Lucie Tosca

Risk of tumorigenicity in mesenchymal stromal cell–based therapies—Bridging scientific observations and regulatory viewpoints

In the past decade, the therapeutic value of mesenchymal stromal cells (MSCs) has been studied in various indications, thereby taking advantage of their immunosuppressive properties. Easy procurement from bone marrow, adipose tissue or other sources and conventional in vitro expansion culture have made their clinical use attractive. Bridging the gap between current scientific knowledge and regulatory prospects on the transformation potential and possible tumorigenicity of MSCs, the Cell Products Working Party and the Committee for Advanced Therapies organized a meeting with leading European experts in the field of MSCs. This meeting elucidated the risk of potential tumorigenicity related to MSC-based therapies from two angles: the scientific perspective and the regulatory point of view. The conclusions of this meeting, including the current regulatory thinking on quality, nonclinical and clinical aspects for MSCs, are presented in this review, leading to a clearer way forward for the development of such products.

Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience

AIM There is now compelling evidence that cells committed to a cardiac lineage are most effective for improving the function of infarcted hearts. This has been confirmed by our pre-clinical studies entailing transplantation of human embryonic stem cell (hESC)-derived cardiac progenitors in rat and non-human primate models of myocardial infarction. These data have paved the way for a translational programme aimed at a phase I clinical trial. METHODS AND RESULTS The main steps of this programme have included (i) the expansion of a clone of pluripotent hESC to generate a master cell bank under good manufacturing practice conditions (GMP); (ii) a growth factor-induced cardiac specification; (iii) the purification of committed cells by immunomagnetic sorting to yield a stage-specific embryonic antigen (SSEA)-1-positive cell population strongly expressing the early cardiac transcription factor Isl-1; (iv) the incorporation of these cells into a fibrin scaffold; (v) a safety assessment focused on the loss of teratoma-forming cells by in vitro (transcriptomics) and in vivo (cell injections in immunodeficient mice) measurements; (vi) an extensive cytogenetic and viral testing; and (vii) the characterization of the final cell product and its release criteria. The data collected throughout this process have led to approval by the French regulatory authorities for a first-in-man clinical trial of transplantation of these SSEA-1(+) progenitors in patients with severely impaired cardiac function. CONCLUSION Although several facets of this manufacturing process still need to be improved, these data may yet provide a useful platform for the production of hESC-derived cardiac progenitor cells under safe and cost-effective GMP conditions.

Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia.

To explore how RUNX1 mutations predispose to leukemia, we generated induced pluripotent stem cells (iPSCs) from 2 pedigrees with germline RUNX1 mutations. The first, carrying a missense R174Q mutation, which acts as a dominant-negative mutant, is associated with thrombocytopenia and leukemia, and the second, carrying a monoallelic gene deletion inducing a haploinsufficiency, presents only as thrombocytopenia. Hematopoietic differentiation of these iPSC clones demonstrated profound defects in erythropoiesis and megakaryopoiesis and deregulated expression of RUNX1 targets. iPSC clones from patients with the R174Q mutation specifically generated an increased amount of granulomonocytes, a phenotype reproduced by an 80% RUNX1 knockdown in the H9 human embryonic stem cell line, and a genomic instability. This phenotype, found only with a lower dosage of RUNX1, may account for development of leukemia in patients. Altogether, RUNX1 dosage could explain the differential phenotype according to RUNX1 mutations, with a haploinsufficiency leading to thrombocytopenia alone in a majority of cases whereas a more complete gene deletion predisposes to leukemia.

Genotype-phenotype correlation in 13q13.3-q21.3 deletion.

Pure interstitial deletions of the long arm of chromosome 13 are correlated with variable phenotypes according to the size and the location of the deleted region. Deletions involving the 13q13q21 region are rare. In order to establish interstitial 13q genotype-phenotype correlation, we used high resolution 244K oligonucleotide array in addition to conventional karyotype and molecular (fluorescent in situ hybridization, microsatellite markers analysis) techniques in two independent probands carrying a deletion 13q13 to 13q21. First patient was a 3-year-old girl with mental retardation and dysmorphy carrying a 13q13.3q21.31 de novo deletion diagnosed post-natally. The second one was a fetus with de novo del(13)(q14q21.2) associated with first trimester increased nuchal translucency. We showed that specific dysmorphic features (macrocephaly, high forehead, hypertelorism, large nose, large and malformed ears and retrognathia) were correlated to the common 13q14q21 chromosomal segment. Physical examination revealed overgrowth with global measurement up to the 95th percentile in both probands. This is the second description of overgrowth in patients carrying a 13q deletion. Haploinsufficiency of common candidates genes such as CKAP2, SUGT1, LECT1, DCLK1 and SMAD9, involved in cell division and bone development, is a possible mechanism that could explain overgrowth in both patients. This study underlines also that cytogenetic analysis could be performed in patients with overgrowth.

Recurrent 70.8 Mb 4q22.2q32.3 duplication due to ovarian germinal mosaicism.

