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ABSTRACT: Spermatogonial stem cells divide throughout life, maintaining their own population and giving rise to differentiated gametes. The unstable regulatory protein Geminin is thought to be one of the factors that determine whether stem cells continue to divide or terminally differentiate. Geminin regulates the extent of DNA replication and is thought to maintain cells in an undifferentiated state by inhibiting various transcription factors and chromatin remodeling proteins. To examine how Geminin might regulate spermatogenesis, we developed two conditional mouse models in which the Geminin gene (Gmnn) is deleted from either spermatogonia or meiotic spermatocytes. Deleting Geminin from spermatogonia causes complete sterility in male mice. Gmnn(-/-) spermatogonia disappear during the initial wave of mitotic proliferation that occurs during the first week of life. Gmnn(-/-) spermatogonia exhibit more double-stranded DNA breaks than control cells, consistent with a defect in DNA replication. They maintain expression of genes associated with the undifferentiated state and do not prematurely express genes characteristic of more differentiated spermatogonia. In contrast, deleting Geminin from spermatocytes does not disrupt meiosis or the differentiation of spermatids into mature sperm. In females, Geminin is not required for meiosis, oocyte differentiation, or fertility after the embryonic period of mitotic proliferation has ceased. We conclude that Geminin is absolutely required for mitotic proliferation of spermatogonia but does not regulate their differentiation. Our results suggest that Geminin maintains replication fidelity during the mitotic phase of spermatogenesis, ensuring the precise duplication of genetic information for transmission to the next generation.
Developmental Biology 08/2012; 371(1):35-46. · 4.07 Impact Factor
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Alexander R Mackie,
Ekaterina Klyachko,
Tina Thorne, Kathryn M Schultz,
Meredith Millay,
Aiko Ito,
Christine E Kamide,
Ting Liu,
Rajesh Gupta,
Susmita Sahoo,
Sol Misener,
Raj Kishore,
Douglas W Losordo
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ABSTRACT: Ischemic cardiovascular disease represents one of the largest epidemics currently facing the aging population. Current literature has illustrated the efficacy of autologous, stem cell therapies as novel strategies for treating these disorders. The CD34+ hematopoetic stem cell has shown significant promise in addressing myocardial ischemia by promoting angiogenesis that helps preserve the functionality of ischemic myocardium. Unfortunately, both viability and angiogenic quality of autologous CD34+ cells decline with advanced age and diminished cardiovascular health.
To offset age- and health-related angiogenic declines in CD34+ cells, we explored whether the therapeutic efficacy of human CD34+ cells could be enhanced by augmenting their secretion of the known angiogenic factor, sonic hedgehog (Shh).
When injected into the border zone of mice after acute myocardial infarction, Shh-modified CD34+ cells (CD34(Shh)) protected against ventricular dilation and cardiac functional declines associated with acute myocardial infarction. Treatment with CD34(Shh) also reduced infarct size and increased border zone capillary density compared with unmodified CD34 cells or cells transfected with the empty vector. CD34(Shh) primarily store and secrete Shh protein in exosomes and this storage process appears to be cell-type specific. In vitro analysis of exosomes derived from CD34(Shh) revealed that (1) exosomes transfer Shh protein to other cell types, and (2) exosomal transfer of functional Shh elicits induction of the canonical Shh signaling pathway in recipient cells.
Exosome-mediated delivery of Shh to ischemic myocardium represents a major mechanism explaining the observed preservation of cardiac function in mice treated with CD34(Shh) cells.
Circulation Research 05/2012; 111(3):312-21. · 9.49 Impact Factor
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Amy Sasman,
Carey Nassano-Miller,
Kyoo Seok Shim,
Hyun Young Koo,
Ting Liu, Kathryn M Schultz,
Meredith Millay,
Atsushi Nanano,
Myengmo Kang,
Takashi Suzuki,
Tsutomu Kume
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ABSTRACT: The Forkhead box transcription factors, Foxc1 and Foxc2, are crucial for development of the eye, cardiovascular network, and other physiological systems, but their cell-type specific and postdevelopmental functions are unknown, in part because conventional (i.e., whole-organism) homozygous-null mutations of either factor result in perinatal death. Here, we describe the generation of mice with conditional-null Foxc1(flox) and Foxc2(flox) mutations that are induced via Cre-mediated recombination. Mice homozygous for the unrecombined alleles are viable and fertile, indicating that the conditional alleles retain their wild-type function. The embryos of Foxc1(flox) or Foxc2(flox) mice crossed with Cre-deleter mice that are homozygous for the recombined allele (i.e., Foxc1(Δ/Δ) or Foxc2(Δ/Δ) embryos) lack expression of the corresponding gene and show the same developmental defects observed in conventional homozygous mutant embryos. We expect these conditional mutations to enable characterization of the cell-type specific functions of Foxc1 and Foxc2 in development, disease, and adult animals. genesis 50:766-774, 2012. © 2012 Wiley Periodicals, Inc.
genesis 04/2012; 50(10):766-74. · 2.53 Impact Factor
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ABSTRACT: In many organisms early development is under control of the maternal genome and zygotic gene expression is delayed until the mid-blastula transition (MBT). As zygotic transcription initiates, cell cycle checkpoints become activated and the tempo of cell division slows. The mechanisms that activate zygotic transcription at the MBT are incompletely understood, but they are of interest because they may resemble mechanisms that cause stem cells to stop dividing and terminally differentiate. The unstable regulatory protein Geminin is thought to coordinate cell division with cell differentiation. Geminin is a bi-functional protein. It prevents a second round of DNA replication during S and G2 phase by binding and inhibiting the essential replication factor Cdt1. Geminin also binds and inhibits a number of transcription factors and chromatin remodeling proteins and is thought to keep dividing cells in an undifferentiated state. We previously found that the cells of Geminin-deficient Xenopus embryos arrest in G2 phase just after the MBT then disintegrate at the onset of gastrulation. Here we report that they also fail to express most zygotic genes. The gene expression defect is cell-autonomous and is reproduced by over-expressing Cdt1 or by incubating the embryos in hydroxyurea. Geminin deficient and hydroxyurea-treated blastomeres accumulate DNA damage in the form of double stranded breaks. Bypassing the Chk1 pathway overcomes the cell cycle arrest caused by Geminin depletion but does not restore zygotic gene expression. In fact, bypassing the Chk1 pathway by itself induces double stranded breaks and abolishes zygotic transcription. We did not find evidence that Geminin has a replication-independent effect on transcription. We conclude that Geminin is required to maintain genome integrity during the rapid cleavage divisions, and that DNA damage disrupts zygotic gene transcription at the MBT, probably through activation of DNA damage checkpoint pathways.
PLoS ONE 01/2012; 7(5):e38009. · 4.09 Impact Factor
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ABSTRACT: Normal vision requires the precise control of vascular growth to maintain corneal transparency. Here we provide evidence for a unique mechanism by which the Forkhead box transcription factor FoxC1 regulates corneal vascular development. Murine Foxc1 is essential for development of the ocular anterior segment, and in humans, mutations have been identified in Axenfeld-Rieger syndrome, a disorder characterized by anterior segment dysgenesis. We show that FOXC1 mutations also lead to corneal angiogenesis, and that mice homozygous for either a global (Foxc1(-/-)) or neural crest (NC)-specific (NC-Foxc1(-/-)) null mutation display excessive growth of corneal blood and lymphatic vessels. This is associated with disorganization of the extracellular matrix and increased expression of multiple matrix metalloproteinases. Heterozygous mutants (Foxc1(+/-) and NC-Foxc1(+/-)) exhibit milder phenotypes, such as disrupted limbal vasculature. Moreover, environmental exposure to corneal injury significantly increases growth of both blood and lymphatic vessels in both Foxc1(+/-) and NC-Foxc1(+/-) mice compared with controls. Notably, this amplification of the angiogenic response is abolished by inhibition of VEGF receptor 2. Collectively, these findings identify a role for FoxC1 in inhibiting corneal angiogenesis, thereby maintaining corneal transparency by regulating VEGF signaling.
Proceedings of the National Academy of Sciences 12/2011; 109(6):2015-20. · 9.68 Impact Factor
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Susmita Sahoo,
Ekaterina Klychko,
Tina Thorne,
Sol Misener, Kathryn M Schultz,
Meredith Millay,
Aiko Ito,
Ting Liu,
Christine Kamide,
Hemant Agrawal,
Harris Perlman,
Gangjian Qin,
Raj Kishore,
Douglas W Losordo
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ABSTRACT: Transplantation of human CD34(+) stem cells to ischemic tissues has been associated with reduced angina, improved exercise time, and reduced amputation rates in phase 2 clinical trials and has been shown to induce neovascularization in preclinical models. Previous studies have suggested that paracrine factors secreted by these proangiogenic cells are responsible, at least in part, for the angiogenic effects induced by CD34(+) cell transplantation.
Our objective was to investigate the mechanism of CD34(+) stem cell-induced proangiogenic paracrine effects and to examine if exosomes, a component of paracrine secretion, are involved.
Exosomes collected from the conditioned media of mobilized human CD34(+) cells had the characteristic size (40 to 90 nm; determined by dynamic light scattering), cup-shaped morphology (electron microscopy), expressed exosome-marker proteins CD63, phosphatidylserine (flow cytometry) and TSG101 (immunoblotting), besides expressing CD34(+) cell lineage marker protein, CD34. In vitro, CD34(+) exosomes replicated the angiogenic activity of CD34(+) cells by increasing endothelial cell viability, proliferation, and tube formation on Matrigel. In vivo, the CD34(+) exosomes stimulated angiogenesis in Matrigel plug and corneal assays. Interestingly, exosomes from CD34(+) cells but not from CD34(+) cell-depleted mononuclear cells had angiogenic activity.
Our data demonstrate that human CD34(+) cells secrete exosomes that have independent angiogenic activity both in vitro and in vivo. CD34(+) exosomes may represent a significant component of the paracrine effect of progenitor cell transplantation for therapeutic angiogenesis.
Circulation Research 08/2011; 109(7):724-8. · 9.49 Impact Factor
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ABSTRACT: Neural stem cells (NSCs) are the progenitors of neurons and glial cells during both embryonic development and adult life. The unstable regulatory protein Geminin (Gmnn) is thought to maintain neural stem cells in an undifferentiated state while they proliferate. Geminin inhibits neuronal differentiation in cultured cells by antagonizing interactions between the chromatin remodeling protein Brg1 and the neural-specific transcription factors Neurogenin and NeuroD. Geminin is widely expressed in the CNS during throughout embryonic development, and Geminin expression is down-regulated when neuronal precursor cells undergo terminal differentiation. Over-expression of Geminin in gastrula-stage Xenopus embryos can expand the size of the neural plate. The role of Geminin in regulating vertebrate neurogenesis in vivo has not been rigorously examined. To address this question, we created a strain of Nestin-Cre/Gmnn(fl/fl) mice in which the Geminin gene was specifically deleted from NSCs. Interestingly, we found no major defects in the development or function of the central nervous system. Neural-specific Gmnn(Δ/Δ) mice are viable and fertile and display no obvious neurological or neuroanatomical abnormalities. They have normal numbers of BrdU(+) NSCs in the subgranular zone of the dentate gyrus, and Gmnn(Δ/Δ) NSCs give rise to normal numbers of mature neurons in pulse-chase experiments. Gmnn(Δ/Δ) neurosphere cells differentiate normally into both neurons and glial cells when grown in growth factor-deficient medium. Both the growth rate and the cell cycle distribution of cultured Gmnn(Δ/Δ) neurosphere cells are indistinguishable from controls. We conclude that Geminin is largely dispensable for most of embryonic and adult mammalian neurogenesis.
PLoS ONE 01/2011; 6(3):e17736. · 4.09 Impact Factor
