (x +/- SD, n = 12) were found in erythrocytes from all patients, with the exception of a girl with a remarkably high enzyme concentration of 149% of controls and a very low specific activity of 4%. In consequence, all patients had cross-reacting immunological material (CRIM)-positive mutations. CRIM-ratios varied between 3.2 and 24.5. The CRIM-positive nature of the gene defect indicated that the mutations altered the activity of URO-III-S. The different CRIM ratios implied the presence of various mutations, which is further evidence for the heterogeneity in the genetic defect found in CEP. URO-III-S activity was determined in erythrocyte lysates by a coupled enzyme assay. Erythrocyte URO-III-S activities of patients were reduced to 4-33% of the normal value (1.72+/-0.14 pkat/mg protein). An increase of urinary coproporphyrin isomer I (40-61%, norm = 17-31%) and a halved URO-III-S activity can serve as a biochemical test for asymptomatic heterozygous gene carriers of CEP.

A novel point mutation in congenital erythropoietic porphyria in two members of Japanese family.

The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in two members of a Japanese family. This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Gnther's disease. The first patient was homoallelic for a novel missense mutation: a T to C transition of nucleotide 634 that predicted a serine to proline substitution at residue 212 (S212P). The second patient appeared heteroallelic, carrying the same missense mutation and a nonsense mutation: a C to T change at nucleotide 745, resulting in a premature stop at codon 249, instead of a glutamine (Q249X). The corresponding mutated proteins were expressed in Escherichia coli and no residual activity was observed. A family study was also performed to determine the carrier status.