SPIOMET4HEALTH-efficacy, tolerability and safety of lifestyle intervention plus a fixed dose combination of spironolactone, pioglitazone and metformin (SPIOMET) for adolescent girls and young women with polycystic ovary syndrome: study protocol for a multicentre, randomised, double-blind, placebo-controlled, four-arm, parallel-group, phase II clinical trial.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most prevalent, chronic endocrine-metabolic disorder of adolescents and young women (AYAs), affecting 5-10% of AYAs worldwide. There is no approved pharmacological therapy for PCOS. Standard off-label treatment with oral contraceptives (OCs) reverts neither the underlying pathophysiology nor the associated co-morbidities. Pilot studies have generated new insights into the pathogenesis of PCOS, leading to the development of a new treatment consisting of a fixed, low-dose combination of two so-called insulin sensitisers [pioglitazone (PIO), metformin (MET)] and one mixed anti-androgen and anti-mineralocorticoid also acting as an activator of brown adipose tissue [spironolactone (SPI)], within a single tablet (SPIOMET). The present trial will evaluate the efficacy, tolerability and safety of SPIOMET, on top of lifestyle measures, for the treatment of PCOS in AYAs. METHODS: In this multicentre, randomised, double-blind, placebo-controlled, four-arm, parallel-group, phase II clinical trial, AYAs with PCOS will be recruited from 7 clinical centres across Europe. Intention is to randomise a total of 364 eligible patients into four arms (1:1:1:1): Placebo, PIO, SPI + PIO (SPIO) and SPI + PIO + MET (SPIOMET). Active treatment over 12 months will consist of lifestyle guidance plus the ingestion of one tablet daily (at dinner time); post-treatment follow-up will span 6 months. Primary endpoint is on- and post-treatment ovulation rate. Secondary endpoints are clinical features (hirsutism, menstrual regularity); endocrine-metabolic variables (androgens, lipids, insulin, inflammatory markers); epigenetic markers; imaging data (carotid intima-media thickness, body composition, abdominal fat partitioning, hepatic fat); safety profile; adherence, tolerability and acceptability of the medication; and quality of life in the study participants. Superiority (in this order) of SPIOMET, SPIO and PIO will be tested over placebo, and if present, subsequently the superiority of SPIOMET versus PIO, and if still present, finally versus SPIO. DISCUSSION: The present study will be the first to evaluate-in a randomised, double-blind, placebo-controlled way-the efficacy, tolerability and safety of SPIOMET treatment for early PCOS, on top of a lifestyle intervention. TRIAL REGISTRATION: EudraCT 2021-003177-58. Registered on 22 December 2021. https://www.clinicaltrialsregister.eu/ctr-search/search?query=%092021-003177-58 .

Effects of half-dose spiomet treatment in girls with early puberty and accelerated bone maturation: a multicenter, randomized, placebo-controlled study protocol.

BACKGROUND: A "mismatch" sequence of less prenatal weight gain and more postnatal weight gain may lead to ectopic lipid accumulation, and trigger the development of early adrenarche/pubarche and the activation of the gonadotropic axis resulting in early puberty and ending up in full-blown adolescent polycystic ovary syndrome (PCOS). In the present study, we assess whether a low-dose combination of generics that collectively reduce ectopic fat through different pathways can slow down the accelerated maturation in "mismatch" girls with early puberty. METHODS: Randomized, placebo-controlled, multicenter, phase 2a, study in 64 girls [age, 8.0-9.3 years; birthweight (BW) for gestational age in lower tertile (-1.96< Z-score <-0.44), body mass index (BMI) in upper tertile (+0.44< Z-score < +1.96) and early progressive puberty (Tanner B2 at 7.7-9.0 years)]. Pharmacological intervention will be with a half-dose version of SPIOMET (mini-spiomet), a combination that reverts the PCOS phenotype in "mismatch" adolescents; mini-spiomet will contain spironolactone (25 mg/day, to raise brown adipose tissue activity), pioglitazone (3.75 mg/day, to raise adiponectin and insulin sensitivity), and metformin (425 mg/day, to raise AMPK activity and GDF15). Recruitment: 1 year; double-blind treatment: 1 year; open follow-up: 1 year; analyses and reporting: 1 year. INTERVENTIONS: randomization (1:1) for placebo vs mini-spiomet. PRIMARY OUTCOME: annualized bone age advancement (0-1 year) by BoneXpert; secondary outcomes: insulin, IGF-I, high-molecular-weight adiponectin (HMW-adip), sex hormone binding globulin (SHBG), ultra-sensitive C-reactive protein (usCRP), androgens, luteinizing hormone (LH), follicle-stimulating hormone (FSH), oestradiol, growth-and-differentiation factor 15 (GDF15), C-X-C motif chemokine ligand-14 (CXCL14), safety parameters, and quantification of hepato-visceral fat. DISCUSSION: The present study, if successful, may provide a first proof of the concept that the rapid maturation of girls with an upward mismatch between pre- and post-natal weight gain can be slowed down with a fixed low-dose combination of old and safe generics jointly targeting a reduction of ectopic fat without necessarily lowering body weight. TRIAL REGISTRATION: EudraCT 2021-006766-21. Registered on May 30, 2022.

Posterior Cervical Brown Fat and CXCL14 Levels in the First Year of Life: Sex Differences and Association With Adiposity.

CONTEXT: Brown adipose tissue (BAT) is particularly abundant in neonates, but its association with measures of adiposity and metabolic health in early infancy is poorly delineated. Besides sustaining nonshivering thermogenesis, BAT secretes brown adipokines that act on systemic metabolism. The chemokine CXCL14 has been identified as a brown adipokine in experimental studies. OBJECTIVE: To determine the relationships among BAT activity, adiposity, and circulating CXCL14 levels in the first year of life in girls and boys. METHODS: Indices of fat accretion, circulating endocrine-metabolic parameters and serum CXCL14 levels were assessed longitudinally in a cohort of infants at birth and at 4 and 12 months. BAT activity was estimated using infrared thermography only at age 12 months.The main outcome measures were weight and length Z-scores, total and abdominal fat content (by dual X-ray absorptiometry), BAT activity at the posterior cervical and supraclavicular regions, serum levels of glucose, insulin, insulin-like growth factor-I, high-molecular-weight adiponectin, and CXCL14; CXCL14 transcript levels in neonatal BAT and liver. RESULTS: Posterior cervical BAT was more active in girls than in boys (P = .02). BAT activity was negatively associated with adiposity parameters only in girls. CXCL14 levels were higher in girls than in boys at age 12 months and correlated positively with the area of active posterior cervical BAT in girls. Neonatal BAT showed high CXCL14 gene expression levels. CONCLUSION: BAT activity and the levels of CXCL14-a potential surrogate of BAT activity-are sex specific in the first year of life. Posterior cervical BAT activity associates negatively with indices of adiposity only in girls.

microRNAs in newborns with low birth weight: relation to birth size and body composition.

