Cytometry in stem cell research and therapy
The recent Nobel Prize in medicine was awarded to two stem cell researchers, John Gurdon and Shinya Yamanaka, for their achievements in stem cell research and reprogramming of somatic cells. Flow cytometry is by nature the ideal tool to identify, characterize, and isolate stem and progenitor cells for research and potential clinical use (1). The major strength of flow cytometry is its ability to rapidly perform highly multiplexed quantitative measurements on single cells within a heterogeneous cell population. However, when the cell type of interest is extremely rare, as most stem and progenitor cells are, several sources of artifact must be addressed. The importance of flow cytometry as a driving force for stem cell research was demonstrated in a focused issue of the journal exactly three years ago (1). This current focus issue of Cytometry A is devoted to the topic of stem cells due to numerous current innovations and discoveries.
Applications of stem cells include several disciplines, from embryogenesis, adult tissue maintenance, and repair, and more recently, cancer as well as for toxicity screening and disease modeling. All of these topics are represented in this issue, with special emphasis on the role of analytic and preparative flow cytometry in the elucidation of stem cell phenotype and function, and best laboratory practices as they apply to flow cytometry. Image and flow cytometry together with cell sorting have revolutionized the study of stem cell biology and the implications of these cells and their progeny in developmental biology, tissue engineering, and cellular therapy. The number of parameters and the speed of their simultaneous measurements in single cells has continued to increase with advances in hardware, reagents, and analytical software (2).
Generating Stem Cells: The iPS
Multilineage-Differentiating Stress Enduring Cells and the Elite Model of iPS Cells
Induced pluripotent stem cells have attracted a great deal of attention, although the mechanism by which they are generated is still not fully understood. Quantitative analysis of the reprogramming factors helps to improve iPS yield but also demonstrates that stoichiometry of these factors is relevant for the fate of the iPS (3). In their review, Wakao and coworkers (this issue, page 18) summarize the current and opposing concepts of iPS generation: the elite versus the stochastic model for iPS generation. The authors present compelling evidence that naïve sort-purified multilineage-differentiating stress enduring (MUSE) cells are a unique candidate sub-population of mesenchymal stem cells (MSC) which are more prone to iPS induction.
Efficient and Simultaneous Generation of Hematopoietic and Vascular Progenitors from Human Induced Pluripotent Stem Cells
Park and coworkers (this issue, page 114) provide feeder-free methods for the in vitro generation and differentiation of human pluripotent stem cells as well as human pluripotent stem cells induced from fibroblasts and differentiated along the hematopoietic and vascular lineages. This methodology recapitulates an orderly hematopoiesis similar to human yolk sac development via the generation of mesodermal-hematoendothelial progenitor cells that give rise to endothelium followed by primitive and definitive hematopoietic cells. The authors clarify the relationship between endothelial and hematopoietic lineages and outline opportunities for elucidating the mechanisms of hematopoietic development.
Disease in a Dish: Induced Pluripotent Stem Cells as a Novel Model for Developmental Disorders
Reprogramming of adult specialized cells back into embryonic stem cell-like cells (named induced stem cells), has been a contribution of enormous importance for basic stem cell research and possible applications in regenerative. Beltrão-Braga and coworkers (this issue, page 11) review the reprogramming of patients' cells suffering from different diseases with special focus on the neurodevelopmental disorder autism. The authors argue that the obtained results in addition to revealing mechanisms of disease development may offer clues for personal medicine strategies during the patient's life.
Hematopoietic Stem Cells
Mouse Hematopoietic Stem Cell Identification and Analysis
Hematopoietic stem cells remain one of the best-characterizedadult stem cell populations in terms of phenotypic markers and assays to assess their functional potential. A useful polychromatic immunofluorescence panel was recently used to identify and characterize human cord blood hematopoietic stem cells (4). For the mouse model, Mayle and coworkers (this issue, page 27) provide a thorough overview of recent experimental and technical advances in the identification and isolation techniques for hematopoietic progenitor populations. The authors describe preparative methods and compare current analytical strategies for rapid flow cytometric analysis of rare peripheral blood cell subsets including the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining.
