Search

Many drugs have progressed through preclinical and clinical trials and have been available - for years in some cases - before being recalled by the FDA for unanticipated toxicity in humans. One reason for such poor translation from drug candidate to successful use is a lack of model systems that accurately recapitulate normal tissue function of human organs and their response to drug compounds. Moreover, tissues in the body do not exist in isolation, but reside in a highly integrated and dynamically interactive environment, in which actions in one tissue can affect other downstream tissues. Few engineered model systems, including the growing variety of organoid and organ-on-a-chip platforms, have so far reflected the interactive nature of the human body. To address this challenge, we have developed an assortment of bioengineered tissue organoids and tissue constructs that are integrated in a closed circulatory perfusion system, facilitating inter-organ responses. We describe a three-tissue organ-on-a-chip system, comprised of liver, heart, and lung, and highlight examples of inter-organ responses to drug administration. We observe drug responses that depend on inter-tissue interaction, illustrating the value of multiple tissue integration for in vitro study of both the efficacy of and side effects associated with candidate drugs.

The inadequacy of animal models in correctly predicting drug and biothreat agent toxicity in humans has resulted in a pressing need for in vitro models that can recreate the in vivo scenario. One of the most important organs in the assessment of drug toxicity is liver. Here, we report the development of a liver-on-a-chip platform for long-term culture of three-dimensional (3D) human HepG2/C3A spheroids for drug toxicity assessment. The bioreactor design allowed for in situ monitoring of the culture environment by enabling direct access to the hepatic construct during the experiment without compromising the platform operation. The engineered bioreactor could be interfaced with a bioprinter to fabricate 3D hepatic constructs of spheroids encapsulated within photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. The engineered hepatic construct remained functional during the 30 days culture period as assessed by monitoring the secretion rates of albumin, alpha-1 antitrypsin, transferrin, and ceruloplasmin, as well as immunostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.01.005 Byline: Ming Zhao (a), Jan Rohozinski (b), Manju Sharma (c), Jun Ju (a), Robert E. Braun (c), Colin E. Bishop (b)(d), Marvin L. Meistrich (a) Keywords: Spermatogenesis; Retroposons; Retrotransposition; Utp14; Juvenile spermatogonial depletion; X-chromosome; Spermatocytes; Spermatogonia; Male germ cells Abstract: The mouse retrogene Utp14b is essential for male fertility, and a mutation in its sequence results in the sterile juvenile spermatogonial depletion (jsd) phenotype. It is a retrotransposed copy of the Utp14a gene, which is located on the X chromosome, and is inserted within an intron of the autosomal acyl-CoA synthetase long-chain family member 3 (Acsl3) gene. To elucidate the roles of the Utp14 genes in normal spermatogenic cell development as a basis for understanding the defects that result in the jsd phenotype, we analyzed the various mRNAs produced from the Utp14b retrogene and their expression in different cell types. Two classes of transcripts were identified: variant 1, a transcript driven by the host gene promoter, that is predominantly found in germ cells but is ubiquitously expressed at low levels; and variants 2-5, a group of alternatively spliced transcripts containing some unique untranslated exons that are transcribed from a novel promoter that is germ-cell-specific. Utp14b (predominantly variant 1) is expressed at moderately high levels in pachytene spermatocytes, the developmental stage at which the expression of the X-linked Utp14a is suppressed. The levels of both classes of Utp14b transcripts were highest in round spermatids despite the transcription of Utp14a in these cells. We propose that when Utp14b initially inserted into Acsl3, it utilized the Acsl3 promoter to drive expression in pachytene spermatocytes to compensate for inactivation of Utp14a expression. The novel cell-type-specific promoter for Utp14b likely evolved later, as the protein may have acquired a germ cell-specific function in spermatid development. Author Affiliation: (a) Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Box 066, 1515 Holcombe Blvd, Houston, TX 77030, USA (b) Department of Obstetrics and Gynecology, Baylor College of Medicine, 1709 Dryden Road, Houston, TX 77030, USA (c) Department of Genome Sciences, University of Washington School of Medicine, Box 357730, 1705 N.E. Pacific Street, Seattle, WA 98195, USA (d) Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Article History: Received 28 September 2006; Revised 9 December 2006; Accepted 3 January 2007

The transgenic insertional mouse mutation Odd Sex (Ods) represents a model for the long-range regulation of Sox9. The mutation causes complete female-to-male sex reversal by inducing a male-specific expression pattern of Sox9 in XX Ods/+ embryonic gonads. We previously described an A/J strain-specific suppressor of Ods termed Odsm[1.sup.A]. Here we show that phenotypic sex depends on a complex interaction between the suppressor and the transgene. Suppression can be achieved only if the transgene is transmitted paternally. In addition, the suppressor itself exhibits a maternal effect, suggesting that it may act on chromatin in the early embryo.

[Quaking.sup.viable] ([qk.sup.v]) is a recessive neurological mouse mutation with severe dysmyelination of the CNS and spermiogenesis failure. The molecular lesion in the [qk.sup.v] mutant is a deletion of [approximately equal to] 1 Mb on mouse chromosome 17 that alters the expression of the qk gene in oligodendrocytes. Complementation analysis between the [qk.sup.v] mutation and qk mutant alleles generated through chemical mutagenesis showed that the male sterility is a distinctive feature of the [qk.sup.v] allele. This observation suggested that the sperm differentiation defect in [qk.sup.v] is due to the deletion of a gene(s) distinct from qk. Here, we demonstrate that the deletion of Pacrg is the cause of male sterility in the [qk.sup.v] mutant. Pacrg is the mouse homologue of the human PARKIN-coregulated gene (PACRG), which encodes for a protein whose biochemical function remains unclear. We show that Pacrg is highly expressed in the testes in both mice and humans. In addition, the expression pattern of Pacrg during spermiogenesis suggests that it plays a role in sperm differentiation. In support of this hypothesis, we show that transgenic expression of Pacrg in testes restores spermiogenesis and fertility in [qk.sup.v] males. This finding provides the first in vivo evidence, to our knowledge, for the function of Pacrg in a model organism. Immunolocalization experiments on isolated spermatozoa show that the Pacrg protein is present in mature sperm. Remarkably, the mammalian Pacrg protein shares significant sequence similarities with gene products from flagellated protozoans, suggesting that Pacrg may be necessary for proper flagellar formation in many organisms.

How memory T cells are maintained in vivo is poorly understood. To address this problem, a male-specific peptide (H-Y) was identified and used to activate female anti-H-Y T cells in vitro. Anti-H-Y T cells survived in vivo for at least 70 days in the absence of antigen. This persistence was not because of the intrinsic ability of memory T cells to survive in vivo. Instead, the survival and function of adoptively transferred memory cells was found to require transporter of antigen protein 1-dependent expression of self-peptide/major histocompatibility complex class I molecules in recipient animals. Therefore, it appears that the level of T cell receptor engagement provided by transporter of antigen protein 1-dependent, self-peptide/major histocompatibility complexes is sufficient to maintain the long-term survival and functional phenotype of memory cells in the absence of persistent antigen. These data suggest that positive selection plays a role not only in T cell development but also in the maintenance of T cell memory.

The discovery of a functional marsupial Y-linked homologue gene of the murine Ube1y gene proves that the Metatherian (marsupial) and Eutherian (placental) infraclasses of the mammalian subclass Theria evolved from a single ancestor, probably between 120 and 150 million years before present. This Ube1y gene contributes to the development of males among both marsupials and placentals by triggering spermatogenesis. This finding confirms the molecular and genetic link between marsupials and placentals.