BACKGROUND: Children with low birth weight (LBW) have a higher risk of developing endocrine-metabolic disorders later in life. Deregulation of specific microRNAs (miRNAs) could underscore the programming of adult pathologies. We analyzed the miRNA expression pattern in both umbilical cord serum samples from LBW and appropriate-for-gestational-age (AGA) newborns and maternal serum samples in the 3rd trimester of gestation, and delineated the relationships with fetal growth, body composition, and markers of metabolic risk. METHODS: Serum samples of 12 selected mother-newborn pairs, including 6 LBW and 6 AGA newborns, were used for assessing miRNA profile by RNA-sequencing. The miRNAs with differential expression were validated in a larger cohort [49 maternal samples and 49 umbilical cord samples (24 LBW, 25 AGA)] by RT-qPCR. Anthropometric, endocrine-metabolic markers and body composition (by DXA) in infants were determined longitudinally over 12 months. RESULTS: LBW newborns presented reduced circulating concentrations of miR-191-3p (P = 0.015). miR-191-3p levels reliably differentiated LBW from AGA individuals (ROC AUC = 0.76) and were positively associated with anthropometric and body composition measures at birth and weight Z-score at 12 months (P < 0.05). CONCLUSIONS: miR-191-3p was reliably different in LBW individuals, and could be a new player in the epigenetic mechanisms linking LBW and future endocrine-metabolic adverse outcomes. IMPACT: Children with low birth weight (LBW) have a higher risk of developing endocrine-metabolic disorders. Deregulation of specific microRNAs (miRNAs) could underscore the programming of those pathologies. miR-191-3p is downregulated in serum of LBW newborns, and its concentrations associate positively with neonatal anthropometric measures, with lean mass and bone accretion at age 15 days and with weight Z-score at age 12 months. miR-191-3p was reliably different in individuals with LBW, and could be a new player in the epigenetic mechanisms connecting LBW and future endocrine-metabolic adverse outcomes.

Gut microbiota in adolescent girls with polycystic ovary syndrome: Effects of randomized treatments.

BACKGROUND: Girls with obesity and polycystic ovary syndrome (PCOS) and women with PCOS have altered gut microbiota. OBJECTIVE: To study the gut microbiota composition of girls with PCOS without obesity (age, 15.8 years; body mass index [BMI] 25 kg/m2 ) and the effects of randomized treatments with an oral contraceptive (OC, N = 15) or with spironolactone-pioglitazone-metformin (SPIOMET, N = 15) for 1 year. Thirty-one age-matched girls served as controls. METHODS: 16S ribosomal subunit gene amplicon sequencing was performed in stool samples from all subjects; samples from 23 out of 30 girls with PCOS (OC, N = 12; SPIOMET, N = 11) were available for analysis post-treatment. Clinical and endocrine-metabolic variables were measured before and after intervention. RESULTS: Girls with PCOS had decreased diversity alpha, altered microbiota pattern and taxonomic profile with more abundance of Family XI (P = .002), and less abundance of family Prevotellaceae (P = .0006) the genus Prevotella (P = .0001) and Senegalimassilia (P < .0001), as compared to controls. Family XI abundance related positively to hepato-visceral fat (R = 0.453; P = .0003). SPIOMET treatment, but not OC, normalized the abundance of Family XI. Prevotellaceae, Prevotella and Senegalimassilia abundance remained unchanged after either treatment. CONCLUSION: SPIOMET's spectrum of normalizing effects in girls with PCOS is herewith broadened as to include Family XI abundance in gut microbiota.

Toward a Treatment Normalizing Ovulation Rate in Adolescent Girls With Polycystic Ovary Syndrome.

Adolescent polycystic ovary syndrome (PCOS) is characterized by androgen excess and oligomenorrhea, and commonly driven by hepato-visceral fat excess ("central obesity") ensuing from a mismatch between prenatal and postnatal nutrition, on a background of genetic susceptibility. There is no approved treatment for adolescent PCOS. We report the pooled results of 2 pilot studies in nonobese girls with PCOS (N = 62, age 15.8 years) that compared the effects of randomized treatment for 1 year, either with an oral estro-progestogen contraceptive (OC), or with a low-dose combination of spironolactone-pioglitazone-metformin (SPIOMET, targeting the excess of ectopic fat). Auxological and endocrine-metabolic variables (including fasting insulin, androgens, high-molecular-weight adiponectin [HMW-adiponectin], and microRNA [miR]-451a), body composition (dual x-ray absorptiometry) and hepato-visceral fat (magnetic resonance imaging) were assessed on- and posttreatment. Data from menstrual diaries were combined with weekly salivary progesterone measurements to infer ovulation rates during the second and fourth quarter of the posttreatment year. OC and SPIOMET treatment reduced the androgen excess comparably, and had no differential effects on total-body lean or fat mass. However, SPIOMET was accompanied by more broadly normalizing effects, including on hepato-visceral fat and on circulating insulin, HMW-adiponectin, and miR-451a. On average, there were 3-fold more ovulations post-SPIOMET than post-OC; normovulation was only observed after SPIOMET; anovulation was >10-fold more prevalent post-OC. Pooled results of randomized studies in nonobese adolescent girls with PCOS indicate that SPIOMET treatment leads to an overall healthier, more insulin-sensitive condition-with less ectopic fat-than OC treatment, and to a more normal posttreatment ovulation rate.

