Relationship between fear of progression and symptom burden, disease factors and social/family factors in patients with stage-IV breast cancer in Shandong, China.

OBJECTIVE: To assess fear of progression (FoP)'s relationship with symptom burden and disease and social/family factors, as well as, determine the status of FoP in women with stage-IV breast cancer in Shandong, China. METHODS: Two hundred and sixteen women were recruited from the department of breast cancer internal medicine, Shandong Cancer Hospital and Institute. Data for this observational study were collected between October 2020 and January 2021 using the MD Anderson Symptom Inventory, the Fear of Progression Questionnaire-Short Form (FoP-Q-SF) and a participant information scale. SPSS 23.0 was used for statistical analysis. RESULTS: After excluding invalid responses, the data of 200 participants were analysed. The average total FoP-Q-SF score was 29.39 ± 9.39 (95% confidence interval, 21.81-27.64). The FoP level among the participants was relatively low. For disease and social/family factors, FoP statistically significantly differed by satisfaction with family emotional support and the Eastern Cooperative Oncology Group (ECOG) score. The ECOG score was positively correlated with FoP. Furthermore, symptom burden was positively correlated with FoP. CONCLUSIONS: Among patients with stage-IV breast cancer, satisfaction with family emotional support, ECOG score and symptom burden play key roles in FoP. Interventions, including providing appropriate emotional support from family, improving physical fitness and relieving symptom burden, must be considered in future studies, which may improve patients' overall physical and mental status and provide a supportive therapeutic environment.

The Potential Effect of General Anesthetics in Cancer Surgery: Meta-Analysis of Postoperative Metastasis and Inflammatory Cytokines.

Metastasis or recurrence following curative surgery is the main indicator of tumor progress and is the main cause of patient death. For more than three decades, the potential for general anesthesia to affect cancer outcomes has been a subject of concern with considerable research interest. Here, we conducted this systematic review and meta-analysis to summarize the effect of inhalational anesthesia (IHNA) vs. propofol-based total intravenous anesthesia (TIVA) on metastasis and recurrence after cancer surgery from clinical and pre-clinical studies. The relative risk for metastasis/recurrence in TIVA is 0.61 (95% confidence interval (95% CI) 0.46 to 0.82, p = 0.0009) compared to IHNA. Inflammatory cytokines have been implicated in cancer metastasis following cancer surgery, thus we analyzed inflammatory cytokines levels after surgery under IHNA or TIVA. Based on pooled analysis, a lower IL-6 level was noticed in TIVA in comparison to IHNA (standardized mean difference (SMD) = 0.77, 95% CI = 0.097 to 1.44, I2 = 92%, p = 0.02) but not TNF-α or IL-10. Preclinical animal model studies show that inhalational anesthetics increase the risk of breast cancer metastasis compared to propofol. In conclusion, the current evidence suggests intravenous anesthetic propofol is associated with less metastasis/recurrence and lower postoperative IL-6 level over inhaled anesthetics in the oncological surgery. We urge more well-designed clinical and preclinical studies in this field.

Comparison of endocrine therapy and chemotherapy as different systemic treatment modes for metastatic luminal HER2-negative breast cancer patients -A retrospective study.

Purpose: The purpose of this study was to evaluate endocrine therapy and chemotherapy for first-line, maintenance, and second-line treatment of hormone receptor-positive HER-2-negative metastatic breast cancer (HR+HER-2-MBC) and the relationship between different treatment options and survival. Patients and methods: The patients included in this study were all diagnosed with metastatic breast cancer (MBC) at Shandong Cancer Hospital from January 2013 to June 2017. Of the 951 patients with MBC, 307 patients with HR+HER-2-MBC were included in the analysis. The progression-free survival (PFS) and overall survival (OS) of the various treatment modes were evaluated using Kaplan-Meier analysis and the log-rank test. Because of the imbalance in data, we used the synthetic minority oversampling technique (SMOTE) algorithm to oversample the data to increase the balanced amount of data. Results: This retrospective study included 307 patients with HR+HER-2-MBC; 246 patients (80.13%) and 61 patients (19.87%) were treated with first-line chemotherapy and first-line endocrine therapy, respectively. First-line endocrine therapy was better than first-line chemotherapy in terms of PFS and OS. After adjusting for known prognostic factors, patients receiving first-line chemotherapy had poorer PFS and OS outcomes than patients receiving first-line endocrine therapy. In terms of maintenance treatment, the endocrine therapy-endocrine therapy maintenance mode achieved the best prognosis, followed by the chemotherapy-endocrine therapy maintenance mode and chemotherapy-chemotherapy maintenance mode, and the no-maintenance mode has resulted in the worst prognosis. In terms of first-line/second-line treatment, the endocrine therapy/endocrine therapy mode achieved the best prognosis, while the chemotherapy/chemotherapy mode resulted in the worst prognosis. The chemotherapy/endocrine therapy mode achieved a better prognosis than the endocrine therapy/chemotherapy mode. There were no significant differences in the KI-67 index (<15%/15-30%/≥30%) among the patients receiving first-line treatment modes, maintenance treatment modes, and first-line/second-line treatment modes. There was no statistical evidence in this study to support that the KI-67 index affected survival. However, in the first-line/second-line model, after SMOTE, we could see that KI-67 ≥ 30% had a poor prognosis. Conclusions: Different treatment modes for HR+HER-2-MBC were analyzed. Endocrine therapy achieved better PFS and OS outcomes than chemotherapy. Endocrine therapy should be the first choice for first-line, maintenance, and second-line treatment of HR+HER-2-MBC.

Transcriptomic Changes of Photoperiodic Response in the Hypothalamus Were Identified in Ovariectomized and Estradiol-Treated Sheep.

Accurate timing of seasonal changes is an essential ability for an animal's survival, and the change in the photoperiod is the key factor affecting reproductive seasonality in mammals. Emerging evidence has suggested that multiple hypothalamic genes participate in the photoperiod-induced regulation of reproductive activities in sheep, but the mechanism is still unclear. In this study, we initially examined the plasma level of two major reproductive hormones, namely, follicle-stimulating hormone (FSH) and prolactin (PRL), under different photoperiods in ovariectomized and estradiol-treated (OVX + E2) sheep using radioimmunoassay (RIA). Of the two hormones, the concentration of PRL significantly increased with the extension of the photoperiod, while FSH showed the opposite trend. Subsequently, an examination of the transcriptomic variation between the short photoperiod (SP) and long photoperiod (LP) was conducted. Differential expression analyses and functional annotation showed that several key genes in the insulin secretion (VAMP2, PRKACB, PRKCG, and PLCB1), GnRH (MAPK13, CGA, CDC42, ATF4, and LHB) pathways, and circadian entrainment (KCNJ5, PER1, GNB2, MTNR1A, and RASD1), as well as numerous lncRNAs, including XR_173257.3, XR_173415.3, XR_001435315.1, XR_001024596.2, and XR_001023464.2, were shown potentially vital for the hypothalamic photoperiodic response. Four of the differentially expressed mRNAs and lncRNAs were validated by qPCR. The constructed mRNA-mRNA interaction networks further revealed that transcripts potentially participated in hypothalamic thyroid hormone synthesis, endocrine resistance, and neuroactive ligand-receptor interactions. The interactome analysis of lncRNAs and their targets implied that XR_173257.3 and its target arylalkylamine N-acetyltransferase (AANAT) and XR_173415.3 and its target TH might participate in the regulation of seasonal reproduction. Together, the changes in reproductive hormones and transcriptome will help to determine the important photoperiod-induced lncRNAs and mRNAs and provide a valuable resource for further research on reproductive seasonality in sheep.

No Difference Among Inhaled Anesthetics on the Growth and Metastasis of Murine 4T1 Breast Cancers in a Mouse Model of Spontaneous Metastasis.

Objective: This study evaluates the effect of the commonly used inhaled anesthetics isoflurane, sevoflurane, and desflurane on the viability and migration of murine 4T1 breast cancer cells, the growth, and lung metastasis in a syngenetic model of spontaneous metastasis. Methods: The murine 4T1 breast cancer cells were exposed to isoflurane (2%), sevoflurane (3.6%), or desflurane (10.3%) for 3 h. Cell viability was measured using the MTT assay. The migratory capacity of 4T1 cells was assessed using a scratch assay after 24 h incubation. Female balb/c mice were subjected to orthotopic implantation of 4T1 cells under anesthesia with one of the inhaled anesthetics: 2% isoflurane, 3.6% sevoflurane, or 10.3% desflurane. Subsequently, resection of primary tumors was performed under the identical anesthetic used during implantation for 3 h. Three weeks later, the mice were euthanized to harvest lungs for ex vivo bioluminescent imaging and histological analysis. Blood was collected for serum cytokine assays by ELISA. Results: There was no difference in cell viability among isoflurane, sevoflurane, desflurane, and control groups (n = 180 for each group, P = 0.648). Sevoflurane but not isoflurane or desflurane significantly increased the migration of 4T1 cells compared to the control group (n = 18, P = 0.024). There was no difference in the growth of the orthotopically implanted primary tumors (n = 12 for the isoflurane group, n = 11 for the sevoflurane group, and for the desflurane group, P = 0.879). Surgical dissection of primary tumors in mice under anesthesia with isoflurane, sevoflurane, or desflurane led to no difference in lung metastasis following surgery (P = 0.789). No significant difference was observed among isoflurane, sevoflurane, and desflurane groups in the serum levels of IL-6 (P = 0.284), CCL-1 (P = 0.591), MCP-1 (P = 0.135), and VEGF (P = 0.354). Conclusion: Our study demonstrated that sevoflurane increased the migration of 4T1 breast cancer cells in vitro. Inhaled anesthetics isoflurane, sevoflurane, and desflurane had no difference on the growth of primary tumor and the lung metastasis of 4T1 cells in the mouse model of spontaneous metastasis with surgical removal of primary tumors.

Effects of FecB Mutation on Estrus, Ovulation, and Endocrine Characteristics in Small Tail Han Sheep.

The Booroola fecundity gene (FecB) has a mutation that was found to increase the ovulation rate and litter size in Booroola Merino sheep. This mutation is also associated with the fecundity of small-tail han (STH) sheep, an important maternal breed used to produce hybrid offspring for mutton production in China. Previous research showed that the FecB gene affects reproduction in STH sheep, based on litter size records. However, the effects of this gene on estrus, ovulation, and endocrine characteristics in these sheep remain unclear. Here, we analyzed the traits mentioned earlier and compared them among the three FecB genotypes of STH ewes using estrus synchronization. Overall, 53 pluriparous ewes were selected from among 890 STH ewes and subjected to FecB genotyping for experiments to characterize estrous and ovulation rates. FecB heterozygous (+B) ewes presented an earlier onset of estrus (42.9 ± 2.2 h) and a shorter estrous cycle (17.2 ± 0.2 days) (P ≤ 0.05). The ovulation rates increased with the increasing copy number of the B allele (P ≤ 0.01). Ovulation time showed no significant differences among the three FecB genotypes. The serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone, estrogen (E2), and progesterone (P4) were measured in 19 of the ewes. Serum concentrations of E2 and FSH dramatically varied around the time of behavioral estrus. In FecB mutant homozygous (BB) ewes, E2 concentration had two peaks, which were higher (P ≤ 0.05) than those of ++ genotypes. FSH concentration of BB ewes was higher (P ≤ 0.05) than that of the ++ ewes just after estrus. The expression of the estrogen receptor 1 (ESR1) gene in the +B genotype was higher than in the other genotypes. Based on the data for the reproductive performance of STH ewes with the three FecB genotypes, our study suggests that the development of follicles in ewes with the B allele is dependent on the response to FSH regulated by E2 in the early stage. +B ewes, exhibiting moderate ovulation and litter size and a shorter estrous cycle, can be highly recommended in sheep crossbreeding systems for commercial mutton production. Moreover, this study provides useful information to conserve better and use the genetic resources of STH sheep in China.

