Cochlear repair by transplantation of human cord blood CD133+ cells to nod-scid mice made deaf with kanamycin and noise.

We investigated the fate of human cord blood CD133+ hematopoietic stem cells (HSC) transplanted intravenously (IV) into irradiated nod-scid mice previously made deaf by ototoxic treatment with kanamycin and/ or intense noise, to verify whether HSC engraft the cochlea and contribute to inner ear restoration, in vivo. We tested the presence of HLA.DQalpha1 by PCR, used for traceability of engrafted cells, finding evidence that HSC migrated to various host tissues, including the organ of Corti (OC).

Prolonged human/sheep cellular chimerism following transplantation of human hemopoietic stem cells into the ewe celomic cavity.

We evaluated the possibility of prolonged chimerism formation in fetus and lamb, following human cord blood-selected CD133+ hemopoietic stem cell (HSC) transplantation into the celomic cavity of ewes at a pre-immune fetal age (44-45 days of pregnancy). Nineteen ewes were injected with HSC and 5 controls with a saline solution. By PCR, HLA-DQ alpha 1 and 6 human microsatellites (CODIS) were used for HSC traceability.

Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture.

We report on an in vitro organ culture method to investigate human conjunctival epithelial basal precursor cells and their progeny within a more natural three-dimensional microenvironment. Conjunctival fragments were cultured on gelatin sponges in medium with 10% FBS. The conjunctival phenotype of the epithelium was confirmed by the expression and distribution of a panel of markers (p63, CK-13/CK-10, CK-19, Ki-67, PAS for goblet cells, CD45 for infiltrating interlamellar leukocytes and nestin for mesenchymal and ocular epithelial precursor cells).

Establishment of an organotypic in vitro culture system and its relevance to the characterization of human prostate epithelial cancer cells and their stromal interactions.

Human prostatic adenocarcinoma fragments (1-6mm) were cultured on collagen sponges in medium supplemented or not supplemented with 4,5alpha-dihydrotesterone (DHT) until 3 weeks, maintaining the three-dimensional (3D) epithelial and stromal organization present in the tumor in vivo. With time, in the presence of DHT, locally progressive cribriform nests of neoplastic cells with proliferative rates higher than those inside the fragment developed on the surface, while the stroma became more dissociated, and fibrosis replaced the muscular component. The 3D-culture provides a promising approach for studying the development and phenotype of prostate epithelial tumor progenitor cells and their stromal interactions.

Selective growth and expansion of human corneal epithelial basal stem cells in a three-dimensional-organ culture.

We report on a three dimensional (3D)-organotypic culture in vitro for selective growth and expansion of human corneal epithelial stem cells. Limbal corneal explants were cultured on porous collagen sponges submerged in Epilife medium containing 10% fetal bovine serum. The fragments were analyzed by immunohistochemistry for the expression and distribution of a spectrum of corneal epithelium markers: p63, CK-19, CK-3, Ki-67, pan-cytokeratins and vimentin.

A three-dimensional organotypic culture of the human uterine exocervix for studying mucosal epithelial differentiation and migrating leukocytes.

We report on a three-dimensional organotypic culture in vitro of explants from the human uterine exocervix. Exocervical fragments (2-3 mm3) from pre-menopausal women were cultured on sponges submerged in Dulbecco's Modified Eagle's Medium containing p-nonylphenol and 10% fetal bovine serum for up to 3 weeks and the viability and cellular responses were assayed. The fragments were analyzed by immunohistochemistry for the expression and distribution of a broad spectrum of cellular markers: p63, Ki-67, involucrin, high molecular weight cytokeratins, estrogen receptor-alpha, vimentin, CD45, and CD31.

Selective growth of epithelial basal cells from human prostate in a three-dimensional organ culture.

Background: A three-dimensional organotypic culture method has been developed for selectively growing epithelial basal cells from human benign prostate.

Methods: Tissue fragments were cultured on sponges for several weeks and the viability of luminal and basal epithelium and cellular responses to 4,5alpha-dihydrotesterone (DHT) stimulation were studied.

Results: The gland tissue could be successfully maintained showing preservation of ducts and lobules as in vivo.

Isolation and clonal analysis of human epidermal keratinocyte stem cells in long-term culture.

We developed a procedure for growing normal epidermal keratinocyte stem cells isolated from a single punch biopsy of adult human skin in long-term culture. Primary skin epithelial cells were maintained in collagen-coated plates with irradiated human neonatal foreskin fibroblasts (line HPI.1) as a feeder for more than 120 days, approximately 115 population doublings, without signs of replicative senescence.

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems.

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges).