RESUMEN
Photoreception is essential for the development of the visual system, shaping vision's first synapse to cortical development. Here, we find that the lighting environment controls developmental rod apoptosis via Opn4-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Using genetics, sensory environment manipulations, and computational approaches, we establish a pathway where light-dependent glutamate released from ipRGCs is detected via a transiently expressed glutamate receptor (Grik3) on rod precursors within the inner retina. Communication between these cells is mediated by hybrid neurites on ipRGCs that sense light before eye opening. These structures span the ipRGC-rod precursor distance over development and contain the machinery for photoreception (Opn4) and neurotransmitter release (Vglut2 & Syp). Assessment of the human gestational retina identifies conserved hallmarks of an ipRGC-to-rod axis, including displaced rod precursors, transient GRIK3 expression, and ipRGCs with deep-projecting neurites. This analysis defines an adaptive retrograde pathway linking the sensory environment to rod precursors via ipRGCs prior to eye opening.
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Purpose: CYP3A5 polymorphisms have been associated with variations in the pharmacokinetics of tacrolimus (Tac) in kidney transplant patients. Our study aims to quantify how the CYP3A5 genotype influences tacrolimus trough concentrations (C0) in a Vietnamese outpatient population by selecting an appropriate population pharmacokinetic model of Tac for our patients. Patients and Methods: The external dataset was obtained prospectively from 54 data of adult kidney transplant recipients treated at the 103 Military Hospital. All published Tac population pharmacokinetic models were systematically screened from PubMed and Scopus databases and were selected based on our patient's available characteristics. Mean absolute prediction error (MAPE), mean prediction error, and goodness-of-fit plots were used to identify the appropriate model for finding the formula that identifies the influence of CYP3A5 genotype on the pharmacokinetic data of Vietnamese patients. Results: The model of Zhu et al had a good predictive ability with MAPE of 19.29%. The influence of CYP3A5 genotype on tacrolimus clearance was expressed by the following formulas: CL/F=27,2×[(WT/70)0,75]×[(HCT/0,35)-0,501]×[(POD/180)0,0306]×CYP3A5(L/h). The simulation result showed that Tac C0 was significantly higher in patients not expressing CYP3A5 (p< 0.001). Conclusion: The incorporation of the CYP3A5 phenotype into Zhu's structural model has significantly enhanced our ability to predict Tacrolimus trough levels in the Vietnamese population. This study's results underscore the valuable role of CYP3A5 phenotype in optimizing the forecast of Tac concentrations, offering a promising avenue to assist health-care practitioners in their clinical decision-making and ultimately advance patient care outcomes.
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Almost all life forms can detect and decode light information for adaptive advantage. Examples include the visual system, in which photoreceptor signals are processed into virtual images, and the circadian system, in which light entrains a physiological clock. Here we describe a light response pathway in mice that employs encephalopsin (OPN3, a 480 nm, blue-light-responsive opsin) to regulate the function of adipocytes. Germline null and adipocyte-specific conditional null mice show a light- and Opn3-dependent deficit in thermogenesis and become hypothermic upon cold exposure. We show that stimulating mouse adipocytes with blue light enhances the lipolysis response and, in particular, phosphorylation of hormone-sensitive lipase. This response is Opn3 dependent. These data establish a key mechanism in which light-dependent, local regulation of the lipolysis response in white adipocytes regulates energy metabolism.
Asunto(s)
Adipocitos Marrones/metabolismo , Adipocitos Marrones/efectos de la radiación , Adipocitos Blancos/metabolismo , Adipocitos Blancos/efectos de la radiación , Luz , Opsinas de Bastones/metabolismo , Termogénesis/efectos de la radiación , Animales , Frío , Metabolismo Energético/efectos de la radiación , Perfilación de la Expresión Génica , Lipólisis/efectos de la radiación , Ratones Endogámicos C57BL , Fenotipo , Fotones , Termogénesis/genéticaRESUMEN
Hydrogels are favored materials in tissue engineering as they can be used to imitate tissues, provide scaffolds, and guide cell behavior. Recent advances in the field of optogenetics have created a need for biocompatible optical waveguides, and hydrogels have been investigated to meet these requirements. However, combining favorable waveguiding characteristics, high biocompatibility, and controllable bioactivity in a single device remains challenging. Here, we investigate the use of poly(ethylene glycol) hydrogels as carriers and illumination systems for in vitro cell culture. We present a comprehensive and reproducible protocol for selective bioactivation of the hydrogels, achieving high proliferation rates and strong cell adhesion on the treated surface. A cell model expressing the photoconvertible fluorescent protein Dendra2 confirmed that light-cell interactions occur at the hydrogel surface. Monte Carlo simulations were performed as a tool to predict the extent of these interactions. This study demonstrates a hydrogel-based waveguiding system for targeted cell stimulation in vitro and potentially in vivo environments.
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During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.
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Dopamina/metabolismo , Ojo/irrigación sanguínea , Luz , Proteínas de la Membrana/metabolismo , Opsinas/metabolismo , Animales , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Endotelio Vascular/metabolismo , Ojo/enzimología , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Opsinas/genética , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/efectos de la radiación , Treonina/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/fisiología , Cuerpo Vítreo/metabolismoRESUMEN
The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength-sensitive cone pigment [S-pigment or cone opsin (OPN1SW)] nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.
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Córnea/fisiología , Proteínas de la Membrana/fisiología , Opsinas/fisiología , Retina/fisiología , Núcleo Supraquiasmático/fisiología , Animales , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Opsinas/genética , Rayos UltravioletaRESUMEN
Conjunctival goblet cells primarily synthesize mucins to lubricate the ocular surface, which is essential for normal vision. Notch signaling has been known to associate with goblet cell differentiation in intestinal and respiratory tracts, but its function in ocular surface has yet to be fully characterized. Herein, we demonstrate that conditional inhibition of canonical Notch signaling by expressing dominant negative mastermind-like 1 (dnMaml1) in ocular surface epithelia resulted in complete suppression of goblet cell differentiation during and subsequent to development. When compared with the ocular surface of wild-type mice (OS(Wt)), expression of dnMaml1 at the ocular surface (OS(dnMaml1)) caused conjunctival epithelial hyperplasia, aberrant desquamation, failure of Mucin 5ac (Muc5ac) synthesis, subconjunctival inflammation and epidermal metaplasia in cornea. In addition, conditional deletion of Notch1 from the ocular surface epithelia partially recapitulated OS(dnMaml1) phenotypes. We have demonstrated that N1-ICD (Notch1 intracellular domain) transactivated the mouse Krüppel-like factor 4 (Klf) promoter and that Klf4 directly bound to and significantly potentiated the Muc5ac promoter. By contrast, OS(dnMaml1) dampened Klf4 and Klf5 expression, and diminished Muc5ac synthesis. Collectively, these findings indicated that Maml-mediated Notch signaling plays a pivotal role in the initiation and maintenance of goblet cell differentiation for normal ocular surface morphogenesis and homeostasis through regulation of Klf4 and Klf5.
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Conjuntiva/metabolismo , Epitelio Corneal/patología , Receptor Notch1/metabolismo , Transducción de Señal , Activación Transcripcional , Animales , Diferenciación Celular , Proliferación Celular , Conjuntiva/embriología , Conjuntiva/patología , Córnea/embriología , Córnea/metabolismo , Córnea/patología , Epitelio Corneal/embriología , Epitelio Corneal/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Células Caliciformes/metabolismo , Células Caliciformes/patología , Hiperplasia/genética , Hiperplasia/patología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Aparato Lagrimal/metabolismo , Aparato Lagrimal/patología , Metaplasia/metabolismo , Metaplasia/patología , Ratones , Ratones Transgénicos , Mucina 5AC/genética , Mucina 5AC/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Receptor Notch1/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cre-mediated apoptosis has been observed in many contexts in mice expressing Cre-recombinase and can confound the analysis of genetically engineered conditional mutant or transgenic alleles. Several mechanisms have been proposed to explain this phenomenon. We find that the degree of apoptosis induced correlates roughly with the copy number of loxP sites present in the genome and that some level of increased apoptosis accompanies the presence of even only a few loxP sites, as occurs in conditional floxed alleles. Cre-induced apoptosis in this context is completely p53-dependent, suggesting that the apoptosis is stimulated by p53 activation in response to DNA damage incurred during the process of Cre-mediated recombination.
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Apoptosis , Daño del ADN , Integrasas/metabolismo , Recombinación Genética , Proteínas Supresoras de Tumor/genética , Alelos , Animales , Dosificación de Gen , Integrasas/genética , Ratones , Ratones Transgénicos , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Systemic derangements and perinatal death of generalized insulin-like growth factor 1 (IGF-1) and IGF-1 receptor (IGF-1R) knockout mice preclude definitive assessment of IGF-1R actions in growth-plate (GP) chondrocytes. We generated cartilage-specific Igf1r knockout ((Cart) Igf1r(-/-)) mice to investigate local control of chondrocyte differentiation in the GP by this receptor. These mice died shortly after birth and showed disorganized chondrocyte columns, delayed ossification and vascular invasion, decreased cell proliferation, increased apoptosis, and increased expression of parathyroid hormone-related protein (Pthrp) RNA and protein in their GPs. The increased Pthrp expression in the knockout GPs likely was due to an increase in gene transcription, as determined by the increased activity of a LacZ reporter that was inserted downstream of the endogenous PTHrP promoter and bred into the knockout mice. To circumvent the early death of (Cart) Igf1r(-/-) mice and investigate the role of IGF-1R during postnatal growth, we made tamoxifen (Tam)-inducible, cartilage-specific Igf1r knockout ((TamCart) Igf1r(-/-)) mice. At 2 weeks of age and 7 to 8 days after Tam injection, the (TamCart) Igf1r(-/-) mice showed growth retardation with a disorganized GP, reduced chondrocyte proliferation, decreased type 2 collagen and Indian Hedgehog (Ihh) expression, but increased expression of PTHrP. Consistent with in vivo observations, in vitro knockout of the Igf1r gene by adenoviral expression of Cre recombinase suppressed cell proliferation, promoted apoptosis, and increased Pthrp expression. Our data indicate that the IGF-1R in chondrocytes controls cell growth, survival, and differentiation in embryonic and postnatal GPs in part by suppression of Pthrp expression.
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Condrocitos/metabolismo , Placa de Crecimiento/crecimiento & desarrollo , Proteínas Hedgehog/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Animales , Animales Recién Nacidos , Apoptosis , Desarrollo Óseo , Huesos/embriología , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Condrocitos/citología , Eliminación de Gen , Regulación de la Expresión Génica , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Meox1 and Meox2 are two related homeodomain transcription factor genes that together are essential for the development of all somite compartments. Here we show that mice homozygous for Meox1 mutations alone have abnormalities that are restricted to the sclerotome and its derivatives. A prominent and consistent phenotype of these mutations is a remodeling of the cranio-cervical joints whose major feature is the assimilation of the atlas into the basioccipital bone so that the skull rests on the axis. These abnormalities can be traced back to changes in the relative rates of cell proliferation in the rostral and caudal sclerotome compartments, and they are associated with alterations in the expression of at least three transcription factor genes, Tbx18, Uncx, and Bapx1. As previously observed for Bapx1, MEOX1 protein occupies evolutionarily conserved promoter regions of Tbx18 and Uncx, suggesting that Meox1 regulates these genes at least in part directly. Hence, Meox1 is part of a regulatory circuit that serves an essential, non-redundant function in the maintenance of rostro-caudal sclerotome polarity and axial skeleton formation.
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Tipificación del Cuerpo/fisiología , Vértebras Cervicales/embriología , Proteínas de Homeodominio/metabolismo , Articulaciones/embriología , Mesodermo/metabolismo , Cráneo/embriología , Animales , Biomarcadores/metabolismo , Vértebras Cervicales/anomalías , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Hibridación in Situ , Articulaciones/anomalías , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Regiones Promotoras Genéticas , Cráneo/anomalías , Somitos/citología , Somitos/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The ability to generate conditional mutant alleles in mice using Cre-lox technology has facilitated analysis of genes playing critical roles in multiple developmental processes at different times. We used a transgenic Hoxb6 promoter to drive tamoxifen-dependent Cre recombinase expression in several developing systems that serve as major models for elucidating inductive interactions and mechanisms of morphogenesis, including lateral plate mesoderm and descendant limb buds, neural crest progenitors of the neural tube, tailbud, and CNS isthmic organizer. The Hoxb6CreER(T) line gives very rapid and complete recombination over a short time window after a single tamoxifen dose, allowing precise time requirements for gene function to be assessed accurately. Embryonic cells cultured from the Hoxb6CreER(T) line also display rapid recombination ex vivo after tamoxifen exposure. Hence, the Hoxb6CreER(T) line provides a valuable tool for analyzing gene function, as well as lineage tracing studies using genetic cell marking, in several developing systems.
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Extremidades/embriología , Tracto Gastrointestinal/embriología , Proteínas de Homeodominio/metabolismo , Integrasas/genética , Mesodermo/embriología , Cresta Neural/embriología , Cola (estructura animal)/embriología , Animales , Tipificación del Cuerpo , Células Cultivadas , Embrión de Mamíferos/metabolismo , Extremidades/fisiología , Tracto Gastrointestinal/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Cresta Neural/metabolismo , Regiones Promotoras Genéticas , Recombinación Genética , Cola (estructura animal)/metabolismo , Tamoxifeno/farmacologíaRESUMEN
Sonic hedgehog (Shh), which regulates proliferation in many contexts, functions as a limb morphogen to specify a distinct pattern of digits. How Shh's effects on cell number relate to its role in specifying digit identity is unclear. Deleting the mouse Shh gene at different times using a conditional Cre line, we find that Shh functions to control limb development in two phases: a very transient, early patterning phase regulating digit identity, and an extended growth-promoting phase during which the digit precursor mesenchyme expands and becomes recruited into condensing digit primordia. Our analysis reveals an unexpected alternating anterior-posterior sequence of normal mammalian digit formation. The progressive loss of digits upon successively earlier Shh removal mirrors this alternating sequence and highlights Shh's role in cell expansion to produce the normal digit complement.
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Embrión de Mamíferos , Extremidades , Proteínas Hedgehog/metabolismo , Esbozos de los Miembros/fisiología , Morfogénesis , Animales , Tipificación del Cuerpo , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Extremidades/anatomía & histología , Extremidades/embriología , Extremidades/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Hedgehog/genética , Esbozos de los Miembros/anatomía & histología , Esbozos de los Miembros/efectos de los fármacos , Ratones , Ratones Transgénicos , Fenotipo , Tamoxifeno/efectos adversosRESUMEN
Indian hedgehog (Ihh) is essential for chondrocyte and osteoblast proliferation/differentiation during prenatal endochondral bone formation. The early lethality of various Ihh-ablated mutant mice, however, prevented further analysis of its role in postnatal bone growth and development. In this study, we describe the generation and characterization of a mouse model in which the Ihh gene was successfully ablated from postnatal chondrocytes in a temporal/spatial-specific manner; postnatal deletion of Ihh resulted in loss of columnar structure, premature vascular invasion, and formation of ectopic hypertrophic chondrocytes in the growth plate. Furthermore, destruction of the articular surface in long bones and premature fusion of growth plates of various endochondral bones was evident, resulting in dwarfism in mutant mice. More importantly, these mutant mice exhibited continuous loss of trabecular bone over time, which was accompanied by reduced Wnt signaling in the osteoblastic cells. These results demonstrate, for the first time, that postnatal chondrocyte-derived Ihh is essential for maintaining the growth plate and articular surface and is required for sustaining trabecular bone and skeletal growth.
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Condrocitos/metabolismo , Placa de Crecimiento/crecimiento & desarrollo , Placa de Crecimiento/metabolismo , Proteínas Hedgehog/metabolismo , Modelos Animales , Animales , Cartilla de ADN , Eliminación de Gen , Proteínas Hedgehog/genética , Inmunohistoquímica , Ratones , Ratones Transgénicos , Osteoblastos/metabolismo , Reacción en Cadena de la Polimerasa , Estadísticas no ParamétricasRESUMEN
Vertebrate retinal pigment epithelium (RPE) cells are derived from the multipotent optic neuroepithelium, develop in close proximity to the retina, and are indispensible for eye organogenesis and vision. Recent advances in our understanding of RPE development provide evidence for how critical signaling factors operating in dorso-ventral and distal-proximal gradients interact with key transcription factors to specify three distinct domains in the budding optic neuroepithelium: the distal future retina, the proximal future optic stalk/optic nerve, and the dorsal future RPE. Concomitantly with domain specification, the eye primordium progresses from a vesicle to a cup, RPE pigmentation extends towards the ventral side, and the future ciliary body and iris form from the margin zone between RPE and retina. While much has been learned about the molecular networks controlling RPE cell specification, key questions concerning the cell proliferative parameters in RPE and the subsequent morphogenetic events still need to be addressed in greater detail.
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Regulación del Desarrollo de la Expresión Génica/fisiología , Nervio Óptico/embriología , Epitelio Pigmentado Ocular/embriología , Pigmentación/fisiología , Retina/embriología , Transducción de Señal/fisiología , Animales , Diferenciación Celular/fisiología , Humanos , RatonesRESUMEN
Cartilage differentiation occurs over a broad time range from early embryonic development, when the mesenchymal condensations that give rise to cartilage models for future bone first appear, and continuing through adult life, when there is ongoing maintenance of articular joint surfaces and re-activation of cartilage formation after fracture. The chondrogenic response also figures in the pathogenesis of degenerative and inflammatory joint diseases. We have generated a transgenic line expressing tamoxifen-dependent Cre recombinase that gives efficient recombination in the chondrogenic lineage, both during embryogenesis and postnatally, and provides a valuable tool for analysis of gene function selectively in chondrogenic cells using conditional genetic approaches. Because the cartilage model of the limb skeleton forms progressively in a proximodistal order during discrete, well-defined time periods, evaluation of the spatial extent of tamoxifen-induced recombination along the limb axis during these time windows has also enabled us to examine the pharmacokinetics of single-dose tamoxifen injections during pregnancy.
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Cartílago/embriología , Cartílago/enzimología , Extremidades/embriología , Integrasas/metabolismo , Tamoxifeno/farmacología , Animales , Animales Recién Nacidos , Secuencia de Bases , Cartílago/efectos de los fármacos , Cartílago/crecimiento & desarrollo , Condrogénesis/efectos de los fármacos , Colágeno Tipo II/genética , Cartilla de ADN/genética , Extremidades/crecimiento & desarrollo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Edad Gestacional , Integrasas/administración & dosificación , Cinética , Operón Lac , Ratones , Ratones Transgénicos , Embarazo , Recombinación Genética/efectos de los fármacos , Tamoxifeno/farmacocinéticaRESUMEN
During vertebrate eye development, the cells of the optic vesicle (OV) become either neuroretinal progenitors expressing the transcription factor Chx10, or retinal pigment epithelium (RPE) progenitors expressing the transcription factor Mitf. Chx10 mutations lead to microphthalmia and impaired neuroretinal proliferation. Mitf mutants have a dorsal RPE-to-neuroretinal phenotypic transformation, indicating that Mitf is a determinant of RPE identity. We report here that Mitf is expressed ectopically in the Chx10(or-J/or-J) neuroretina (NR), demonstrating that Chx10 normally represses the neuroretinal expression of Mitf. The ectopic expression of Mitf in the Chx10(or-J/or-J) NR deflects it towards an RPE-like identity; this phenotype results not from a failure of neuroretinal specification, but from a partial loss of neuroretinal maintenance. Using Chx10 and Mitf transgenic and mutant mice, we have identified an antagonistic interaction between Chx10 and Mitf in regulating retinal cell identity. FGF (fibroblast growth factor) exposure in a developing OV has also been shown to repress Mitf expression. We demonstrate that the repression of Mitf by FGF is Chx10 dependent, indicating that FGF, Chx10 and Mitf are components of a pathway that determines and maintains the identity of the NR.
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Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/fisiología , Neuronas/metabolismo , Retina/embriología , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Animales , Linaje de la Célula , Ojo/embriología , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Transcripción Asociado a Microftalmía , Microscopía Fluorescente , Modelos Biológicos , Mutación , Fenotipo , Plásmidos/metabolismo , Retina/citología , Retina/metabolismo , Factores de Tiempo , TransgenesRESUMEN
Human stem cells derived from human fertilized oocytes, fetal primordial germ cells, umbilical cord blood, and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However, the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach, employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D), we established stem cell lines homozygous for H-2-B and H-2-D, respectively. The undifferentiated cells retained a normal karyotype, expressed stage-specific embryonic antigen-1 and Oct4, and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition, these cells demonstrated the potential for in vitro differentiation into endoderm, neuronal, and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes, and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible "homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation, while decreasing the ethical concerns surrounding human embryonic stem cell research.