Optimizing the Cell Culture Microenvironment.

The survival, proliferation, and differentiation of cells in culture are determined not only by their intrinsic potential but also by cues provided by the permissive or restrictive microenvironment in which they reside. The robustness and reproducibility of cell culture assays and endpoints relies on the stability of that microenvironment and vigilant attention to the control of variables that affect cell behavior during culture. These often underappreciated variables include, but are not limited to, medium pH and buffering, osmolarity, composition of the gas phase, the timing and periodicity of refeeding and subculture, and the impact of fluctuations in temperature and gas phase composition on frequent opening and closing of incubator doors.

Detection, Labeling, and Culture of Lung Stem and Progenitor Cells.

Identification, isolation, and clonal culture of stem cells is essential for understanding their proliferative and differentiation potential, and the cellular and molecular mechanisms that regulate their fate. Akin to development in vivo, the in vitro growth of adult lung epithelial stem cells requires support of mesenchymal-derived growth factors. In the adult mouse lung, epithelial stem/progenitor cells are defined by the phenotype CD45neg CD31neg EpCAMpos CD104pos CD24low, and mesenchymal cells are defined by the phenotype CD45neg CD31neg EpCAMneg Sca-1hi.

Epithelial disruption: a new paradigm enabling human airway stem cell transplantation.

Background: Airway disease is a primary cause of morbidity and early mortality for patients with cystic fibrosis (CF). Cell transplantation therapy has proven successful for treating immune disorders and may have the potential to correct the airway disease phenotype associated with CF. Since in vivo cell delivery into unconditioned mouse airways leads to inefficient engraftment, we hypothesised that disrupting the epithelial cell layer using the agent polidocanol (PDOC) would facilitate effective transplantation of cultured stem cells in mouse nasal airways.

Role of Basal Cells in Producing Persistent Lentivirus-Mediated Airway Gene Expression.

Cystic fibrosis (CF) lung disease is an ideal candidate for a genetic therapy. It has been shown previously that preconditioning with lysophosphatidylcholine (LPC) prior to lentiviral (LV) vector delivery results in long-term in vivo gene expression in the airway epithelium of CF mice. It was hypothesized that this outcome is largely due to transduction of airway basal cells that in turn pass the transgene onto their progeny.

Properties of Adult Lung Stem and Progenitor Cells.

The last decade has seen significant progress in understanding the organisation of regenerative cells in the adult lung. Cell-lineage tracing and in vitro clonogenic assays have enabled the identification and characterisation of endogenous lung epithelial stem and progenitor cells. Selective lung injury models, and genetically engineered mice have revealed highly conserved gene networks, factors, signalling pathways, and cellular interactions important in maintaining lung homeostasis and regulating lung regeneration and repair following injury.

Clonal culture of adult mouse lung epithelial stem/progenitor cells.

Clonal culture of stem cells is crucial for their identification, and the characterization of the cellular and molecular mechanisms that regulate their proliferation and differentiation. In the adult mouse lung, epithelial stem/progenitor cells are defined by the phenotype CD45(neg) CD31(neg) EpCAM(pos) CD104(pos) CD24(low). Here we describe a tissue dissociation and flow cytometry strategy for the detection and isolation of adult mouse lung epithelial stem/progenitor cells, and a three-dimensional colony-forming assay for their clonal culture in vitro.

Are clinical trials with mesenchymal stem/progenitor cells too far ahead of the science? Lessons from experimental hematology.

The cells referred to as mesenchymal stem/progenitor cells (MSCs) are currently being used to treat thousands of patients with diseases of essentially all the organs and tissues of the body. Strikingly positive results have been reported in some patients, but there have been few prospective controlled studies. Also, the reasons for the beneficial effects are frequently unclear.

LysoTracker is a marker of differentiated alveolar type II cells.

Background: LysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.

Methods: Mouse lung cells were sorted on the basis of CD45(neg)CD31(neg)EpCAM(pos)LysoTracker(pos) expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay.

TGF-β signaling in stromal cells acts upstream of FGF-10 to regulate epithelial stem cell growth in the adult lung.

Tissue resident mesenchymal stromal cells (MSCs) contribute to tissue regeneration through various mechanisms, including the secretion of trophic factors that act directly on epithelial stem cells to promote epithelialization. However, MSCs in tissues constitute a heterogeneous population of stromal cells and different subtypes may have different functions. In this study we show that CD166(neg) and CD166(pos) lung stromal cells have different proliferative and differentiative potential.

Mesenchymal stromal cell turnover in the normal adult lung revisited.

We have employed a simple and robust noninvasive method of continuous in vivo long-term bromodeoxyuridine (BrdU) labeling to analyze lung mesenchymal stromal cell turnover in adult mice in the steady state. Mathematical modeling of BrdU uptake in flow cytometrically sorted CD45(neg)CD31(neg)Sca-1(pos) lung cells following long-term feeding of BrdU to mice in their drinking water reveals that lung mesenchymal stromal cells cycle continuously throughout life. Analysis of BrdU incorporation during long-term feeding and during chasing (delabeling) following replacement of BrdU-water with normal water shows that the CD45(neg)CD31(neg)Sca-1(pos) lung mesenchymal stromal cell compartment turns over at a rate of ∼2.

Identification of unsafe human induced pluripotent stem cell lines using a robust surrogate assay for pluripotency.

Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation.

CSF-1 receptor-dependent colon development, homeostasis and inflammatory stress response.

The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species.

Lung stem cells: do they exist?

Recognition of the potential of stem cell-based therapies for alleviating intractable lung diseases has provided the impetus for research aimed at identifying regenerative cells in the adult lung, understanding how they are organized and regulated, and how they could be harnessed in lung regenerative medicine. In this review, we describe the attributes of adult stem and progenitor cells in adult organs and how they are regulated by the permissive or restrictive microenvironment in which they reside. We describe the power and limitations of experimental models, cell separative strategies and functional assays used to model the organization and regulation of adult airway and alveolar stem cells in the adult lung.

Concise review: Deconstructing the lung to reveal its regenerative potential.

Despite burgeoning interest in the potential of cellular therapies in lung regenerative medicine, progress in delivering these therapies has been confounded by a lack of knowledge about the identity of appropriate targets which can be harnessed to repair the lung, and the cellular and molecular factors which regulate their regenerative potential. While systematic analysis of lung development and cell lineage tracing studies in normal and perturbed animal models provides a framework for understanding the complex interplay of the multiple cell types, biomatrix elements and soluble and insoluble cytokines and factors that regulate lung structure and function, a reductionist approach is also required to analyze the organization of regenerative cells in the adult lung and identify the factors and molecular pathways which regulate their capacity to generate descendent lineages. In this review we describe recent progress in identifying and characterizing endogenous epithelial, mesenchymal and endothelial stem/progenitor cells in the adult lung using multiparameter cell separative strategies and functional in vitro clonogenic assays.

Cell-based therapies for lung disease.

Introduction Or Background: The adult lung is a complex organ whose large surface area interfaces extensively with both the environment and circulatory system. Yet, in spite of the high potential for exposure to environmental or systemic harm, epithelial cell turnover in adult lung is comparatively slow. Moreover, loss of lung function with advancing age is becoming an increasingly costly healthcare problem.

The relationship between bone, hemopoietic stem cells, and vasculature.

A large body of evidence suggests hemopoietic stem cells (HSCs) exist in an endosteal niche close to bone, whereas others suggest that the HSC niche is intimately associated with vasculature. In this study, we show that transplanted hemopoietic stem and progenitor cells (HSPCs) home preferentially to the trabecular-rich metaphysis of the femurs in nonablated mice at all time points from 15 minutes to 15 hours after transplantation. Within this region, they exist in an endosteal niche in close association with blood vessels.

Stem cells and cell therapies in lung biology and lung diseases.

The University of Vermont College of Medicine and the Vermont Lung Center, with support of the National Heart, Lung, and Blood Institute (NHLBI), the Alpha-1 Foundation, the American Thoracic Society, the Emory Center for Respiratory Health,the Lymphangioleiomyomatosis (LAM) Treatment Alliance,and the Pulmonary Fibrosis Foundation, convened a workshop,‘‘Stem Cells and Cell Therapies in Lung Biology and Lung Diseases,’’ held July 26-29, 2009 at the University of Vermont,to review the current understanding of the role of stem and progenitor cells in lung repair after injury and to review the current status of cell therapy approaches for lung diseases. These are rapidly expanding areas of study that provide further insight into and challenge traditional views of the mechanisms of lung repair after injury and pathogenesis of several lung diseases. The goals of the conference were to summarize the current state of the field, discuss and debate current controversies, and identify future research directions and opportunities for both basic and translational research in cell-based therapies for lung diseases.

Isolation and clonal assay of adult lung epithelial stem/progenitor cells.

Adult mouse lung epithelial stem/progenitor cells (EpiSPC) can be defined in vitro as epithelial colony-forming units that are capable of self-renewal, and which when co-cultured with lung mesenchymal stromal cells (MSC) are able to give rise to differentiated progeny comprising mature lung epithelial cells. This unit describes a protocol for the prospective isolation and in vitro propagation and differentiation of adult mouse lung EpiSPC. The strategy used for selection of EpiSPC and MSC from adult mouse lung by enzymatic digestion and flow cytometry is based on the differential expression of CD45, CD31, Sca-1, EpCAM, and CD24.

Rad21-cohesin haploinsufficiency impedes DNA repair and enhances gastrointestinal radiosensitivity in mice.

Approximately half of cancer-affected patients receive radiotherapy (RT). The doses delivered have been determined upon empirical experience based upon average radiation responses. Ideally higher curative radiation doses might be employed in patients with genuinely normal radiation responses and importantly radiation hypersensitive patients would be spared the consequences of excessive tissue damage if they were identified before treatment.

Functional analysis of two distinct bronchiolar progenitors during lung injury and repair.

Air spaces of the mammalian lung are lined by a specialized epithelium that is maintained by endogenous progenitor cells. Within bronchioles, the abundance and distribution of progenitor cells that contribute to epithelial homeostasis change as a function of maintenance versus repair. It is unclear whether functionally distinct progenitor pools or a single progenitor cell type maintain the epithelium and how the behavior is regulated in normal or disease states.

Endogenous lung stem cells: what is their potential for use in regenerative medicine?

Advances in stem cell technologies in recent years have generated considerable interest in harnessing the potential of adult and embryonic stem cells in regenerative medicine. Stem cell-based therapies are a particularly attractive option for the treatment of intractable lung diseases for which current therapies are essentially palliative. Proof-of-principle experiments in animal models demonstrate the efficacy of exogenous stem cells in mediating lung repair by attenuating fibrotic responses to injury, but also suggest that their ability to contribute to lung epithelial regeneration and repair is limited.

Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung.

The role of lung epithelial stem cells in maintenance and repair of the adult lung is ill-defined, and their identity remains contentious because of the lack of definitive markers for their prospective isolation and the absence of clonogenic assays able to measure their stem/progenitor cell potential. In this study, we show that replication of epithelial-mesenchymal interactions in a previously undescribed matrigel-based clonogenic assay enables the identification of lung epithelial stem/progenitor cells by their colony-forming potential in vitro. We describe a population of EpCAM(hi) CD49f(pos) CD104(pos) CD24(low) epithelial cfus that generate colonies comprising airway, alveolar, or mixed lung epithelial cell lineages when cocultured with EpCAM(neg) Sca-1(pos) lung mesenchymal cells.

An innovative triple immunogold labeling method to investigate the hemopoietic stem cell niche in situ.

The ultrastructural study of rare cells within their niche in situ is very difficult. We have developed a method for locating individual transplanted cells and simultaneously identifying and analyzing the molecules and cellular phenotypes surrounding them in situ using transmission electron microscopy. This innovative method involves triple immunogold labeling combined with serial ultrathin sectioning.

Identification of human embryonic stem cell surface markers by combined membrane-polysome translation state array analysis and immunotranscriptional profiling.

Surface marker expression forms the basis for characterization and isolation of human embryonic stem cells (hESCs). Currently, there are few well-defined protein epitopes that definitively mark hESCs. Here we combine immunotranscriptional profiling of hESC lines with membrane-polysome translation state array analysis (TSAA) to determine the full set of genes encoding potential hESC surface marker proteins.

Colony stimulating factor-1 dependence of paneth cell development in the mouse small intestine.

Background & Aims: Paneth cells (PCs) secrete defensins and antimicrobial enzymes that contribute to innate immunity against pathogen infections within the mucosa of the small intestine. We examined the role of colony stimulating factor-1 (CSF-1) in PC development.

Methods: CSF-1-deficient and CSF-1 receptor (CSF-1R)-deficient mice and administration of neutralizing anti-CSF-1R antibody were used to study the requirement of CSF-1 for the development of epithelial cells of the small intestine.