Gabriel Gonzalez

Regeneration and experimental orthotopic transplantation of a bioengineered kidney

Approximately 100,000 individuals in the United States currently await kidney transplantation, and 400,000 individuals live with end-stage kidney disease requiring hemodialysis. The creation of a transplantable graft to permanently replace kidney function would address donor organ shortage and the morbidity associated with immunosuppression. Such a bioengineered graft must have the kidneys architecture and function and permit perfusion, filtration, secretion, absorption and drainage of urine. We decellularized rat, porcine and human kidneys by detergent perfusion, yielding acellular scaffolds with vascular, cortical and medullary architecture, a collecting system and ureters. To regenerate functional tissue, we seeded rat kidney scaffolds with epithelial and endothelial cells and perfused these cell-seeded constructs in a whole-organ bioreactor. The resulting grafts produced rudimentary urine in vitro when perfused through their intrinsic vascular bed. When transplanted in an orthotopic position in rat, the grafts were perfused by the recipients circulation and produced urine through the ureteral conduit in vivo.

Dicer is required for female reproductive tract development and fertility in the mouse

Dicer encodes a riboendonuclease required for microRNA biosynthesis. Dicer was inactivated in Müllerian duct mesenchyme‐derived tissues of the reproductive tract of the mouse, using an Amhr2‐Cre allele. Although Amhr2‐Cre; Dicer conditional mutant males appeared normal and were fertile, mutant females were infertile. In adult mutant females, there was a reduction in the size of the oviducts and uterine horns. The oviducts were less coiled compared to controls and cysts formed at the isthmus near the uterotubal junction. Unfertilized, degenerate oocytes were commonly found within these cysts, indicating a defect in embryo transit. Beads transferred into the mutant oviduct failed to migrate into the uterus. In addition, blastocysts transferred directly into the mutant uterus did not result in pregnancy. Histological analysis demonstrated that the mutant uterus contained less glandular tissue and often the few glands that remained were found within the myometrium, an abnormal condition known as adenomyosis. In adult mutants, there was ectopic expression of Wnt4 and Wnt5a in the luminal epithelium (LE) and glandular epithelium (GE) of the uterus, and Wnt11 was ectopically expressed in GE. These results demonstrate that Dicer is necessary for postnatal differentiation of Müllerian duct mesenchyme‐derived tissues of the female reproductive tract, suggesting that microRNAs are important regulators of female reproductive tract development and fertility. Mol. Reprod. Dev. 76: 678–688, 2009.

Perfusion decellularization of human and porcine lungs: Bringing the matrix to clinical scale

BACKGROUND Organ engineering is a theoretical alternative to allotransplantation for end-stage organ failure. Whole-organ scaffolds can be created by detergent perfusion via the native vasculature, generating an acellular matrix suitable for recellularization with selected cell types. We aimed to up-scale this process, generating biocompatible scaffolds of a clinically relevant scale. METHODS Rat, porcine, and human lungs were decellularized by detergent perfusion at constant pressures. Collagen, elastin, and glycosaminoglycan content of scaffolds were quantified by colorimetric assays. Proteomic analysis was performed by microcapillary liquid chromatography tandem mass spectrometry. Extracellular matrix (ECM) slices were cultured with human umbilical vein endothelial cells (HUVEC), small airway epithelial cells (SAEC), or pulmonary alveolar epithelial cells (PAECs) and evaluated by time-lapse live cell microscopy and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Whole-organ culture was maintained under constant-pressure media perfusion after seeding with PAECs. RESULTS Rat lungs were decellularized using: (1) sodium dodecyl sulfate (SDS), (2) sodium deoxycholate (SDC), or (3) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Resulting scaffolds showed comparable loss of DNA but greatest preservation of ECM components in SDS-decellularized lungs. Porcine (n = 10) and human (n = 7) lungs required increased SDS concentration, perfusion pressures, and time to achieve decellularization as determined by loss of DNA, with preservation of intact matrix composition and lung architecture. Proteomic analysis of human decellularized lungs further confirmed ECM preservation. Recellularization experiments confirmed scaffold biocompatibility when cultured with mature cell phenotypes and scaffold integrity for the duration of biomimetic culture. CONCLUSIONS SDS-based perfusion decellularization can be applied to whole porcine and human lungs to generate biocompatible organ scaffolds with preserved ECM composition and architecture.

Bioengineering Human Myocardium on Native Extracellular Matrix

RATIONALE More than 25 million individuals have heart failure worldwide, with ≈4000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only ≈2500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. OBJECTIVE The objective of this study is to translate previous work to human scale and clinically relevant cells for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human induced pluripotent stem cell-derived cardiomyocytes. METHODS AND RESULTS To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiomyocytes derived from nontransgenic human induced pluripotent stem cells and generated tissues of increasing 3-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole-heart scaffolds with human induced pluripotent stem cell-derived cardiomyocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue and showed electrical conductivity, left ventricular pressure development, and metabolic function. CONCLUSIONS Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human induced pluripotent stem cell-derived cardiomyocytes and enable the bioengineering of functional human myocardial-like tissue of multiple complexities.