The Functional Duality of HoxB4 in Hematopoietic Reconstituting Cells
Kaplan and coworkers (this issue, page 127) have performed high-resolution immunophenotyping of several marker proteins on a flow cytometric platform following transplantation of CD34+ hematopoietic reconstituting cells (HRC). The transplanted cells need to be able to replicate for long periods and to readily differentiate as assayed by their capability. The authors conclude that among the studied proteins, increased HoxB4 expression enhanced proliferation, but reduced the capacity of the stem cell for short-term differentiation. Moreover, the authors conclude that HoxB4 provides a molecular marker for the appropriateness of these cells for transplantation following myeloablation.
Mesenchymal Stem Cells
Human Mesenchymal Stem Cells: From Immunophenotyping by Flow Cytometry Toward Clinical Applications
Regenerative medicine is one of the fastest growing specializations of modern medicine. Nery and coworkers (this issue, page 48) summarize how flow cytometry and imaging have been used in clinical cell isolation, immunophenotyping, and studies of differentiation of mesenchymal stem cells of diverse origins. The authors argue the critical role of multi-dimensional cytometry in the characterization of cellular products tested in almost two hundred clinical trials treating a very broad range of conditions.
Mesenchymal Markers on Human Adipose Stem/Progenitor Cells
The stromal-vascular fraction of adipose tissue contains multiple cell types with a multipotent differentiation potential. Zimmerlin and coworkers (this issue, page 134) have described the phenotypes of these populations and their resemblance to CD45−/CD34−/CD73+/CD105+/CD90+ bone marrow-derived mesenchymal stem cells. Based on gain or loss of markers, and the fraction of proliferating cells, the authors hypothesize a progenitor/progeny relationship between CD34−/CD146+ pericytes, a transitional CD34+/CD146+ population, and the mesenchymal-like supra-adventitial adipose stromal cells.
Neuronal Stem Cells and Regeneration
Isolation and Differentiation Properties of Neural Crest Stem Cells
Several stem cell types including aortic pericytes can be induced to differentiate neuronal lineages (5). In their review, Dupin and Coelho-Aguiar (this issue, page 38) provide a detailed and timely compendium of markers used for the identification and isolation of neural crest stem cells. The authors summarize the current neural crest stem cell markers in human, chick, and murine tissues, and discuss the procedures for isolating these cells.
Functions of Neurotrophins and Growth Factors in Neurogenesis and Brain Repair
Multipotent neural stem and progenitor cells in the brain give rise to neurons, astrocytes, and oligodendrocytes. Their discovery permitted studies on the basic mechanisms of endogenous neurogenesis and repair mechanisms of the nervous system, and has facilitated novel therapeutic strategies for cellular regeneration therapies in brain disease. Oliveira and coworkers (this issue, page 76) discuss the effects of neurotrophic factors, including their intracellular signal transduction, on fate determination and neuroprotective mechanisms. The understanding of neural stem cell proliferation and differentiation will provide critical insights into the use of neural stem cell transplantation and neural tissue repair.
Cancer Stem Cells
The Hitchhikers Guide to Cancer Stem Cell Theory: Markers, Pathways, and Therapy
Cancer stem cell (CSC) biology has become an integral part of cancer research and their analysis and purification has been reported by various authors (6). CSCs are postulated to be a unique cell population exclusively capable of self-renewal, multilineage differentiation, and having the ability to evade conventional cytotoxic cancer therapy. Fábián and coworkers (this issue, page 62) review the current methodologies for the identification and isolation of cancer stem-like cells and argue how the understanding of intracellular signaling pathways in CSCs and their interaction with the surrounding niche affects their phenotype and function. The authors also discuss potential CSC-targetedtreatment strategies and their future role in cancer therapy.
The Cancer Stem Cell: Cell Type or Cell State?
Cancer is often viewed as a caricature of normal development, but the degree to which tumors recapitulate cellular multilineage differentiation is unknown. Donnenberg and coworkers (this issue, page 5) argue that despite the well-established unidirectional differentiation scheme of the adult hematopoietic lineages, not all tissues follow this differentiation paradigm. The authors draw on the de-differentiation potential of induced pluripotent stem cells and propose that “stemness” may be an inducible cell state rather than a cell type. They extrapolate that CSCs may not be a unique cell type, but rather an interchangeable cell state that can be conditionally re-expressed in response to environmental cues. They also propose that rapidly dividing, self-renewing, treatment-resistant, undifferentiated cancer cells may attain their stem-like phenotype by loss of functional changes in potential differentiation pathways. The authors underscore the importance of multi-dimensional cytometry and single cell sorting as an integral part of molecular and functional analysis of cancer cell states.
Special Types and Sources of Stem Cells
Flow Cytometric Determination of Stem/Progenitor Content in Epithelial Tissues: An Example From Non-Small Lung Cancer and Normal Lung
Donnenberg and coworkers (this issue, page 141) discuss concerns unique to performing flow cytometry on disaggregated solid tissues, using lung tumors and normal lung as examples. The manuscript has several unique features including a supplement with a detailed SOP (Standard Operating Procedure) for preparation of single cell suspension from solid tissue and a commentary regarding the methods specific to that example. This manuscript reflects the evolving understanding of best practices developed over more than a decade of working with solid tissues.
Implication of Hepatic Stem Cells in Functional Liver Repopulation
The liver is known for its enormous regeneration capacity; however, there is yet a controversial debate on the existence of hepatic stem cells. Christ and Pelz (this issue, page 90) emphasize that such liver stem cells exist as a heterogeneous population in the fetal and adult liver in normal and injury conditions in addition to adult stem cells from non-hepatic sources which are also able to give rise to cells with characteristics of functional hepatocytes. Phenotypic characteristics of such cells are provided. Furthermore, endogenous regeneration mechanisms following partial hepatectomy and massive liver injury are discussed. Functional repopulation of the host liver by hepatic stem cell and hepatocyte transplants provide a clinical perspective for cell therapy for the treatment of human liver diseases, where endogenous repair mechanisms are not sufficient.
Equine Cellular Therapy—From Bench to Stall to Bedside?
Ground-breaking clinical stem cell research and cellular therapy is being performed in the horse, a recipient of cutting edge veterinary medicine as well as a unique animal model, paving the way for human medical applications. Immunophenotyping of equine leukocytes became easier because several anti-human CD-markers cross-react with them (7). Burk and coworkers (this issue, page 103) summarize the progress that has been made on the veterinary front and in the characterization of equine multipotent mesenchymal stromal cells and equine embryonic stem cells.
Murine and Human VSEL Cells: Comparing Apples and Oranges
In 2006, very small embryonic-like (VSEL) stem cells were described as a pluripotent population isolated from adult murine bone marrow and human umbilical cord blood (8). Several years later a rigorous proof of pluripotency is still lacking. In their review, Heider and coworkers (this issue, page 72) argue on the basis of rigorous experimental evidence that there is very little similarity between murine VSEL cells, their reportedly equivalent population in human umbilical cord blood, and pluripotent embryonic stem cells. On the contrary, their data strongly suggest that human VSEL cells are an aberrant, inactive population, incapable of in vitro expansion, and exhibiting neither embryonic nor adult stem cell-like properties.
Technical Aspects of Stem Cell Analysis
Classification of Multivariate Cytometry Data
Normolle and coworkers explore the use of multivariate classification strategies to explore the multidimensional dataset from lung tumor and adjacent lung (this issue, page 150). Unlike previous efforts which use classification methods to discern populations within individual data files, the authors begin with the results of conventional region and gate analysis, which generated 86 analytical variables, many of them highly correlated. Several different methodologies, commonly used in gene expression analysis where the number of variables often exceeds the number of samples, are compared and their assumptions and relative merits are discussed. The detailed supplement includes an in-depth discussion of the statistical methods and the data transformation code, enabling straight-forward reproducibility by investigators having multi-dimensional data.
Kinetic Analysis of Intracellular Hoechst 33342—DNA Interactions: Misinterpretation of Side Population Status?
Smith et al. (this issue, page 161) outline a simple approach including instrument set-up and calibration for the analysis of Hoechst dye 33342-loading in human cell lines for exploring heterogeneity in dye efflux efficiency and the status of the side population phenotype. They demonstrate how a kinetic gating strategy in fixed permeabilized reference samples permits reproducible instrument set-up, guides gate boundaries, and offers a simple side population comparison across laboratory sites and instrument platforms. The authors clearly show how live cell kinetic sorting of cells with the same dye to DNA loading ratio, but with differences in efflux capacity, can be used to explore drug resistance while eliminating artifactual toxicity due to dye intercalation in efflux negative cells.
In summary, this focus issue presents a state of the art upgrade of the whole plethora and richness of stem cell research. Demonstrating how cytometry, may it be flow or image, pushes the boundaries to yet undiscovered fields and warrants highest quality in quantitative cell science.