Transcriptome Analysis of Neuroendocrine Regulation of Ovine Hypothalamus-Pituitary-Ovary Axis during Ovine Anestrus and the Breeding Season.

Most sheep are seasonal estrus, and they breed in autumn when the days get shorter. Seasonal estrus is an important factor that affects the productivity and fertility of sheep. The key point to solve this problem is to explore the regulation mechanism of estrus in sheep. Therefore, in this study, transcriptomic sequencing technology was used to identify differentially expressed mRNAs in the hypothalamus, pituitary and ovary of Small Tail Han sheep (year-round estrus) and tan sheep (seasonal estrus) among luteal, proestrus and estrus stages. There were 256,923,304,156 mRNAs being identified in the hypothalamus, pituitary and ovary, respectively. Functional analysis showed that the photosensor, leucine and isoleucine biosynthesis pathways were enriched significantly. It is speculated that photoperiod may initiate estrus by stimulating the corresponding pathways in hypothalamus. ODC1, PRLH, CRYBB2, SMAD5, OPN1SW, TPH1 are believed to be key genes involved in the estrogen process. In conclusion, this study expanded the database of indigenous sheep breeds, and also provided new candidate genes for future genetic and molecular studies on the seasonal estrus trait in sheep.

Whole Genome Sequencing Reveals the Effects of Recent Artificial Selection on Litter Size of Bamei Mutton Sheep.

Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. Here, we focused on detecting candidate genes associated with the increasing of the litter size in this breeds under recent artificial selection to improve the efficiency of mutton production. We selected five high- and five low-fecundity Bamei mutton sheep for whole-genome resequencing to identify candidate genes for sheep prolificacy. We used the FST and XP-EHH statistical approach to detect the selective sweeps between these two groups. Combining the two selective sweep methods, the reproduction-related genes JUN, ITPR3, PLCB2, HERC5, and KDM4B were detected. JUN, ITPR3, and PLCB2 play vital roles in GnRH (gonadotropin-releasing hormone), oxytocin, and estrogen signaling pathway. Moreover, KDM4B, which had the highest FST value, exhibits demethylase activity. It can affect reproduction by binding the promoters of estrogen-regulated genes, such as FOXA1 (forkhead box A1) and ESR1 (estrogen receptor 1). Notably, one nonsynonymous mutation (p.S936A) specific to the high-prolificacy group was identified at the TUDOR domain of KDM4B. These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. The identified genomic regions that are responsible for litter size can in turn be used for further selection.

Expression and functional analysis of the Follistatin-like 3 (FSTL3) gene in the sheep ovary during the oestrous cycle.

Follistatin-like 3 (FSTL3) is a regulator of cellular apoptosis and was previously identified via RNA-Seq to be associated with follicular development in mammalian ovaries. However, the mechanism underlying the FSTL3 regulation of oestrus in sheep remained poorly understood. In this study, the oestrogen (E2) and progesterone (P4) concentrations in blood were detected, and the expression level and functional analysis of FSTL3 in the ovary were studied during the different reproductive stage in Aohan fine wool sheep (seasonal breeding breed in China). The concentrations of E2 and P4 at the anestrus were significantly lower compared to dioestrus, proestrus and oestrus stages. Higher expression levels of FSTL3 were observed in the sheep ovary, hypothalamus, and thyroid. During different reproductive stages, higher expression levels were found during the stages of dioestrus and proestrus, while lower levels were found during the oestrus and anestrus stages. Functional analysis of FSTL3 was performed in primary granulosa cells (GCs) of sheep. The concentration of E2 increased significantly after RNAi interference of FSTL3, while the P4 level decreased. FSTL3 can decrease P4 levels, which might be involved in mediating oestrous cycle in sheep.