Long-term persistence and development of induced pancreatic beta cells generated by lineage conversion of acinar cells

In the version of this article initially published, the first three bars in the histogram in Figure 1a should have read “No vehicle,” “No Dz” and “Dz13” instead of “No Dz,” “Dz13” and “Dz13scr.” The legend of Figure 1a should have included the sentences: “‘No vehicle’ represents the normoxia control without vehicle (transfection agent) or DNAzyme or siRNA. All other groups contain vehicle.” The H&E-stained images in Figure 1a should have read “Dz13 in hyperoxia-normoxia” and “Dz13scr in hyperoxia-normoxia” instead of “Normoxia” and “Hyperoxia-normoxia.” None of the conclusions is affected by the errors. The errors have been corrected in the HTML and PDF versions of the article.

A Requirement for ERK-Dependent Dicer Phosphorylation in Coordinating Oocyte-to-Embryo Transition in C. elegans

Signaling pathways and small RNAs direct diverse cellular events, but few examples are known of defined signaling pathways directly regulating small RNA biogenesis. We show that ERK phosphorylates Dicer on two conserved residues in its RNase IIIb and double-stranded RNA (dsRNA)-binding domains and that phosphorylation of these residues is necessary and sufficient to trigger Dicers nuclear translocation in worms, mice, and human cells. Phosphorylation of Dicer on either site inhibits Dicer function in the female germline and dampens small RNA repertoire. Our data demonstrate that ERK phosphorylates and inhibits Dicer during meiosis I for oogenesis to proceed normally in Caenorhabditis elegans and that this inhibition is released before fertilization for embryogenesis to proceed normally. The conserved Dicer residues, their phosphorylation by ERK, and the consequences of the resulting modifications implicate an ERK-Dicer nexus as a fundamental component of the oocyte-to-embryo transition and an underlying mechanism coupling extracellular cues to small RNA production.

CTNNB1 in Mesenchyme Regulates Epithelial Cell Differentiation during Müllerian Duct and Postnatal Uterine Development

Müllerian duct differentiation and development into the female reproductive tract is essential for fertility, but mechanisms regulating these processes are poorly understood. WNT signaling is critical for proper development of the female reproductive tract as evident by the phenotypes of Wnt4, Wnt5a, Wnt7a, and β-catenin (Ctnnb1) mutant mice. Here we extend these findings by determining the effects of constitutive CTNNB1 activation within the mesenchyme of the developing Müllerian duct and its differentiated derivatives. This was accomplished by crossing Amhr2-Cre knock-in mice with Ctnnb1 exon (ex) 3(f/f) mice. Amhr2-Cre(Δ/+); Ctnnb1 ex3(f/+) females did not form an oviduct, had smaller uteri, endometrial gland defects, and were infertile. At the cellular level, stabilization of CTNNB1 in the mesenchyme caused alterations within the epithelium, including less proliferation, delayed uterine gland formation, and induction of an epithelial-mesenchymal transition (EMT) event. This EMT event is observed before birth and is complete within 5 days after birth. Misexpression of estrogen receptor α in the epithelia correlated with the EMT before birth, but not after. These studies indicate that regulated CTNNB1 in mesenchyme is important for epithelial cell differentiation during female reproductive tract development.

Identification and frequency of CCR5Δ32/Δ32 HIV-resistant cord blood units from Houston area hospitals

The use of umbilical cord blood (CB) that is genetically resistant to HIV infection has been proposed as a novel stem cell therapy for the treatment of patients with AIDS. These genetically unique CB units (CBUs) should be present in public CB banks at a predicted frequency.

Cord blood stem cell therapy for acquired immune deficiency syndrome.

Cord blood stem cell transplantation is routinely used to treat hematopoietic diseases. Individuals who are homozygous for the 32 polymorphism of the CCR5 locus, encoding a co-receptor for HIV-1, are normal and are resistant to HIV infection. Here we suggest that public cord blood repositories are likely to contain CCR5 homozygous units that could be used as a therapy for HIV-infected individuals.

Illuminating Testis Morphogenesis in the Mouse

The mammalian testis is a complex organ composed of multiple cell types that are organized into seminiferous tubules and an interstitium, producing spermatozoa and hormones, respectively. During embryogenesis, the testis forms from the genital ridge associated with the embryonic kidney called the mesonephros. After germ cells migrate into the genital ridge, the Sertoli cell-germ cell mass forms and undergoes morphogenetic changes to generate testis cords, the precursors of the seminiferous tubules. Static images of the fetal testis at sequential stages of development provide structural information about cord formation. Transgenic mice that express fluorescent protein reporters offer new opportunities for time-lapse imaging to visualize live cells and their behaviors during testis differentiation and morphogenesis.