Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis.

Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived in vitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT.

Knockout and Replacement Gene Surgery to Treat Rhodopsin-Mediated Autosomal Dominant Retinitis Pigmentosa.

Mutations in the rhodopsin (RHO) gene are the predominant causes of autosomal dominant retinitis pigmentosa (adRP). Given the diverse gain-of-function mutations, therapeutic strategies targeting specific sequences face significant challenges. Here, we provide a universal approach to conquer this problem: we have devised a CRISPR-Cas12i-based, mutation-independent gene knockout and replacement compound therapy carried by a dual AAV2/8 system. In this study, we successfully delayed the progression of retinal degeneration in the classic mouse disease model RhoP23H, and also RhoP347S, a new native mouse mutation model we developed. Our research expands the horizon of potential options for future treatments of RHO-mediated adRP.

Therapeutic In Vivo Gene Editing Achieved by a Hypercompact CRISPR-Cas12f1 System Delivered with All-in-One Adeno-Associated Virus.

CRISPR-based gene therapies are making remarkable strides toward the clinic. But the large size of most widely used Cas endonucleases including Cas9 and Cas12a restricts their efficient delivery by the adeno-associated virus (AAV) for in vivo gene editing. Being exceptionally small, the recently engineered type V-F CRISPR-Cas12f1 systems can overcome the cargo packaging bottleneck and present as strong candidates for therapeutic applications. In this study, the pairwise editing efficiencies of different engineered Cas12f1/sgRNA scaffold combinations are systemically screened and optimized, and the CasMINI_v3.1/ge4.1 system is identified as being able to significantly boost the gene editing activity. Moreover, packaged into single AAV vectors and delivered via subretinal injection, CasMINI_v3.1/ge4.1 achieves remarkably high in vivo editing efficiencies, over 70% in transduced retinal cells. Further, the efficacy of this Cas12f1 system-based gene therapy to treat retinitis pigmentosa in RhoP23H mice is demonstrated by the therapeutic benefits achieved including rescued visual function and structural preservation. And minimal bystander editing activity is detected. This work advances and expands the therapeutic potential of the miniature Cas12f1 system to support efficient and accurate in vivo gene therapy.

Gamma distribution based predicting model for breast cancer drug response based on multi-layer feature selection.

In the pursuit of precision medicine for cancer, a promising step is to predict drug response based on data mining, which can provide clinical decision support for cancer patients. Although some machine learning methods for predicting drug response from genomic data already exist, most of them focus on point prediction, which cannot reveal the distribution of predicted results. In this paper, we propose a three-layer feature selection combined with a gamma distribution based GLM and a two-layer feature selection combined with an ANN. The two regression methods are applied to the Encyclopedia of Cancer Cell Lines (CCLE) and the Cancer Drug Sensitivity Genomics (GDSC) datasets. Using ten-fold cross-validation, our methods achieve higher accuracy on anticancer drug response prediction compared to existing methods, with an R 2 and RMSE of 0.87 and 0.53, respectively. Through data validation, the significance of assessing the reliability of predictions by predicting confidence intervals and its role in personalized medicine are illustrated. The correlation analysis of the genes selected from the three layers of features also shows the effectiveness of our proposed methods.

Continuous expression of reprogramming factors induces and maintains mouse pluripotency without specific growth factors and signaling inhibitors.

OBJECTIVES: Derivation and maintenance of pluripotent stem cells (PSCs) generally require optimized and complex culture media, which hinders the derivation of PSCs from various species. Expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) can reprogram somatic cells into induced PSCs (iPSCs), even for species possessing no optimal culture condition. Herein, we explored whether expression of OSKM could induce and maintain pluripotency without PSC-specific growth factors and signaling inhibitors. METHODS: The culture medium of Tet-On-OSKM/Oct4-GFP mouse embryonic stem cells (ESCs) was switched from N2B27 with MEK inhibitor, GSK3ß inhibitor, and leukemia inhibitory factor (LIF) (2iL) to N2B27 with doxycycline. Tet-On-OSKM mouse embryonic fibroblast (MEF) cells were reprogrammed in N2B27 with doxycycline. Cell proliferation was traced. Pluripotency was assessed by expression of ESC marker genes, teratoma, and chimera formation. RNA-Seq was conducted to analyze gene expression. RESULTS: Via continuous expression of OSKM, mouse ESCs (OSKM-ESCs) and the resulting iPSCs (OSKM-iPSCs) reprogrammed from MEF cells propagated stably, expressed pluripotency marker genes, and formed three germ layers in teratomas. Transcriptional landscapes of OSKM-iPSCs resembled those of ESCs cultured in 2iL and were more similar to those of ESCs cultured in serum/LIF. Furthermore, OSKM-iPSCs contributed to germline transmission. CONCLUSIONS: Expression of OSKM could induce and maintain mouse pluripotency without specific culturing factors. Importantly, OSKM-iPSCs could produce gene-modified animals through germline transmission, with potential applications in other species.

Reconstruction of functional uterine tissues through recellularizing the decellularized rat uterine scaffolds by MSCs in vivo and in vitro.

Infertile people who suffered from loss of uterine structures and/or functions can be treated through gestational surrogacy or uterus transplantation, which remains challenging due to the ethical and social issues, the lack of donor organs as well as technical and safety risks. One promising solution is to regenerate and reconstruct a bioartificial uterus for transplantation through the engineering of uterine architecture and appropriate cellular constituents. Here, we developed a well-defined system to regenerate a functional rat uterine through recellularization of the decellularized uterine matrix (DUM) patches reseeded with human mesenchymal stem cells (hMSCs). Engraftment of the recellularized DUMs on the partially excised uteri yielded a functional rat uterus with a pregnancy rate and number of fetuses per uterine horn comparable to that of the control group with an intact uterus. Particularly, the recellularized DUMs enhanced the regeneration of traumatic uterine in vivo because of MSC regulation. The established system here will shed light on the treatment of uterine infertility with heterogeneous DUMs/cell resources through tissue engineering in the future.

Derivation of Mouse Haploid Trophoblast Stem Cells.

Trophoblast stem (TS) cells are increasingly used as a model system for studying placentation and placental disorders. However, practical limitations of genetic manipulation have posed challenges for genetic analysis using TS cells. Here, we report the generation of mouse parthenogenetic haploid TS cells (haTSCs) and show that supplementation with FGF4 and inhibition of Rho-associated protein kinase (ROCK) enable the maintenance of their haploidy and developmental potential. The resulting haTSCs have 20 chromosomes, exhibit typical expression features of TS cells, possess the multipotency to differentiate into specialized trophoblast cell types, and can chimerize E13.5 and term placentas. We also demonstrate the capability of the haTSCs to undergo genetic manipulation and facilitate genome-wide screening in the trophoblast lineage. We expect that haTSCs will offer a powerful tool for studying functional genomics and placental biology.

PR-Set7 deficiency limits uterine epithelial population growth hampering postnatal gland formation in mice.

Formation of secretary endometrial glands in the uterus known as adenogenesis is a typical process of branching morphogenesis involving dynamic epithelial growth and differentiation. Unsuccessful adenogenesis often leads to female infertility. However, it remains largely unexplored so far regarding the epigenetic machinery governing normal endometrial gland formation. Here, we demonstrated that PR-Set7, an epigenetic regulator for H4K20me1 modification, was extensively expressed in the postnatal uteri, and its conditional deletion resulted in a complete lack of endometrial glands and infertility in mice. Subsequent analysis revealed that uterine PR-Set7 deficiency abolishes the dynamic endometrial epithelial population growth during the short span of gland formation from postnatal days 3 to 9. This markedly reduced epithelial population growth in PR-Set7-null mutant uteri is well associated with DNA damage accumulation and massive apoptotic death in the epithelium, due to blockade of 53BP1 recruitment to DNA damage sites upon reduced levels of H4K20me1/2. Using PgrCre/+/Rosa26DTA/+ mouse line and postnatal progesterone injection mouse model, we further confirmed that an impaired epithelial cell population growth either by inducing epithelial death in the diphtheria toxin-A (DTA)-mouse model or attenuating epithelial growth upon postnatal progesterone treatment similarly hampers uterine adenogenesis. Collectively, we establish here a novel 'epithelial population growth threshold' model for successful gland development. Besides further shedding light on the regulatory machinery governing uterine gland formation, our findings raise a safety concern on progesterone supplementation to prevent preterm birth in women bearing a female fetus, as exogenous progesterone may hamper uterine adenogenesis via attenuating epithelial population growth.