A mosaicism is defined by the presence of two or more populations of cells with different genotypes in one individual. Chromosomal germinal mosaicism occurs in germ cells before the onset of meiosis. Previously, few studies have described germinal mosaicism. In this study, we report on two siblings who carried identical pure and direct interstitial 4q22.2q32.3 duplication. Procedure investigations included complete clinical description, conventional cytogenetic analysis, fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) array experiments and microsatellite study searching for parental origin of the duplication. Microarray CGH and further FISH experiments with BAC clones showed the same 70.8 Mb direct duplication, dup(4)(q22.2q32.3). Molecular studies of the 4q duplication were consistent with maternal origin associated with mitotic or meiotic rearrangements. This structural chromosomal aberration was associated in both cases with increased nuchal translucency, growth retardation and dysmorphy. Cardiopathy and lung malformations were only evident in the first case. These clinical manifestations are similar to those previously reported in previous studies involving pure 4q trisomy of the same region, except for thumb and renal abnormalities that were not obvious in the presented cases. The amplified region included genes involved in neurological development (NEUROG2, MAB21L2, PCDH10/18 and GRIA2). The recurrent 4q duplication in these siblings is consistent with a maternal ovarian germinal mosaicism. This is the first description of germinal mosaicism for a large chromosomal duplication and highlights that genetic counselling for apparently de novo chromosome aberration should be undertaken with care.

Confined blood chimerism in a monochorionic dizygotic sex discordant twin pregnancy conceived after induced ovulation.

BACKGROUND Monochorionic twins are generally considered as a monozygotic twin pregnancy. However, several cases of monochorial dizygotic twin pregnancies have been reported. CASE REPORT We report on a rare case of monochorionic dizygotic twin pregnancy conceived after induced ovulation in a 32-year-old woman. The diagnosis was made on morphological ultrasound examination at 18+4 weeks of gestation, showing two fetuses with discordant sex. The amniocentesis was declined by the patient. RESULTS The monochorionic status was confirmed after a histopathalogical study of the placenta. At delivery, both a phenotypically normal boy and a phenotypically normal girl without sexual abnormality were observed. This analysis also revealed the presence of vascular anastomoses between both fetal circulations. Postnatal cytogenetic analyses indicated the presence of a chimerism in peripheral blood lymphocytes. This chimerism was not observed in cells obtained from a buccal swab. Molecular determination of zygosity confirmed the existence of the confined peripheral blood chimerism with the presence of four parental alleles. CONCLUSION We report on a case of monochorionic dizygotic twin pregnancy. This observation underlies the need to carefully assess twin pregnancies, especially when obtained after assisted reproductive technology.

Small Supernumerary Marker Chromosomes in Human Infertility.

Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes that cannot be unambiguously identified by banding cytogenetics. The objective of this study was to provide an overview of sSMC frequency and characterization in a context of infertility and to review the literature describing sSMC in relation with male and female infertility. Therefore, a systematic literature review on sSMC associated with infertility was conducted by means of a PubMed literature and a sSMC database (http://ssmc-tl.com/sSMC.html) search. A total of 234 patients with infertility were identified as carriers of sSMC. All chromosomes, except chromosomes 10, 19 and the X, were involved in sSMC, and in 72% the sSMC originated from acrocentric chromosomes. Euchromatic imbalances were caused by the presence of sSMC in 30% of the cases. Putative genes have been identified in only 1.2% of sSMC associated with infertility. The implication of sSMC in infertility could be due to a partial trisomy of some genes but also to mechanical effects perturbing meiosis. Further precise molecular and interphase-architecture studies on sSMC are needed in the future to characterize the relationship between this chromosomal anomaly and human infertility.

Adrenal GIPR expression and chromosome 19q13 microduplications in GIP-dependent Cushing’s syndrome

GIP-dependent Cushings syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Here we performed molecular analyses on adrenocortical adenomas and bilateral macronodular adrenal hyperplasias obtained from 14 patients with GIP-dependent adrenal Cushings syndrome and one patient with GIP-dependent aldosteronism. GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had a 19q duplication only. We demonstrated that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in adenoma cells. Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushings syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.

Transplantation of Macaca cynomolgus iPS-derived hematopoietic cells in NSG immunodeficient mice

The use of iPSCs to generate hematopoietic stem/progenitor cells (HSPC) is of considerable therapeutic interest, as allogeneic HSPC transplantation is limited by the lack of compatible donors, a high risk of engraftment failure and GVHD. Efficacy and safety assessments are required and non-human

Tetrasomy 13q31.1qter due to an inverted duplicated neocentric marker chromosome in a fetus with multiple malformations

Small supernumerary marker chromosome (sSMC) lacking alpha satellite DNA or endogenous centromere regions are rare and contain fully functional centromeres, called neocentromeres. We report on a woman with a 14‐week gestation pregnancy with a cystic hygroma and cerebellar hypoplasia at ultrasound examination. Cytogenetic studies showed a karyotype 47,XY,+mar dn. This sSMC was observed in chorionic villi, lung, and muscle tissue. Array Comparative Genomic Hybridization showed a gain from 13q31.1 to 13qter region. Fluorescent in situ hybridization with pan alpha satellite probe and probes specific for chromosome 13 showed a marker corresponding to an inversion duplication of the 13q distal chromosomal region without alpha satellite DNA sequence, suggesting the presence of a neocentromere. Examination of the fetus showed dysmorphic features, cystic cervical hygroma, postaxial polydactyly of the right hand and left foot with short fingers, malrotation of the gut, and a micropenis with hypospadias. Genotype–phenotype correlation in tetrasomy 13q is discussed according to the four 13q chromosomal breakpoints reported (13q32, 13q31, 13q21, 13q14) for chromosome 13 supernumerary markers.