Activated and nonactivated MSCs increase survival in humanized mice after acute liver injury through alcohol binging.

The ability of the liver to regenerate after injury makes it an ideal organ to study for potential therapeutic interventions. Mesenchymal stem cells (MSCs) possess self-renewal and differentiation properties, as well as anti-inflammatory properties that make them an ideal candidate for therapy of acute liver injury. The primary aim of this study is to evaluate the potential for reversal of hepatic injury using human umbilical cord-derived MSCs. Secondary aims include comparison of various methods of administration as well as comparison of activated versus nonactivated human umbilical cord stem cells. To induce liver injury, humanized mice were fed high-cholesterol high-fat liquid diet with alcohol binge drinking. Mice were then treated with either umbilical cord MSCs, activated umbilical cord MSCs, or a placebo and followed for survival. Blood samples were obtained at the end of the binge drinking and at the time of death to measure alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Histology of all mouse livers was reported at time of death. Activated MSCs that were injected intravenously, intraperitoneally, or both routes had superior survival compared with nonactivated MSCs and with placebo-treated mice. AST and ALT levels were elevated in all mice before treatment and improved in the mice treated with stem cells. Conclusion: Activated stem cells resulted in marked improvement in survival and in recovery of hepatic chemistries. Activated umbilical cord MSCs should be considered an important area of investigation in acute liver injury.

Pyrophosphate Supplementation Prevents Chronic and Acute Calcification in ABCC6-Deficient Mice.

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6-/- mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6-/- mice over several months prevented the development of pseudoxanthoma elasticum-like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6-/- mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.

Presence of the Paternal Pronucleus Assists Embryo in Overcoming Cycloheximide Induced Abnormalities in Zygotic Mitosis.

After fertilization, the maternal and paternal chromosomes independently proceed through pronuclear formation. These chromatin reconfigurations occur within a shared cytoplasm thus exposing both gametes to the same factors. Here, we report that continuous cycloheximide [40 µg/mL] treatment of parthenogenotes, androgenotes, and ICSI embryos reveals ORC2 pronuclear instability in the maternal (MPN) but not the paternal pronucleus (PPN). When released from CHX after 8 h, the MPN can recover ORC2 and proceed through replication, however, parthenogenotes encounter severe mitotic defects while both ICSI embryos and androgenotes are able to recover and develop at significantly higher rates. Taken together, these data suggest cycloheximide treatment promotes an environment that asymmetrically affects the stability of ORC2 on the MPN, and the ability of the MPN to develop. Furthermore, the presence of the PPN in the zygote can ameliorate both effects. These data suggest further evidence for crosstalk between the two pronuclei during the first cell cycle of the embryo. J. Cell. Biochem. 117: 1806-1812, 2016. © 2016 Wiley Periodicals, Inc.

ORC4 surrounds extruded chromatin in female meiosis.

Six proteins, ORC1-6, make up the origin recognition complex (ORC) that initiates licensing of DNA replication origins. We have previously reported that subunit ORC2 is localized between the separating maternal chromosomes at anaphase II just after fertilization and is present in zygotic pronuclei at G1. Here, we found that ORC1, 3, and 5 all localize between the chromosomes at anaphase II, but could not be detected in zygotic G1. ORC6 localized to the periphery of the nucleoli at all zygotic stages. We identified an unexpected potential role for ORC4 in polar body formation. We found that in both female meiotic divisions, ORC4 surrounds the set of chromosomes, as a sphere-like structure, that will eventually be discarded in the polar bodies, but not the chromosomes that segregate into the oocyte. None of the other five ORC proteins are involved in this structure. In Zygotic G1, ORC4 surrounds the nuclei of the polar bodies, but was not detectable in the pronuclei. When the zygote entered mitosis ORC4 was only detected in the polar body. However, ORC4 appeared on both sets of separating chromosomes at telophase. At this point, the ORC4 that was in the polar body also migrated into the nuclei, suggesting that ORC4 or an associated protein is modified during the first embryonic cell cycle to allow it to bind DNA. Our results suggest that ORC4 may help identify the chromosomes that are destined to be expelled in the polar body, and may play a role in polar body extrusion. ORC4 surrounds the chromatin that will be extruded in the polar body in both female meiotic divisions, then makes a transition from the cytoplasm to the chromosomes at zygotic anaphase, suggesting multiple roles for this replication licensing protein.

Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development.

Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis.

We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community.

Unique pattern of ORC2 and MCM7 localization during DNA replication licensing in the mouse zygote.