Regulation of cytochrome P450 4F11 expression by liver X receptor alpha.

Cytochrome P450 4F (CYP4F) enzymes are responsible for the metabolism of eicosanoids, which play important roles in inflammation. Nuclear receptor liver X receptor alpha (LXRα) is a critical signal node connecting inflammation and lipid metabolism. Studies revealed that the release of cytokines and nuclear factor-κB (NF-κB) can change the CYP4F11 expression in HepG2 cells. However, the effect of LXRα on the CYP4F family and the underlying mechanism remain unclear. This study found that CYP4F11 is a target gene of LXRα. Luciferase assays and siRNA transfection showed that LXRα increased the transcription of CYP4F11 and LXRα agonist GW3965 could induce the expression of CYP4F11 by activating the LXRα-CYP4F11 pathway. Besides, overexpression of CYP4F11 could decrease TNF-α and IL-1ß in lipopolysaccharide (LPS)-induced THP-1 cells. The finding of the regulation of CYP4F11 may contribute to the anti-inflammatory activity of LXRα agonists.

Highly efficient cascading synergy of cancer photo-immunotherapy enabled by engineered graphene quantum dots/photosensitizer/CpG oligonucleotides hybrid nanotheranostics.

Currently, photoimmunotherapy based on a theranostic nanoplatform emerges as a promising modality in advanced cancer therapy. In this study, a new type of versatile nanoassemblies (denoted as PC@GCpD(Gd)) was rationally designed by integrating the polydopamine stabilized graphene quantum dots (GQD)-photosensitizer nanocomposites (denoted as GCpD), immunostimulatory polycationic polymer/CpG oligodeoxynucleotide (CpG ODN) nanoparticles (denoted as PC) and Gd3+/Cy3 imaging probes for dual magnetic resonance/fluorescence imaging-guided photoimmunotherapy. PC@GCpD(Gd) effectively killed the tumor cells through the amplified photothermal and photodynamic effects mediated by GCpD, and contemporaneously delivered CpG ODN to the targeted endosomal Toll-like receptor 9 (TLR9) to continuously stimulate the secretion of proinflammatory cytokines and the maturation of dendritic cells, thereby resulting in the activation and infiltration of T lymphocytes. As a result, PC@GCpD(Gd) achieved robust inhibition efficiency to almost completely suppress the EMT6 murine mammary cancer model under laser irradiation, implying the superior synergy of combined photoimmunotherapy. Moreover, the in vivo delivery and biodistribution of PC@GCpD(Gd) could be tracked using the high-quality bimodal magnetic resonance imaging/fluorescence imaging. This study highlighted the potent prospect of hybrid PC@GCpD(Gd) nanoassemblies for precise cancer photoimmunotherapy with a cascading effect.

"Triple-Punch" Anticancer Strategy Mediated by Near-Infrared Photosensitizer/CpG Oligonucleotides Dual-Dressed and Mitochondria-Targeted Nanographene.

Nanomedicine-based combination therapy has sparked a growing interest in clinical disease treatment and pharmaceutical industry. In this study, a mitochondria-targeted and near-infrared (NIR) light-activable multitasking nanographene (i.e., GT/IR820/DP-CpG) was engineered to in situ trigger highly efficient "triple-punch" strategy of cancer photodynamic therapy, photothermal therapy, and immunotherapy. Modification of triphenylphosphonium on graphene made the vehicle specifically guide the NIR dye IR820 home to mitochondria, followed by lysosomes escape in a time-dependent manner. The photoactive nanocomplex generated an abundant reactive oxygen species as well as photothermal heat to ultimately kill cancer cells by inducing mitochondrial collapse and irreversible cell apoptosis upon the NIR laser irradiation. Further introduction of an immunostimulatory conjugate DP-CpG significantly promoted the secretion of proinflammatory cytokines (i.e., interleukin-6, tumor necrosis factor-α, and interferon-γ) and thus improved the immunogenicity of tumors. In vivo studies demonstrated that GT/IR820/DP-CpG remarkably inhibited tumor growth (tumor inhibition rate, ∼88%) resulting from the combinational phototherapeutic effect of IR820 and immunostimulatory activity of DP-CpG, thereby causing negligible toxic effects on mice. Our work provides a new paradigm of architecting organelle-targeted and stimulative nanocomplex for highly efficient cancer photoimmunotherapy.

Metabolic Effects of FecB Gene on Follicular Fluid and Ovarian Vein Serum in Sheep (Ovis aries).

The FecB gene has been discovered as an important gene in sheep for its high relationship with the ovulation rate, but its regulatory mechanism remains unknown. In the present study, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were adopted to detect the metabolic effects of FecB gene in follicular fluid (FF) and ovarian vein serum (OVS) in Small Tail Han (STH) sheep. ANOVA and random forest statistical methods were employed for the identification of important metabolic pathways and biomarkers. Changes in amino acid metabolism, redox environment, and energy metabolism were observed in FF from the three FecB genotype STH ewes. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) showed that metabolic effects of FecB gene are more pronounced in FF than in OVS. Therefore, the difference of the metabolic profile in FF is also affected by the FecB genotypes. In Spearman correlation analysis, key metabolites (e.g., glucose 6-phosphate, glucose 1-phosphate, aspartate, asparagine, glutathione oxidized (GSSG), cysteine-glutathione disulfide, γ-glutamylglutamine, and 2-hydrosybutyrate) in ovine FF samples showed a significant correlation with the ovulation rate. Our findings will help to explain the metabolic mechanism of high prolificacy ewes and benefit fertility identification.

Molecular cloning and epigenetic change detection of Kiss1 during seasonal reproduction in Chinese indigenous sheep.

Like most seasonal domesticated species, sheep are short-day breeders, which means that the reproduction axis is activated by short days. The annual photoperiodic cycle affects the amount of daylength information that is transmitted to the hypothalamic-pituitary-gonadal (HPG) axis by regulating pulsatile secretion of gonadotrophin-releasing hormone from the hypothalamus. Kisspeptin, which is encoded by Kiss1, plays a major role in reproductive seasonality. Based on results from our previous Solexa sequencing data obtained from Tan (T) and Small Tail Han (STH) sheep during anoestrus and the breeding season, full-length mRNA information for ovine Kiss1 was obtained; 894bp in T sheep and 1145bp in STH sheep. Both encode 135 amino acids. Additionally, T and STH sheep have different transcription start sites of Kiss1. Kiss1 expression during oestrus was significantly higher than that during dioestrus, both in T and STH sheep (P<0.01). We also found a strong relationship between Kiss1 mRNA levels and histone H3 acetylation status in the 5' promoter region of ovine Kiss1. These data indicated that epigenetic modification occurs during reproduction in sheep, and this is the first report that histone H3 deacetylation occurs in the hypothalamus of seasonal sheep breeders during the transition from dioestrus to oestrus.

Characterization and comparative profiling of ovarian microRNAs during ovine anestrus and the breeding season.