MCM2 mediates progesterone-induced endometrial stromal cell proliferation and differentiation in mice.

Uterine decidualization characterized by stromal cell proliferation and differentiation is critical to the establishment of pregnancy in many species. Progesterone is a key factor in regulating endometrial cell decidualization, however, the molecular basis involved in mediating the effects of progesterone during decidualization remains largely unknown. We report here that the DNA replication licensing factor MCM2, one of the conserved set of six-related proteins (MCM complex: MCM2-7) essential for eukaryotic DNA replication, is dynamically expressed in both proliferative and differentiated stromal cells during mouse periimplantation uterus. Applying PR-knockout mouse model and pharmacological strategy, we further found that the expression of Mcm2 is induced by progesterone action in the mouse uterine stroma. Employing a primary cell culture system, we further demonstrated that siRNA-mediated silencing of MCM2 arrests the cell cycle at G1-S transition during stromal cell proliferation. Moreover, the downregulation of Mcm2 could also compromise stromal cell differentiation. Collectively, our studies uncovered the role of a unique DNA replication licensing molecule MCM2 in mediating Progesterone-induced stromal cell decidualization in mouse uterus.

FoxM1 Directs STAT3 Expression Essential for Human Endometrial Stromal Decidualization.

Human endometrium decidualization, which involves endometrial stromal proliferation and differentiation, is a prerequisite for embryo implantation, thus successful pregnancy. The Forkhead Box M1 (FoxM1), previously known as HNF-3, HFH-11, MPP2, Win, and Trident, is a transcriptional factor that plays crucial roles in cell proliferation and cell cycle progression. However, the molecular mechanism of FoxM1 during human endometrial decidualization remains unexplored. In this study, we first found FoxM1 is dynamically expressed in human endometrium during menstrual cycle. Employing a human endometrial stromal cell (HESC) line, we then demonstrated that FoxM1 inhibition downregulates cyclin B1 expression, delaying G2/M phase transition during HESC proliferation. Additionally, loss of FoxM1 expression blocks the differentiation of HESCs in response to estrogen, progesterone, and dbcAMP. Applying chromatin immunoprecipitation (ChIP) technique and luciferase assay, we further approved that FoxM1 can transcriptionally active signal transducer and activator of transcription 3 (STAT3), ensuring normal HESC differentiation. Besides enriching our knowledge on molecular basis underlying stromal decidualization, these findings help to shed light on the potential molecular causes for the endometrial disorders in humans.

Cell-specific distributions of estrogen receptor alpha (ERα) and androgen receptor (AR) in anterior pituitary glands from adult cockerels as revealed by immunohistochemistry.

Estrogens and androgens play important roles in regulating the hormone-secreting functions of the pituitary gland by binding to their corresponding receptors. However, the expression of estrogen receptors (ERs) and the androgen receptor (AR) and the cell types containing ERs and AR in the anterior pituitary gland of adult chickens have not been well-studied. In this study, the distribution of ERα, AR and their corresponding cell types in the anterior pituitary gland of adult cockerels was detected by immunohistochemistry. The results showed that ERα was expressed in 68.63 % of luteinizing hormone (LH) producing cells but was not found in thyrotropes, lactotropes, somatotropes, corticotropes and folliculo-stellate (FS) cells. Pituitary hormone and AR double labeling results showed that about 37 % of LH cells and 50 % of thyroid-stimulating hormone (TSH) producing cells expressed AR, respectively. In contrast, less than 1 % of the somatotropes had an AR positive signal and AR signals were not detected in lactotropes, corticotropes or FS cells. In addition, there were only a few AR and ERα dual-labeled cells observed. These novel results provide evidence for a cell-specific distribution of ERα and AR in the anterior pituitary from adult cockerels by immunohistochemistry. The different distributions of ERα and AR in the LH cells suggest that the feedback-regulating mechanisms of estrogen and androgen on the pituitary hormones secretion are different. The functions and related mechanisms still need to be elucidated further.