Reduced circulating levels of chemokine CXCL14 in adolescent girls with polycystic ovary syndrome: normalization after insulin sensitization.

OBJECTIVE: CXCL14 (C-X-C motif chemokine ligand-14) is a chemokine released by active brown fat, showing protective effects against insulin resistance in experimental models. Polycystic ovary syndrome (PCOS) in adolescent girls is usually related to hepato-visceral fat excess and insulin resistance, and associates with comorbidities such as type 2 diabetes. Treatment with a low-dose combination of one antiandrogen and antimineralocorticoid drug (spironolactone) and two insulin sensitizers (pioglitazone/metformin) (SPIOMET) is particularly effective in improving these metabolic derangements. Adipose tissue may be involved in the metabolic alterations of PCOS, and it is a likely target of therapeutic action. We investigated the alterations in CXCL14 levels and the effects of drugs composing SPIOMET treatment on CXCL14 in human adipocytes. RESEARCH DESIGN AND METHODS: We studied 51 adolescent patients with PCOS and 21 age-matched healthy controls. Thirty-one adolescent patients with PCOS under SPIOMET or oral contraception-based treatment were also studied. For studies in vitro, Simpson Golabi Behmel Syndrome (SGBS) adipose cells were used. Gene expression for CXCL14 and other genes was quantified using quantitative real-time PCR. The levels of CXCL14 and adipokines in serum and cell culture media were determined by ELISA. RESULTS: Serum CXCL14 levels are reduced in patients with PCOS. One-year SPIOMET treatment normalized CXCL14 concentrations and improved the metabolic status of patients with PCOS. Pioglitazone induced CXCL14 expression in differentiating human SGBS adipocytes, in parallel with the induction of marker genes of brown adipogenesis. Spironolactone induced CXCL14 expression and release in differentiated human adipocytes. CONCLUSION: Insulin sensitization with SPIOMET normalizes the abnormally low levels of CXCL14 in girls with PCOS. This is consistent with the effects of pioglitazone and spironolactone inducing CXCL14 expression and promoting a brown-like phenotype in adipocytes. CXCL14 may be a novel biomarker for PCOS as well as a potential mediator of the beneficial effects of the SPIOMET combination and may hold promise as a therapeutic modulator of the disorder. TRIAL REGISTRATION NUMBERS: ISRCTN29234515 and ISCRCTN11062950.

Targeting pancreatic islet PTP1B improves islet graft revascularization and transplant outcomes.

Deficient vascularization is a major driver of early islet graft loss and one of the primary reasons for the failure of islet transplantation as a viable treatment for type 1 diabetes. This study identifies the protein tyrosine phosphatase 1B (PTP1B) as a potential modulator of islet graft revascularization. We demonstrate that grafts of pancreatic islets lacking PTP1B exhibit increased revascularization, which is accompanied by improved graft survival and function, and recovery of normoglycemia and glucose tolerance in diabetic mice transplanted with PTP1B-deficient islets. Mechanistically, we show that the absence of PTP1B leads to activation of hypoxia-inducible factor 1α-independent peroxisome proliferator-activated receptor γ coactivator 1α/estrogen-related receptor α signaling and enhanced expression and production of vascular endothelial growth factor A (VEGF-A) by ß cells. These observations were reproduced in human islets. Together, these findings reveal that PTP1B regulates islet VEGF-A production and suggest that this phosphatase could be targeted to improve islet transplantation outcomes.

