Generation of induced pluripotent stem cells and neural cells from urine-derived cells of Alzheimer disease patients

AIM: In this study, we aim to obtain the induced pluripotent stem cells (iPSCs) from the patients with sporadic Alzheimer disease (AD). METHODS: Three typical Alzheimer's patients were chosen, and the epithelial cells were isolated from their urine. We reprogrammed these cells into induced pluripotent stem cells by transfection of 4 factors (Oct4, Sox2, Klf4 and SV40LT) with the technique of electro-transfection. After getting these iPSCs, we continue to differentiate them into neural cells by a specific method—dual inhibition of Smad signaling. RESULTS: The primary cells from 3 AD patients were successfully reprogrammed to iPSCs, and these patients-derived iPSCs were differentiated into neural cells. There was no significant difference, during iPSCs reprogramming and neural differentiation, between cells from AD patients and normal people. CONCLUSION: The urine cells from AD patients were able to transfer to iPSCs, functional neurons and neurogliocytes.     

Research progress in reprogramming induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) have been first induced from mouse fibroblasts since 2006, and the research on iPSCs has made great progress in the following years. iPS cell lines were established from different somatic cells through DNA, RNA, protein, and small molecule compounds and various methods of transduction, making the induction of iPSCs more secure and effective, and more attractive prospect of clinical application. In this review, different somatic cell reprogramming, different levels of reprogramming, different transduction pathways, and prospect of application are discussed.     

Generation of thalassemia-specific integration-free induced pluripotent stem cells and determination of their differentiation ability

AIM: To generate thalassemia-specific integration-free induced pluripotent stem cells(iPSC) and to detect their ability of differentiation into hematopoietic precursors.METHODS: The plasmids pEB-C5 and pEB-Tg were transfected into the fibroblast cells from hemoglobin Bart's hydrops fetalis's skin by the method of nuclear transfection to reprogramm the cells into iPSC. The ability of the iPSC to differentiate into 3-germ layer cells was determined. The iPSC were cocultured with mouse OP9 cells to differentiate into hematopoietic precursors and the hematopoietic precursor specific antigens were detected. RESULTS: The integration-free iPSC from hemoglobin Bart's hydrops fetalis's skin fibroblasts were successfully derived, and had the ability to differentiate into 3 germ layers. When cocultured with OP9 cells for 9 d, the positive rate of hematopoietic progenitor cell marker CD34 was 18.7%, and the CD34 and CD45 double positive rate was 12.2%. CONCLUSION: Hemoglobin Bart's hydrops fetalis's skin fibroblasts can be successfully induced into "integration-free" iPSC. This cell line has the ability to differentiate into 3 germ layers, and can be differentiated into hematopoietic precursors when cocultured with OP9 cells.     

Effects of maxadilan on proliferation of human induced pluripotent stem cells

AIM: To investigate the promoting effect of maxadilan, which specifically activate the type I receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), on the proliferation of human induced pluripotent stem cells (IPSCs). METHODS: PACAP type I (PAC1) receptor in IPSCs was detected by RT-PCR and Western blotting. maxadilan at various concentrations was added to the medium of IPSCs as experimental groups. The medium in control group was without maxadilan treatment. The effect of maxadilan on theproliferation of IPSCs was measured with Cell Counting Kit-8 (CCK-8). The changes of cell cycle caused by maxadilan in IPSCs were analyzed by flow cytometry. The analysis of karyotype was carried out in IPSCs treated with maxadilan. Proteins and gene expression levels of both Nanog and OCT4 in IPSCs treated with maxadilan were detected by real-time quantitative polymerase chain reaction (real-time-qPCR) and immunofluorescence. The gene expression levels of Nestin and PAX6 in both IPSCs treated with maxadilan and cells of embryonic body, which was birthed from IPSCs with maxadilan treatment, were detected by real-time qPCR. The ability of IPSCs treated with maxadilan differentiating into 3 embryonic layers was evaluated by analyzing the component of embryo using RT-PCR. RESULTS: The PAC1 receptor in IPSCs was identified by RT-PCR and Western blotting. Viability of the IPSCs with 100 nmol/L maxadilan treatment was increased by 16% compared with control group. The differences with statistical significance were found in the cell viability between 100 nmol/L maxadilan treatment group and control group (P<0.05). The average values of proliferation index (PI) in IPSCs with 100 nmol/L maxadilan treatment for 3 h, 6 h and 9 h were 47.23%, 59.70% and 55.67%,respectively, while that in control group was 37.00%. The differences with statistical significance were found in PI between 100 nmol/L maxadilan treatment for 3 h group, 6 h group, 9 h group and control group (P<0.05). Normal karyotype and unchanged pluripotent state in IPSCs treated with maxadilan were observed. Compared with control group, the gene expression levels of Nestin and PAX6 were not significantly different in both IPSCs and the cells of embryonic body birthed from IPSCs with maxadilan treatment. The ability of differentiation into 3 embryonic layers in IPSCs treated with 100 nmol/L maxadilan was found. CONCLUSION: PAC1 receptor presents in IPSCs. Maxadilan promotes the proliferation of IPSCs but does not affect their pluripotent state and karyotype.     

Roles of reactive oxygen species in pluripotent stem cell functions

Pluripotent stem cells are characterized by the properties of self-renewal and the ability to differentiate into multiple cell types. Reactive oxygen species (ROS) are highly reactive metabolites. High levels of ROS are toxic and involved in stem cell senescence and apoptosis. However, regulation of ROS has an important role in maintaining “stemness” and differentiation of the stem cells. The role of ROS in the stem cells varies among different stem cell types. NADPH oxidase is one of the major sources of ROS in stem cells. Excessive amounts of ROS are produced in various pathophysiological states such as atherosclerosis, heart failure, hypertension, diabetes, and aging. Induced pluripotent stem cells have the potential to be used in modeling of ROS-associated diseases.Understanding the molecular mechanisms how ROS regulate the functions of stem cells will greatly enhance their translational applications. In this review, we summarize the recent progress regarding the roles of ROS in regulating the functions of embryonic and induced pluripotent stem cells.     

Differentiation of mouse iPSCs into insulin-producing cells and experimental study on treating diabetes

AIM: To induce mouse induced pluripotent stem cells (iPSCs) to differentiate into insulin-producing cells (IPCs) by a new 3-step method, and to detect the efficiency and maturity for the treatment of diabetic mice. METHODS: We constructed iPSCs from mouse embryonic fibroblasts of male C57/C mouse by piggyBac transposon, then induced the iPSCs into IPCs by a 3-step method. The cell morphological change was traced by microscopy during the process of differentiation. The expression of mRNA and protein associated with islet β cell development was determined by real-time PCR and immunofluorescence staining. Flow cytometry was used to analysis the efficiency of differentiation. Insulin and C-peptide secretions of IPCs in response to glucose at high (25 mmol/L) or low (5.5 mmol/L) level were measured by ELISA. The IPCs were transplanted into the capsul of left kidney in the male C57/C diabetic mouse model. Blood glucose was continuously monitored for 28 day, serum insulin was tested by ELISA in different stages. The glucose tolerance test was performed on the 28th day, and the left kidney was excised. RESULTS: IPCs were obtained from mouse iPSCs by the 3-step method. The cells expressed the marker genes (Pdx1, Ngn3, Pax6 and Ins2) and proteins (Pdx1, Nkx6.1 and insulin) of β cells. The glucose stimulation induced the secretion of insulin and C-peptide. The efficiency of differentiation was 28% detected by flow cytometry. After transplantation of IPCs to the diabetic mice, the blood glucose was decreased to normal level on the 3rd day,and serum insulin level and the ability of regulating glucose were improved. IPCs were still alive after 28 d of transplantation by pathological observation. CONCLUSION: iPSCs is efficiently induced into IPCs by a 3-step method , and the induction time is shortened significantly. The hyperglycemia of diabetes mice is reversed after transplanting IPCs to same sex inbred strain mice.     

Preliminary study on induction of mouse embryonic stem cells into neural stem cells in vitro

AIM:To explore the feasibility of inducing mouse embryonic stem cells into neural stem cells in vitro. METHODS:Embryonic body induced by retinoic acid and retinal müller cells were selected in neural stem cell-defined medium for 7 days, and the morphological changes were observed. The selected cells were stained immunocytochemically with anti-nestin, anti-BrdU antibodies, and their ability of expansion and differentiation were analyzed. RESULTS:Large amounts of neurospheres were derived from embryonic body in the selected medium on the 7th day, which could be passaged and differentiated, stained positive with nestin and BrdU, and expressed nestin, glutaminase and Brn-3 genes. CONCLUSION:Neural stem cells could be derived from embryonic body induced by RA and müller cells in the selected medium, which would offer an alternative to treat neuropathy such as glaucoma and retinal degeneration in the future.     

Purification and differentiation potentiality of fetal liver mesenchymal stem cells from mouse

AIM: To purify and investigate the differentiation potentials of fetal liver mesenchymal stem cells (flMSCs) from murine in vitro.METHODS: flMSCs from mouse fetuses at embryonic and fetal day (ED)13.5 or ED14.5 were isolated by adhering to plastic and passaged by modified method.Cell cycle and phenotype were analyzed by flow cytometry.The cell differentiation was induced by special induction media.The cells differentiated to adipose,cartilaginous and osteoid tissues were identified with oil red O,Toluid blue,alkaline phosphatease (ALP) and von Kossa’s staining.The cells differentiated to neural-like cells were detected by RT-PCR and immuno-staining.RESULTS: Fibroblast-like cells predominated in culture.(83.76±2.88)% of flMSCs stayed in the G₀/G₁ phases.Homogenous cells were positive for mesenchymal lineage markers CD44,CD29,but not for markers of hematopoietic cells CD45,CD11b.flMSCs were able to differentiate into adipogenic,chondrogenic,osteogenic and neurogenic cells.CONCLUSION: flMSCs can be purified by modified plastic-attachment method and have multiple differentiation,which is available to stem cell therapy for various diseases.     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

Comparison of 2 methods for inducing iPSC to differentiate into neural stem cells

AIM: To select an efficient way of promoting induced pluripotent stem cells (iPSC) to differentiate into neural stem cells (NSC) by comparing 2 methods. METHODS: The culture system in method A contained SB431542 (5 mmol/L) and drosomophorin (5 mmol/L) with 100% initial cell density, while that in method B contained SB431542 (5 mmol/L) and drosomophorin (1 mmol/L) with 30%~50% initial cell density. For comparison and identification of the 2 methods, the growth state was observed under microscope, and the expression of Pax6, nestin, Sox1 and Sox2 was quantitatively detected by real-time PCR and flow cytometry. The related protein expression and the ability of spontaneous differentiation were determined by immunofluorescence analysis. RESULTS: The cells derived from method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density achieved the higher expression of Pax6, nestin, Sox1 and Sox2. The growth state was better and the cells differentiated into neurons and astrocytes normally. CONCLUSION: The method A was superior to method B, and we recommend the method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density as the method for differentiating NSC.     

Functional hepatic differentiation from mouse induced pluripotent stem cells in vitro

AIM: To directly differentiate the induced pluripotent stem cells (IPS cells) into hepatocytes in chemically-defined, monolayer conditions in vitro. METHODS: Based on the knowledge of mammalian hepatic development, we differentiated the IPS cells into hepatic lineage efficiently. The present study involves stepwise transition of IPS cells through a series of media designs to first specify cells towards anterior definitive endoderm, and secondly, to induce hepatic maturation. RESULTS: The IPS cells recapitulated the development of liver in vivo at RNA and protein level. Real-time PCR analysis was performed on cell preparations at various times during the culture process to determine the time course and degree to which the IPS cells differentiated toward a hepatocyte phenotype. Analysis included markers for endoderm-specific gene expression (Sox17; FoxA2, CXCR4), hepatocyte-specific gene expression (albumin and AFP), and markers for undifferentiated IPS cells (OCT4 and Nanog). The endoderm-specific gene expression increased early while the expression of OCT4 and Nanog decreased and hepatocyte-specific gene expression progressively increased over the course of the differentiation program. The resultant hepatocyte-like cells(HLCs) showed phenotypic similarities with adult hepatocytes (expressing AFP and albumin) and additionally showed functionality expected to well-differentiated hepatocytes such as indocyanine green(ICG) uptake and release. The P450 activity of cytochromsome was also positive. CONCLUSION: We successfully construct the IPS cells which efficiently differentiate into hepatocytes and recapitulate the hepatocytic development in vivo. The reproducible and efficient generation of HLCs from IPS cells represents a critical step towards the ultimate goal of producing functional HLCs for clinical use including cell transplantation or bioartificial liver support. Moreover, our system provide a valid way to study the liver development since it is monolayer, serum-free and mimic the in vivo liver development.     

Differentiation of embryonic-like stem cells derived from human bone marrow into multinucleated fibers in vitro

AIM: To compare the capacity of in vitro differentiation into multinucleated fibers between embryonic-like stem cells (ELSCs) and mesenchymal stem cells (MSCs) derived from human bone marrow. METHODS: To isolate ELSCs, human bone marrow mononuclear cells were cultured in gelatin-coated flask with serum-free Knockout-DMEM medium designed for the expansion of human embryonic stem cells. MSCs were isolated from the same bone marrow by the traditional method. The morphological characters of both ELSCs and MSCs were observed under inverted phase-contrast microscope, and the expression of their multipotent antigen markers was identified by immunofluorescent staining. ELSCs and MSCs were cultured in myogenic differentiation medium. The protein levels of muscle-specific antigen markers myosin heavy chain (MHC), myogenin and MyoD were detected by the method of immunostaining. The mRNA expression of MHC, myogenin and MyoD was detected by RT-PCR. The capacity of in vitro differentiation into multinucleated fibers was compared between ELSCs and MSCs by calculating the proportion of MHC-positive multinucleated fibers. RESULTS: ELSCs, which weakly expressed the multipotential markers Oct-4, Nanog-3 and Sox-2, were isolated from bone marrow by the method of serum-free medium. ELSCs appeared smaller, slenderer and more homogeneous, and were morphologically different from MSCs derived from the same marrow. No multipotential marker in MSCs was expressed. ELSCs and MSCs were induced into long multinucleated fibers expressing MHC and myogenin at mRNA and protein levels by culturing in the myogenic differentiation medium. However, on the 10th day after induction, the proportion of the MHC-positive fibers in ELSCs was (25.7?4.1)%, and the proportion in MSCs was (15.8?7.6)%.The capacity for differentiation into muscle in ELSCs was significantly higher than that in MSCs (P<0.05). CONCLUSION: Bone marrow ELSCs are induced into multinucleated fibers and have the stronger myogenic differentiation capacity than MSCs derived from the same marrow. ELSCs are a more ideal candidate for muscular disease therapy.     

Ultrastructural characteristics of neural stem cells derived from adipose-derived stromal cells during their differentiation into neurons and astrocytes

AIM: To observe the ultrastructural characteristics of neural stem cells derived from adipose-derived stromal cells (ADSC) differentiating into neurons and astrocytes. METHODS: ADSC were cultured, amplified and induced with β-mercaptoethanol or 3-isobutyl-1-methylxanthine (IBMX). The expression of nestin in the induced cells was determined by the method of immunofluorescence. The ultrastructure of the induced neural stem cells was observed under transmission electron microscope.RESULTS: The expression of nestin reached the peak at 3 h in β-mercaptoethanol group and at 14 d in IBMX group, and the positive rate of the former (86%) was significantly higher than that of the latter (23%). However, the duration of the induction in IBMX group was obviously longer than that in β-mercaptoethanol group. The ultrastructure of the induced neural stem cells in the two groups was similar under the transmission electron microscope, only with some differences in organelles.CONCLUSION: In the process of ADSC differentiating into astrocytes or neurons with the induction of β-mercaptoethanol or IBMX, the different changes of ultrastructure in ADSC-derived neural stem cells occur in some organelles in cytoplasm, not in nucleus.     

Advances in studying the differentiation into neurons of mesenchymal stem cells and their application

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can proliferate and differentiate into marrow and non-marrow cell types, such as adipocytes, chondrocytes, myocytes, and so on. In recent years, many researchers have studied whether MSCs are capable of differentiation into neurons in vivo and ex vivo. The result that MSCs-derived neurons express NSE and NF, but don't express GFAP suggests MSCs can differentiate into neurons, some researchers have achieved success in promoting functional recovery in Pakinsons and transactional spinal cord injury rat models by use of MSCs-derived neurons. Therefore, MSCs-derived neurons will play an important role in the therapy for a variety of diseases of the nervous system.     

Effect of lentiviral vector of microRNA-9-1 on differentiation of mouse bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of microRNA-9 in inducing bone marrow mesenchymal stem cell(MSCs) differentiation into neurons.METHODS: The lentiviral vector of microRNA-9-1(microRNA-9-1-LV) was constructed and transfected into mouse MSCs. The cells were divided into non-transfected group, transfected group(transfected with microRNA-9-1-LV) and negative control group(transfected with FU-RNAi-NC-LV). MSCs were treated with β-mercaptoethanol(β-ME) as an inducer for triggering the cells to differentiate into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The mRNA expression of microtublin-associated protein 2(MAP-2) was detected by RT-PCR. The expression of neuron-specific markers,neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP), were measured by immunocytochemical method. The viability of MSCs was determined by MTT method. RESULTS: The results of PCR confirmed successful construction of mouse microRNA-9-1-LV. The virus titer was 1×10¹² TU/L(TU, transduction unit). The best transfection efficiency(up to 91.3%±4.2%) and survival rate appeared when multiply of infection(MOI)was 20 and on 4th day. β-ME induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group. The expression levels of NSE and MAP-2 in transfected cells were higher than those in the cells of other group(P<0.05).CONCLUSION: MicroRNA-9-1-LV has high transfection efficiency in mouse MSCs. Higher differentiation rate from mouse MSCs to neurons is induced by β-ME after the cells are transfected with microRNA-9-1-LV.     