Association between asymmetry in cataract and asymmetry in age-related macular degeneration. The Beijing Eye Study.

BACKGROUND: To examine in an intra-individual comparison whether cataract is associated with age-related macular degeneration (AMD). METHODS: The population-based Beijing Eye Study included 4,439 subjects (age: 40+ years) out of 5,324 subjects invited to be examined. Using lens and fundus photographs, the amount of AMD was graded according to the Wisconsin Age-Related Maculopathy Grading system and the degree of cataract was graded using the system of the Age-Related Eye Disease Study. RESULTS: Photographs with sufficient quality for bilateral examination of the lens and macula were available for 3,826 (86.2%) participants with a mean age of 55.3 ± 10.0 years (range: 40-90 years) and a mean refractive error of -0.38 ± 2.18 diopters (range: -20.13 diopters to +7.50 diopters). The side difference in presence of early AMD and late AMD respectively was not significantly associated with the inter-eye difference in the amount of nuclear cataract [P = 0.27 and P = 0.28 (r = 0.02) respectively), amount of cortical cataract (P = 0.12 and P = 0.05 respectively), and amount of subcapsular posterior cataract (P = 0.91 and P = 0.85 respectively). In a similar manner, the side difference in the presence of early AMD and late AMD was not significantly associated with the inter-eye difference in the presence of nuclear cataract (P = 0.99 and P = 0.99 respectively), cortical cataract (P = 0.25 and P = 1.00 respectively), and subcapsular posterior cataract (P = 0.59 and P = 0.05 respectively). The side difference in the number of macular drusen was not significantly associated with the inter-eye difference in the amount of nuclear cataract (P = 0.74), amount of cortical cataract (P = 0.19) and amount of subcapsular posterior cataract (P = 0.88). As a corollary, unilateral pseudophakia or aphakia was not significantly associated with inter-eye differences in the count (P = 0.59) of drusen, and overall presence of early AMD (P = 0.99) or late AMD (P = 0.99). CONCLUSIONS: In an intra-individual, inter-eye comparison, avoiding interdependencies of systemic parameters, inter-eye difference was not significantly associated with any characteristics of age-related macular degeneration in either any type of cataract or in pseudophakia. This suggests that the development of cataract or cataract surgery did not markedly influence the development of age-related macular degeneration.

Prevalence and risk factors of lens opacities in urban and rural Chinese in Beijing.