Towards a simple marker of hepato-visceral adiposity and insulin resistance: The Z-score change from weight-at-birth to BMI-in-childhood.

BACKGROUND: Insulin resistance and hepato-visceral (central) fat excess are thought to contribute to an earlier timing of adrenarche/pubarche and puberty/menarche; this earlier timing in turn relates often to a mismatch between prenatal and postnatal weight gain, which can be estimated by calculating the Z-score change from birth weight (BW) to body mass index (BMI) in childhood. METHODS: We tested the hypothesis that this calculation may serve as a proxy of insulin resistance and hepato-visceral adiposity in prepuberty by reappraising a cohort of children (mean age, 8.5 years), born appropriate- (AGA, n = 41) or small-for-gestational age (SGA, n = 45), followed since birth (n = 76) or since the age of 3 years (n = 10). Assessments included anthropometry; fasting glucose and insulin; liver volume; and hepatic fat, subcutaneous fat, and visceral fat in the abdominal region (by magnetic resonance imaging [MRI]). RESULTS: Z-score change BW-BMI closely associated to central fat (R = 0.74; P < .0001) and insulin resistance (R = 0.71; P < .0001). CONCLUSION: These results suggest that Z-score change BW-BMI could be viewed as a simple candidate-marker for hepato-visceral adiposity and insulin resistance in prepubertal children.

Towards a circulating marker of hepato-visceral fat excess: S100A4 in adolescent girls with polycystic ovary syndrome - Evidence from randomized clinical trials.

S100A4 is a marker of subcutaneous adipose tissue dysfunction. Polycystic ovary syndrome (PCOS) is often driven by hepato-visceral adiposity. PCOS phenotypes are normalized more by reduction of central fat with spironolactone/pioglitazone/metformin (SPIOMET) than by oral contraceptive (OC) treatment. We studied whether circulating S100A4 concentrations are high in adolescents with PCOS and, if so, whether they normalize more with OC or SPIOMET. Assessments included circulating S100A4, endocrine markers, body composition, abdominal fat partitioning in controls (n = 12) and girls with PCOS (n = 51; age 15.8 y; body mass index [BMI] 24.5 kg/m2 ), and 1-year changes in girls with PCOS randomized for OC (n = 27) or SPIOMET (n = 24) treatment. Mean S100A4 concentrations were 71% higher (P < 0.001) in girls with PCOS than in controls and associated with hepato-visceral adiposity (r = 0.47; P = 0.001); S100A4 concentrations decreased more (P < 0.01) with SPIOMET, those decreases associating to hepato-visceral fat loss (r = 0.50; P < 0.0001). S100A4 may become a circulating marker of hepato-visceral fat excess in adolescents with PCOS.

Brown adipose tissue in prepubertal children: associations with sex, birthweight, and metabolic profile.

BACKGROUND/OBJECTIVES: Individuals born small-for-gestational age (SGA), especially those who experience postnatal catch-up growth, are at increased risk for developing endocrine-metabolic abnormalities before puberty. In adults, brown adipose tissue (BAT) has been associated with protection against metabolic disorders, such as obesity, type 2 diabetes, and dyslipidaemia. Here, we assessed for the first time whether BAT activation differs between prepubertal children born SGA or appropriate-for-gestational age (AGA). SUBJECTS/METHODS: The study population consisted of 86 prepubertal children [41 AGA and 45 SGA; age (mean ± SEM), 8.5 ± 0.1 years], recruited into two prospective longitudinal studies assessing endocrine-metabolic status and body composition in infancy and childhood. The temperature at the supraclavicular region (SCR) before and after a cold stimulus was measured by infrared thermal imaging, and the area of thermally active SCR (increase after cold challenge, ΔAreaSCR) was calculated as a surrogate index of BAT activation. The results were correlated with clinical, endocrine-metabolic, and inflammation variables, and with visceral and hepatic adiposity (assessed by Magnetic Resonance Imaging). RESULTS: No differences in BAT activation index, as judged by ΔAreaSCR, were found between AGA and SGA children. However, girls showed higher baseline and post-cold induction AreaSCR than boys (both p ≤ 0.01). An interaction between gender and birth weight subgroup was observed for BAT activation; AGA girls increased significantly the ΔAreaSCR as compared to AGA boys; this increase did not occur in SGA girls vs SGA boys. Cold-induced ΔAreaSCR negatively correlated with HOMA-IR, us-CRP, liver volume, and liver fat. CONCLUSIONS: Prepubertal AGA girls had significantly greater BAT activation index as compared to AGA boys; this difference was not observed in SGA subjects. Higher BAT activation associated with a lower amount of visceral fat and with a favorable metabolic profile. Long-term follow-up is needed to determine whether those differences relate to pubertal timing, and to the development of obesity and metabolic disorders.

Body Composition and Circulating Polyunsaturated Fatty Acids at Age 6 Years: A Longitudinal Pilot Study.

Maternal polyunsaturated fatty acid (PUFA) status during pregnancy may influence birth outcomes and offspring adiposity during childhood. Cord blood PUFA levels associate positively with maternal PUFA and negatively with the newborn's abdominal adiposity. However, longitudinal, prospective studies consistently evaluating maternal and cord blood and PUFA levels in childhood and their association with the child's body composition are so far lacking. In a population of 16 apparently healthy children born appropriate for gestational age and followed longitudinally since birth, we assessed circulating PUFA (by gas chromatography) in maternal, cord, and peripheral blood at age 6 years and studied their correlation with body composition (by absorptiometry) and endocrine-metabolic variables at age 6 years. No associations were found among parameters of body composition and endocrine-metabolic variables at age 6 years and maternal, cord blood or circulating PUFA in peripheral blood. Maternal levels of n-6 linoleic acid, total n-6, and the ratio of n-6:n-3 correlated with their corresponding PUFA levels at age 6 years. In conclusion, in this pilot study, maternal, cord blood, and children's circulating PUFA do not appear to have an impact on body composition and endocrine-metabolic status at the age of 6 years. The close association between maternal PUFA and offspring PUFA at that age may reflect a similarity in nutritional habits in the mother and child.

Reduced Prenatal Weight Gain and/or Augmented Postnatal Weight Gain Precedes Polycystic Ovary Syndrome in Adolescent Girls.

OBJECTIVE: Hepato-visceral fat excess is a feature of polycystic ovary syndrome (PCOS). Risk factors for such excess include low prenatal weight gain and high postnatal weight gain. This study examined whether adolescent PCOS was preceded by a relatively low birth weight and/or a relatively high BMI at diagnosis. METHODS: Study participants included 467 girls with PCOS (298 without obesity and 169 with obesity), diagnosed, respectively, in Spain and Germany; 87 healthy girls were controls. Z scores for weight at birth and BMI at PCOS diagnosis were derived, and their differences were calculated. RESULTS: Spanish girls with PCOS and without obesity and German girls with PCOS and obesity had mean birth weight z scores of -0.7 and 0.0, respectively, and mean BMI z scores of + 0.4 and +2.7, respectively, so that mean z score increments amounted to +1.1 and +2.6 (P < 0.001 vs. controls). CONCLUSIONS: PCOS in adolescent girls was preceded by marked z score increments between weight at birth and BMI at PCOS diagnosis, thus corroborating the notion that PCOS development is driven by a mismatch between prenatal weight gain and postnatal weight gain.

Integrative analysis reveals novel pathways mediating the interaction between adipose tissue and pancreatic islets in obesity in rats.

AIMS/HYPOTHESIS: Comprehensive characterisation of the interrelation between the peripancreatic adipose tissue and the pancreatic islets promises novel insights into the mechanisms that regulate beta cell adaptation to obesity. Here, we sought to determine the main pathways and key molecules mediating the crosstalk between these two tissues during adaptation to obesity by the way of an integrated inter-tissue, multi-platform analysis. METHODS: Wistar rats were fed a standard or cafeteria diet for 30 days. Transcriptomic variations by diet in islets and peripancreatic adipose tissue were examined through microarray analysis. The secretome from peripancreatic adipose tissue was subjected to a non-targeted metabolomic and proteomic analysis. Gene expression variations in islets were integrated with changes in peripancreatic adipose tissue gene expression and protein and metabolite secretion using an integrated inter-tissue pathway and network analysis. RESULTS: The highest level of data integration, linking genes differentially expressed in both tissues with secretome variations, allowed the identification of significantly enriched canonical pathways, such as the activation of liver/retinoid X receptors, triacylglycerol degradation, and regulation of inflammatory and immune responses, and underscored interaction network hubs, such as cholesterol and the fatty acid binding protein 4, which were unpredicted through single-tissue analysis and have not been previously implicated in the peripancreatic adipose tissue crosstalk with beta cells. CONCLUSIONS/INTERPRETATION: The integrated analysis reported here allowed the identification of novel mechanisms and key molecules involved in peripancreatic adipose tissue interrelation with beta cells during the development of obesity; this might help the development of novel strategies to prevent type 2 diabetes.

Surface-based cryopreservation strategies for human embryonic stem cells: a comparative study.

Human embryonic stem cells (hESC) hold tremendous potential in the emerging fields of gene and cell therapy as well as in basic scientific research. One of the major challenges regarding their application is the development of efficient cryopreservation protocols for hESC since current methods present poor recovery rates and/or technical difficulties which impair the development of effective processes that can handle bulk quantities of pluripotent cells. The main focus of this work was to compare different strategies for the cryopreservation of adherent hESC colonies. Slow-rate freezing protocols using intact hESC colonies was evaluated and compared with a surface-based vitrification approach. Entrapment within ultra-high viscous alginate was investigated as the main strategy to avoid the commonly observed loss of viability and colony fragmentation during slow-rate freezing. Our results indicate that entrapment beneath a layer of ultra-high viscous alginate does not provide further protection to hESC cryopreserved through slow-rate freezing, irrespectively of the cryomedium used. Vitrification of adherent hESC colonies on culture dishes yielded significantly higher recovery rates when compared to the slow-rate freezing approaches investigated. The pluripotency of hESC was not changed after a vitrification/thawing cycle and during further propagation in culture. In conclusion, from the cryopreservation methods investigated in this study, surface-based vitrification of hESC has proven to be the most efficient for the cryopreservation of intact hESC colonies, reducing the time required to amplify frozen stocks thus supporting the widespread use of these cells in research and clinical applications.

3D cultures of human neural progenitor cells: dopaminergic differentiation and genetic modification. [corrected].

Central nervous system (CNS) disorders remain a formidable challenge for the development of efficient therapies. Cell and gene therapy approaches are promising alternatives that can have a tremendous impact by treating the causes of the disease rather than the symptoms, providing specific targeting and prolonged duration of action. Hampering translation of gene-based therapeutic treatments of neurodegenerative diseases from experimental to clinical gene therapy is the lack of valid and reliable pre-clinical models that can contribute to evaluate feasibility and safety. Herein we describe a robust and reproducible methodology for the generation of 3D in vitro models of the human CNS following a systematic technological approach based on stirred culture systems. We took advantage of human midbrain-derived neural progenitor cells (hmNPCs) capability to differentiate into the various neural phenotypes and of their commitment to the dopaminergic lineage to generate differentiated neurospheres enriched in dopaminergic neurons. Furthermore, we describe a protocol for efficient gene transfer into differentiated neurospheres using CAV-2 viral vectors and stable expression of the transgene for at least 10 days. CAV-2 vectors, derived from canine adenovirus type 2, are promising tools to understand and treat neurodegenerative diseases, in particular Parkinson's disease. CAV-2 vectors preferentially transduce neurons and have an impressive level of axonal retrograde transport in vivo. Our model provides a practical and versatile in vitro approach to study the CNS in a 3D cellular context. With the successful differentiation and subsequent genetic modification of neurospheres we are increasing the collection of tools available for neuroscience research and contributing for the implementation and widespread utilization of 3D cellular CNS models. These can be applied to study neurodegenerative diseases such as Parkinson's disease; to study the interaction of viral vectors of therapeutic potential within human neural cell populations, thus enabling the introduction of specific therapeutic genes for treatment of CNS pathologies; to study the fate and effect of delivered therapeutic genes; to study toxicological effects. Furthermore these methodologies may be extended to other sources of human neural stem cells, such as human pluripotent stem cells, including patient-derived induced pluripotent stem cells.

Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells.

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

Alginate encapsulation as a novel strategy for the cryopreservation of neurospheres.

Primary cultures of brain cell neurospheres are valuable in vitro models for neurotoxicology and brain cell research. Such applications would greatly benefit from the development of efficient cryopreservation protocols that assure the availability of viable and genetically stable stocks of functional neurospheres. In this work we aimed at developing an integrated strategy allowing for long-term culture and cryopreservation of brain cell neurospheres with high viability and reduced recovery time postthawing. Microencapsulation in clinical-grade, ultrahigh viscous, highly purified alginate uniformly cross-linked with Ba(2+) was evaluated as the main strategy to avoid the commonly observed loss of cell-cell and cell-matrix interactions with consequent aggregate's fragmentation and decrease in cell viability that occurs postthawing. Brain cells isolated from 16-day-old fetal rats were cultured in spinner vessels as neurospheres, encapsulated at the 5th day of culture, and cryopreserved at day 19. Culture characterization and assessment of postthawing recovery, concerning cell metabolism, aggregate's cell type composition, and neuron-astrocyte interactions were performed through analysis of membrane integrity, metabolic activity assays, and immunohistochemistry. Our results show that the encapsulation process does not affect cell viability's central metabolism; neither cell differentiation nor cell extensions into cell networks are usually observed between neurons and astrocytes within the neurosphere structure. Neurosphere encapsulation resulted in reduced recovery time postthawing and significantly less fragmentation. Further, the use of serum-free CryoStor™ solution provided further protection for both nonencapsulated and encapsulated aggregates compared with serum-supplemented culture medium as the cryopreservation medium.

Cryopreservation of adherent cells: strategies to improve cell viability and function after thawing.

The commonly applied cryopreservation protocols routinely used in laboratories worldwide were developed for simple cell suspensions, and their application to complex systems, such as cell monolayers, tissues, or biosynthetic constructs, is not straightforward. In particular for monolayer cultures, cell detachment and membrane damage are often observed after cryopreservation. In this work, combined strategies for the cryopreservation of cells attached to Matrigel-coated well plate's surfaces were investigated based on cell entrapment in clinicalgrade, ultra-high viscosity alginate using two cell lines, neuroblastoma N2a and colon adenocarcinoma Caco-2, with distinct structural and functional characteristics. As the cryopreservation medium, serum-free CryoStor solution was compared with serum-supplemented culture medium, both containing 10% DMSO. Using culture medium, entrapment beneath an alginate layer was needed to improve cell recovery by minimizing membrane damage and cell detachment after thawing; nevertheless, up to 50% cell death still occurred within 24 h after thawing. The use of CryoStor solution represented a considerable improvement of the cryopreservation process for both cell lines, allowing the maintenance of high postthaw membrane integrity as well as full recovery of metabolic activity and differentiation capacity within 24 h postthawing; in this case, entrapment beneath an alginate layer did not confer further protection to cryopreserved Caco-2 cells, but was crucial for maintenance of attachment and integrity of N2a neuronal networks.