Differentiation potential of human placenta derived mesenchymal stem cells into vascular endothelial cells

AIM: To investigate the differentiation potential of human placenta derived mesenchymal stem cells (PDMSCs) into endothelial cells (ECs) in vitro. METHODS: PDMSCs were isolated from human placenta tissues, characterized by flow cytometry and induced to differentiate into endothelial cells with 50 μg/L VEGF and 10 μg/L bFGF. To detect the specific markers of ECs during the process of differentiation, the method of immunocytochemistry was performed. The specific structure and function of endothelial cells were observed by transmission electron microscopy and in vitro angiogenesis assay kit, respectively. RESULTS: CD105 and CD106 were positive in PDMSCs, while CD34,CD45 and CD31 were negative.The ECs differentiated from PDMSCs showed cobblestone-like morphology, and expressed early endothelial marker of Flk-1/KDR and mature endothelial markers of CD31, vWF and CD144/VE-cadherin in a time-dependent manner during the endothelial cell differentiation (0 day, 4 days, 8 days and 12 days). The endothelial specific structure, Weibel-palade body, was observed under transmission electron microscope. The inoculation of ECs on the extra cellular matrix gel formed capillary-like structures. CONCLUSION: Plentiful PDMSCs can be isolated from placenta, and differentiate into the cells with functional characteristics of ECs in vitro, indicating that the placenta tissues will become optimal source of seed cells for vascular engineering and regenerative medicine.     

Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro via cardiotrophin 1

AIM: To investigate the effects of cardiotrophin 1 (CT-1) on differentiation of swine bone marrow mesenchymal stem cells (MSCs) into cardiomyocyte-like cells in vitro.METHODS: MSCs were isolated and proliferated from Tibet miniswine. Adipogenic and osteogenic potentials were identified. MSCs were divided into 4 groups for induction: untreated group, 5-azacytidine (5-Aza) group,CT-1 group and 5-Aza combined with CT-1 group. After induction for 4 weeks, the expression of cardiac cell markers including α-actin and cardiac troponin-T (cTnT) was estimated by immunofluorescence staining. Finally, the rates of red fluorescence positive-staining cells were calculated. RESULTS: The expression of α-actin in the 4 groups by red fluorescence staining was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 29.90%±4.76%, significantly higher than that in 5-Aza group (17.73%±2.34%, P<0.01), CT-1 group (6.63%±0.55%, P<0.01) and untreated group (1.62%±0.09%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.05). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01). The expression of cTnT in the 4 groups was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 36.50%±4.09%, significantly higher than that in 5-Aza group (14.37%±1.65%, P<0.01), CT-1 group (7.50%±0.61%, P<0.01) and untreated group (1.12%±0.23%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.01). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01).CONCLUSION: Appropriate concentrations of 5-Aza (10 μmol/L) and CT-1 (0.1 μg/L) induce swine bone marrow MSCs to differentiate into cardiomyocyte-like cells in vitro. CT-1 combined with 5-Aza significantly increases the differentiation rate.     

Neural differentiation of bone marrow mesenchymal stem cells from an Alzheimer disease mouse model

AIM：To estimate the neural differentiation efficiency of bone marrow mesenchymal stem cells (MSCs) derived from amyloid precursor protein (APP) transgenic mice and to investigate the correlation with Notch1 signaling and the autophagy activity during the differentiation. METHODS：The MSCs were divided into APP group (MSCs from APP transgenic mice) and WT group (MSCs from wild-type mice). MSCs were treated with β-mercaptoethanol as an inducer for differentiating into neurons. The levels of Aβ40 and Aβ42 were measured using enzyme-linked immunosorbent assay kits. The expression of neural cell-specific markers, neuron-specific enolase (NSE) and microtubule-associated protein 2 (MAP-2), was measured by immunocytochemistry and Western blotting. The expression levels of Notch1, Notch intracellular domain (NICD), Hes5, LC3 and p62 (a selective substrate of autophagy) were also detected by Western blotting. RESULTS：The neural differentiation capacity and the Aβ expression level of the MSCs in APP group were higher than those in WT group, and stronger inhibition of Notch1 signaling pathway in the MSCs from APP group was observed. However, the process of autophagy, which is essential for the survival and function of the neural cells, was impaired in the neural differentiated counterpart of the MSCs in APP group. CONCLUSION:Over-expression of APP might contribute to the high neural differentiation capacity of MSCs by inhibiting Notch1 signaling pathway in vitro. However, autophagy is impaired in the differentiated MSCs from APP transgenic mice.