This review focuses on current tissue engineering strategies for promoting vascularized bone regeneration. We review the role of angiogenic growth factors in promoting vascularized bone regeneration and discuss the different therapeutic strategies for controlled/sustained growth factor delivery. Next, we address the therapeutic uses of stem cells in vascularized bone regeneration. Specifically, this review addresses the concept of co-culture using osteogenic and vasculogenic stem cells, and how adipose derived stem cells compare to bone marrow derived mesenchymal stem cells in the promotion of angiogenesis. We conclude this review with a discussion of a novel approach to bone regeneration through a cartilage intermediate, and discuss why it has the potential to be more effective than traditional bone grafting methods.

Despite the increasing importance of the pig as a large animal model, little is known about porcine neural precursor cells. To evaluate the markers expressed by these cells, brains were dissected from 60-day fetuses, enzymatically dissociated, and grown in the presence of epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor. Porcine neural precursors could be grown as suspended spheres or adherent monolayers, depending on culture conditions. Expanded populations were banked or harvested for analysis using reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, microarrays, and flow cytometry, and results compared with data from analogous human forebrain progenitor cells. Cultured porcine neural precursors widely expressed neural cell adhesion molecule (NCAM), polysialic acid (PSA)-NCAM, vimentin, Ki-67, and Sox2. Minority subpopulations of cells expressed doublecortin, β-III tubulin, synapsin I, glial fibrillary acidic protein (GFAP), and aquaporin 4 (AQP4) consistent with increased lineage restriction. A human microarray detected porcine transcripts for nogoA (RTN4) and stromal cellderived factor 1 (SDF1), possibly cyclin D2 and Pbx1, but not CD133, Ki-67, nestin, or nucleostemin. Subsequent RT-PCR showed pig forebrain precursors to be positive for cyclin D2, nucleostemin, nogoA, Pbx1, vimentin, and a faint band for SDF1, whereas no signal was detected for CD133, fatty acid binding protein 7 (FABP7), or Ki-67. Human forebrain progenitor cells were positive for all the genes mentioned. This study shows that porcine neural precursors share many characteristics with their human counterparts and, thus, may be useful in porcine cell transplantation studies potentially leading to the application of this strategy in the setting of nervous system disease and injury.

Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials.

The cat has served as an important nonrodent research model for neurophysiology and retinal degenerative disease processes, yet very little is known about feline neural precursor cells. To culture these cells and evaluate marker expression, brains were dissected from 45-day-old fetuses, enzymatically dissociated, and grown in the presence of EGF, bFGF and PDGF. Expanded cells widely expressed nestin, Sox2, Ki-67, fusin (CXCR4) and vimentin, while subpopulations expressed A2B5, GFAP, or beta-III tubulin. Precursors prelabeled with BrdU and/or transduced with a recombinant lentivirus that expresses GFP were transplanted subretinally in five dystrophic Abyssinian cats. Two to 4 weeks following surgery, histology showed survival of grafted cells in three of the animals. Labeled cells were found in the neuroretina and RPE layer, as well as in the vitreous and the vicinity of Bruch's membrane. There was no evidence of an immunologic response in any of the eyes. Neural precursor cells can therefore be cultured from the developing cat brain and survive as allografts for up to 4 weeks without immune suppression. The feasibility of deriving and transplanting feline neural precursor cells, combined with the availability of the dystrophic Abyssinian cat, provide a new feline model system for the study of retinal repair.

The autism spectrum disorders (ASDs) comprise a set of neurodevelopmental disorders that are, at best, poorly understood but are the fastest growing developmental disorders in the United States. Because animal models of polygenic disorders such as the ASDs are difficult to validate, the derivation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming offers an alternative strategy for identifying the cellular mechanisms contributing to ASDs and the development of new treatment options. Access to statistically relevant numbers of ASD patient cell lines, however, is still a limiting factor for the field. We describe a new resource with more than 200 cell lines (fibroblasts, iPSC clones, neural stem cells, glia) from unaffected volunteers and patients with a wide range of clinical ASD diagnoses, including fragile X syndrome. We have shown that both normal and ASD-specific iPSCs can be differentiated toward a neural stem cell phenotype and terminally differentiated into action-potential firing neurons and glia. The ability to evaluate and compare data from a number of different cell lines will facilitate greater insight into the cause or causes and biology of the ASDs and will be extremely useful for uncovering new therapeutic and diagnostic targets. Some drug treatments have already shown promise in reversing the neurobiological abnormalities in iPSC-based models of ASD-associated diseases. The ASD Stem Cell Resource at the Children's Hospital of Orange County will continue expanding its collection and make all lines available on request with the goal of advancing the use of ASD patient cells as disease models by the scientific community.

Culturing human embryonic stem cells (hESCs) requires a significant commitment of time and resources. It takes weeks to establish a culture, and the cultures require daily attention. Once hESC cultures are established, they can, with skill and the methods described, be kept in continuous culture for many years. hESC lines were originally derived using very similar culture medium and conditions as those developed for the derivation and culture of mouse ESC lines. However, these methods were suboptimal for hESCs and have evolved considerably in the years since the first hESC lines were derived. Compared with mouse ESCs, hESCs are very difficult to culture – they grow slowly, and most importantly, since we have no equivalent assays for germline competence, we cannot assume that the cells that we have in our culture dishes are either stable or pluripotent. This makes it far more critical to assay the cells frequently using the characterization methods, such as karyotyping, immunocytochemistry, gene expression analysis, and flow cytometry, provided in this manual.

It has been demonstrated that the presence of cancer results in detectable changes to uninvolved tissues, collectively termed cancer field effects (CFE). In this study, we directly assessed the ability of Raman microspectroscopy to detect CFE via in-vitro study of organotypic tissue rafts approximating human skin. Raman spectra were measured from both epidermis and dermis after transfer of the rafts to dishes containing adherent cultures of either normal human fibroblasts or fibrosarcoma (HT1080) cells. Principal components analyses allowed discrimination between the groups with 86% classification accuracy in the epidermis and 94% in the dermis. These results encourage further study to evaluate the Raman capacity for detecting CFE as a possible tool for noninvasive screening for tumor presence.

This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodeficiency virus (HIV) and provide an effective means for the delivery, integration, and expression of exogenous genes in mammalian cells. Lentiviruses are attractive gene delivery vehicles as they are able to infect both proliferating and nonproliferating cells. Lentiviruses stably integrate into the genome without incurring cellular toxicity and can maintain sustained transgene expression during prolonged host cell proliferation and differentiation. In this protocol, we describe how to prepare lentiviruses, stably transduce human fibroblasts, and identify bona fide iPSC colonies based on morphological similarity to human embryonic stem cell (ESC) colonies and live-cell immunological staining using cell-surface markers of human PSCs such as Tra-1-60 and Tra-1-81.