BACKGROUND: Seasonal estrus is a critical limiting factor of animal fecundity, and it involves changes in both ovarian biology and hormone secretion in different seasons. Previous studies indicate that two classes of small RNAs (miRNAs and piRNAs) play important regulatory roles in ovarian biology. To understand the roles of small RNA-mediated post-transcriptional regulation in ovine seasonal estrus, the variation in expression patterns of ovarian small RNAs during anestrus and the breeding season were analyzed using Solexa sequencing technology. In addition, reproductive hormone levels were determined during ovine anestrus and the breeding season. RESULTS: A total of 483 miRNAs (including 97 known, 369 conserved and 17 predicated novel miRNAs), which belong to 183 different miRNA families, were identified in ovaries of Tan sheep and Small Tail Han (STH) sheep. Compared with the three stages of the breeding season, 25 shared significantly differentially expressed (including 19 up- and six down-regulated) miRNAs were identified in ovine anestrus. KEGG Pathway analysis revealed that the target genes for some of the differentially expressed miRNAs were involved in reproductive hormone related pathways (e.g. steroid biosynthesis, androgen and estrogen metabolism and GnRH signaling pathway) as well as follicular/luteal development related pathways. Moreover, the expression of the differentially expressed miRNAs and most of their target genes were negatively correlated in the above pathways. Furthermore, the levels of estrogen, progesterone and LH in ovine anestrus were significantly lower than those in the breeding season. Combining the results of pathway enrichment analysis, expression of target genes and hormone measurement, we suggest that these differentially expressed miRNAs in anestrus might participate in attenuation of ovarian activity by regulating the above pathways. Besides miRNAs, a large and unexpectedly diverse set of piRNAs were also identified. CONCLUSIONS: The miRNA profiles of ovine ovaries in anestrus were presented for the first time. The identification and characterization of miRNAs that are differentially expressed between ovine anestrus and the breeding season will help understanding of the role of miRNAs in the regulation of seasonal estrus, and provides candidates for determining miRNAs which could be potentially used to regulate ovine seasonal estrus.

Survival and engraftment of dopaminergic neurons manufactured by a Good Manufacturing Practice-compatible process.

BACKGROUND AIMS: We have previously reported a Good Manufacturing Practice (GMP)-compatible process for generating authentic dopaminergic neurons in defined media from human pluripotent stem cells and determined the time point at which dopaminergic precursors/neurons (day 14 after neuronal stem cell [NSC] stage) can be frozen, shipped and thawed without compromising their viability and ability to mature in vitro. One important issue we wished to address is whether dopaminergic precursors/neurons manufactured by our GMP-compatible process can be cryopreserved and engrafted in animal Parkinson disease (PD) models. METHODS: In this study, we evaluated the efficacy of freshly prepared and cryopreserved dopaminergic neurons in the 6-hydroxydopamine-lesioned rat PD model. RESULTS: We showed functional recovery up to 6 months post-transplantation in rats transplanted with our cells, whether freshly prepared or cryopreserved. In contrast, no motor improvement was observed in two control groups receiving either medium or cells at a slightly earlier stage (day 10 after NSC stage). Histologic analysis at the end point of the study (6 months post-transplantation) showed robust long-term survival of donor-derived tyrosine hydroxylase (TH)(+) dopaminergic neurons in rats transplanted with day 14 dopaminergic neurons. Moreover, TH(+) fibers emanated from the graft core into the surrounding host striatum. Consistent with the behavioral analysis, no or few TH(+) neurons were detected in animals receiving day 10 cells, although human cells were present in the graft. Importantly, no tumors were detected in any grafted rats, but long-term tumorigenic studies will need to determine the safety of our products. CONCLUSIONS: Dopaminergic neurons manufactured by a GMP-compatible process from human ESC survived and engrafted efficiently in the 6-OHDA PD rat model.

Optimizing dopaminergic differentiation of pluripotent stem cells for the manufacture of dopaminergic neurons for transplantation.

BACKGROUND AIMS: We have previously described a xeno-free scalable system to generate transplantable dopaminergic neurons from human pluripotent stem cells. However, several important questions remain to be answered about our cell therapy efforts. These include determining the exact time at which cells should be transplanted and whether cells at this stage can be frozen, shipped, thawed and injected without compromising their ability to mature and survive the transplantation procedure. We also needed to determine whether further optimization of the culture process could shorten the development time and reduce variability and whether a current Good Manufacture Practice (CGMP) facility could manufacture cells with fidelity. METHODS: We developed an optimized protocol that included modulating the sonic hedgehog homolog gradient with bone morphogenetic proteins (BMP2) and addition of activin to the culture medium, which shortened the time to generate Lmx1A and FoxA2 immunoreactive cells by 4-6 days. RESULTS: We showed that cells at this stage could be safely frozen and thawed while retaining an excellent ability to continue to mature in vitro and survive transplant in vivo. Importantly, we successfully adapted this process to a CGMP facility and manufactured two lots of transplant-ready dopaminergic neurons (>250 vials) under CGMP-compatible conditions. In vitro characterization, including viability/recovery on thawing, whole genome expression as well as expression of midbrain/dopaminergic markers, showed that the cells manufactured under GMP-compatible conditions were similar to cells produced at lab scale. CONCLUSIONS: Our results suggest that this optimized protocol can be used to generate dopaminergic neurons for Investigational New Drug enabling studies.

Efficient generation of astrocytes from human pluripotent stem cells in defined conditions.

Astrocytes can be generated from various tissue sources including human pluripotent stem cells (PSC). In this manuscript, we describe a chemically defined xeno-free medium culture system for rapidly generating astrocytes from neural stem cells derived from PSC. We show that astrocyte development in vitro, mimics normal development in vivo, and also passes through a CD44(+) astrocyte precursor stage. Astrocytes generated by our method display similar gene expression patterns, morphological characteristics and functional properties to primary astrocytes, and they survive and integrate after xenotransplantation. Whole genome expression profiling of astrocyte differentiation was performed at several time points of differentiation, and the results indicate the importance of known regulators and identify potential novel regulators and stage-specific lineage markers.

Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells: induction, maintenance, and differentiation into functional schwann cells.

The neural crest (NC) is a transient, multipotent, migratory cell population unique to vertebrates that gives rise to diverse cell lineages. Much of our knowledge of NC development comes from studies of organisms such as chicken and zebrafish because human NC is difficult to obtain because of its transient nature and the limited availability of human fetal cells. Here we examined the process of NC induction from human pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). We showed that NC cells could be efficiently induced from hESCs by a combination of growth factors in medium conditioned on stromal cells and that NC stem cells (NCSCs) could be purified by p75 using fluorescence-activated cell sorting (FACS). FACS-isolated NCSCs could be propagated in vitro in five passages and cryopreserved while maintaining NCSC identity characterized by the expression of a panel of NC markers such as p75, Sox9, Sox10, CD44, and HNK1. In vitro-expanded NCSCs were able to differentiate into neurons and glia (Schwann cells) of the peripheral nervous system, as well as mesenchymal derivatives. hESC-derived NCSCs appeared to behave similarly to endogenous embryonic NC cells when injected in chicken embryos. Using a defined medium, we were able to generate and propagate a nearly pure population of Schwann cells that uniformly expressed glial fibrillary acidic protein, S100, and p75. Schwann cells generated by our protocol myelinated rat dorsal root ganglia neurons in vitro. To our knowledge, this is the first report on myelination by hESC- or iPSC-derived Schwann cells.

Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions.

Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. As a first step, critical to clinical applications, we attempted to develop defined culture conditions to expand and differentiate human iPSCs into functional progeny such as dopaminergic neurons for treating or modeling Parkinson's disease (PD). We used a completely defined (xeno-free) system that we previously developed for efficient generation of authentic dopaminergic neurons from human embryonic stem cells (hESCs), and applied it to iPSCs. First, we adapted two human iPSC lines derived from different somatic cell types for the defined expansion medium and showed that the iPSCs grew similarly as hESCs in the same medium regarding pluripotency and genomic stability. Second, by using these two independent adapted iPSC lines, we showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was also similar to hESCs. Importantly, iPSC-derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. In addition, iPSC-derived NSCs and neurons could be efficiently transduced by a baculoviral vector delivering episomal DNA for future gene function study and disease modeling using iPSCs. We also performed genome-wide microarray comparisons between iPSCs and hESCs, and we derived NSC and dopaminergic neurons. Our data revealed overall similarity and visible differences at a molecular level. Efficient generation of functional dopaminergic neurons under defined conditions will facilitate research and applications using PD patient-specific iPSCs.