OBJECTIVE: To determine the prevalence of lens opacities in the elderly Chinese population in an urban and a rural region of Beijing. DESIGN: Population-based cross-sectional study. PARTICIPANTS: The study included 4439 subjects of 5324 subjects invited to participate (response rate, 83.4%). The subjects were divided into a rural part (1973 subjects [44.4%]) and an urban part (2466 subjects [55.6%]). The study was limited to participants age 40 and older, and the mean age was 56.2+/-10.6 years (range, 40-101 years). METHODS: Nuclear, cortical, and posterior subcapsular lens opacities were assessed based on standardized slit-lamp photographs of the lens using a modification of the grading score of the Age-related Eye Disease Study (AREDS). MAIN OUTCOME MEASURES: Grading score of the AREDS. RESULTS: Lens data were provided for 4378 subjects (98.6%) of 4439 persons examined, which consisted of 8724 eyes. Prevalence of any cataract surgery was 1.3% (95% confidence interval [CI]: 1.0-1.7), which was statistically independent of gender (P = 0.51; odds ratio [OR]: 0.86; 95% CI: 1.00-1.34), rural area versus urban region (P = 0.25), and level of education (P = 0.84). Prevalence of any nuclear lens opacity was 82.0% (95% CI: 80.8-83.2); prevalence of any cortical lens opacity was 10.3% (95% CI: 9.4-11.3); and prevalence of any posterior subcapsular opacity was 4.3% (95% CI: 3.7-4.9). If grade 2 of nuclear lens opacity was considered to be normal, prevalence of nuclear cataract was 50.3% (95% CI: 48.8-51.8), and the overall prevalence of any cataract was 53.1% (95% CI: 51.6-54.6), increasing from 6.5% (95% CI: 5.2-7.8) in those subjects 40 to 49 years of age to 52.3% (95% CI: 47.4-55.3) in those who were 50 to 59 years of age, and to 97.8% (95% CI: 96.4-99.2) in those 70 years and older (P<0.001). Frequencies of any cortical cataract and any subcapsular posterior cataract were 10.3% (95% CI: 9.4-11.3) and 4.3% (95% CI: 3.7-4.9), respectively. CONCLUSIONS: Cataracts are common among adult Chinese residents in Beijing, with age as the most important associated factor. In view of the relatively low rate of cataract surgery performed so far, one may expect a marked increase in the number of cataract surgeries to meet the visual needs of the growing elderly population in China.

Refractive error in urban and rural adult Chinese in Beijing.

PURPOSE: To evaluate refractive error and its demographic associations in an urban and rural population in northern China. DESIGN: Epidemiological study. PARTICIPANTS: The Beijing Eye Study is a population-based cohort study in northern China including 4439 subjects. Excluding pseudophakic and aphakic patients, the present study involved 4319 subjects. It was divided into a rural part (1905 [44.1%] subjects) and an urban part (2414 [55.9%] subjects). Mean age was 55.85+/-10.33 years (range, 40-90). METHODS: Standardized ophthalmologic examination. For statistical analysis, the spherical equivalent was converted to binary variables, and logistic regression was used to investigate the association with continuous or categorical independent variables. MAIN OUTCOME MEASURE: Refractive error. RESULTS: Mean refractive error measured -0.33+/-2.22 diopters (D) (range, -20.88 to +7.88). Myopia of >-0.50 D, -1.0 D, >-6.0 D, and >-8 D, respectively, occurred in 22.9% (95% confidence interval [CI], 21.7-24.2), 16.9% (95% CI, 15.8-18.0), 2.6% (95% CI, 2.2-3.1), and 1.5% (95% CI, 1.1-1.9) of the subjects, respectively. Myopic refractive error was associated significantly with younger age (P<0.001), urban region (vs. rural region) (P<0.001), higher educational background (P<0.001), higher degree of nuclear cataract (P<0.001), decreasing uncorrected visual acuity (UCVA) (P<0.001), decreasing best-corrected visual acuity (BCVA) (P<0.001), and female gender (P<0.001). Prevalence of high myopia (myopic refractive error >-8 D) was associated with age (P<0.001), female gender (P = 0.020), urban region (P = 0.023), and lower BCVA (P<0.001). Mean anisometropia was 1.09+/-2.03 D (median, 0.38; range, 0-22.0). Prevalence of anisometropia of > or =1 D was associated significantly with age (P<0.001), refractive error (P<0.001), BCVA (P<0.001), and region (P<0.001). Mean astigmatic error measured 0.62+/-0.90 D (median, 0.25; range, 0-7.50). Astigmatism of > or =1 D was associated significantly with age (P<0.001), lower UCVA (P = 0.003), lower BCVA (P<0.001), urban area (P<0.001), and degree of cortical cataract (P = 0.027). CONCLUSIONS: As in other population-based studies on Chinese, myopia was more prevalent in younger subjects. Myopia was associated with urban region, educational background, female gender, decreasing visual acuity, and nuclear cataract. If longitudinal studies confirm the association of refractive error with age, refractive surgery may achieve emmetropia only for a limited time.