In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the mouse zygotes and two-cell embryos. In somatic cells ORC2 remains bound to DNA replication origins throughout the cell cycle, while MCM7 is one of the last proteins to bind to the licensing complex. We found that MCM7 but not ORC2 was bound to DNA in metaphase II oocytes and remained associated with the DNA until S-phase. Shortly after fertilization, ORC2 was detectable at the metaphase II spindle poles and then between the separating chromosomes. Neither protein was present in the sperm cell at fertilization. As the sperm head decondensed, MCM7 was bound to DNA, but no ORC2 was seen. By 4 h after fertilization, both pronuclei contained DNA bound ORC2 and MCM7. As expected, during S-phase of the first zygotic cell cycle, MCM7 was released from the DNA, but ORC2 remained bound. During zygotic mitosis, ORC2 again localized first to the spindle poles, then to the area between the separating chromosomes. ORC2 then formed a ring around the developing two-cell nuclei before entering the nucleus. Only soluble MCM7 was present in the G2 pronuclei, but by zygotic metaphase it was bound to DNA, again apparently before ORC2. In G1 of the two-cell stage, both nuclei had salt-resistant ORC2 and MCM7. These data suggest that licensing follows a unique pattern in the early zygote that differs from what has been described for other mammalian cells that have been studied.

Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects.

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

Differentiation potential of germ line stem cells derived from the postnatal mouse ovary.

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.

Generation of multipotent cell lines from a distinct population of male germ line stem cells.

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres.

The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being. ES cell lines are conventionally isolated from the inner cell mass of blastocysts and, in a few instances, from cleavage stage embryos. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines-using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects-that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.

The Sall3 locus is an epigenetic hotspot of aberrant DNA methylation associated with placentomegaly of cloned mice.

DNA methylation controls various developmental processes by silencing, switching and stabilizing genes as well as remodeling chromatin. Among various symptoms in cloned animals, placental hypertrophy is commonly observed. We identified the Spalt-like gene3 (Sall3) locus as a hypermethylated region in the placental genome of cloned mice. The Sall3 locus has a CpG island containing a tissue-dependent differentially methylated region (T-DMR) specific to the trophoblast cell lineage. The T-DMR sequence is also conserved in the human genome at the SALL3 locus of chromosome 18q23, which has been suggested to be involved in the 18q deletion syndrome. Intriguingly, larger placentas were more heavily methylated at the Sall3 locus in cloned mice. This epigenetic error was found in all cloned mice examined regardless of sex, mouse strain and the type of donor cells. In contrast, the placentas of in vitro fertilized (IVF) and intracytoplasmic sperm injected (ICSI) mice did not show such hypermethylation, suggesting that aberrant hypermethylation at the Sall3 locus is associated with abnormal placental development caused by nuclear transfer of somatic cells. We concluded that the Sall3 locus is the area with frequent epigenetic errors in cloned mice. These data suggest that there exists at least genetic locus that is highly susceptible to epigenetic error caused by nuclear transfer.

Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning.

Germ cells undergo epigenetic modifications as they develop, which suggests that they may be ideal donors for nuclear transfer (cloning). In this study, nuclei from confirmed embryonic germ cells were used as donors to determine whether they are competent for cloning and at which stage they are most competent. Embryos cloned from migrating 10.5-days-postcoitum (dpc) primordial germ cells (PGCs) showed normal morphological development to midgestation but died shortly thereafter. In contrast, embryos cloned from later-stage germ cells were developmentally delayed at midgestation. Thus, donor germ cell age inversely correlated with the developmental stage attained by cloned embryos. The methylation status of the H19- and Snrpn-imprinting control regions in germ cell clones paralleled that of the donors, and revealed that demethylation, or erasure of imprints, was already initiated in PGCs at 10.5 dpc and was complete by 13.5 dpc. Similarly, clones derived from male 15.5-dpc germ cells showed increased methylation correlating with the initiation of de novo methylation that resets imprints at this stage, and clones from neonatal germ cells showed nearly complete methylation in the H19 imprinting control region. These results indicate that the epigenetic state of the donor nucleus is retained in cloned embryos, and that germ cells are therefore inadequate nuclear donors for cloning because they are either erasing or resetting epigenetic patterns.

Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes.

It has been shown that mature oocytes injected with nuclei from round spermatids collected from mouse testis can generate normal offspring and that round spermatids can develop in vitro. An undetermined issue is whether spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (gamma-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy (http://www.sci.ccny.cuny.edu/ approximately kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins.