Cytometry Part A

Cover image for Vol. 87 Issue 9

Edited By: Attila Tárnok

Impact Factor: 2.928

ISI Journal Citation Reports © Ranking: 2014: 28/79 (BIOCHEMICAL RESEARCH METHODS); 109/184 (Cell Biology)

Online ISSN: 1552-4930

Associated Title(s): Cytometry Part B: Clinical Cytometry

Special Issue on Cytometry in Stem Cell Research - Highlights

Guest editors Vera S. Donnenberg and Henning Ulrich have worked closely with Cytometry Part A’s Editor-in-Chief Attila Tárnok to create this compelling collection of reviews and original research articles that describe the current state of flow cytometry applications in stem cell research.  As they state in their Editorial “The major strength of flow cytometry is its ability to rapidly perform highly multiplexed quantitative measurements on single cells within a heterogeneous cell population.” Below find several highlights from the issue or enjoy the entire Special Issue freely available online.

Regeneration therapy with mesenchymal stem cells

Regenerative medicine may provide cures for millions of patients worldwide. Mesenchymal stem cells (MSCs), isolated from adult tissues, have been extensively used for phenotypic characterization by flow cytometry to define common surface marker expression. Nery and coworkers discuss overlapping and divergent antigen expression patterns of human MSCs from different sources and provide an overview about the use of these cells in ongoing clinical trials for regeneration of bone and cartilage tissue as well as for transplantation therapy in cardiovascular diseases and brain injury.

Nery A A, Nascimento I C, Glaser T, Bassaneze V, Krieger J E. Ulrich H. Human mesenchymal stem cells: From immunophenotyping by flow cytometry to clinical applications. Cytometry A 2013;  83A: 48–61. DOI: 10.1002/cyto.a.22205

Promoting neurogenesis and brain repair

Neural stem cells (NSCs) give rise to neurons and glial cells along brain development. However, stem cells also remain in defined areas of the adult brain and originate new neurons upon appropriate stimulation. In this review, Oliveira and coworkers provide detailed information on marker proteins used in cytometry for the identification of NSCs and their differentiation fate. Furthermore, actions of the principal growth and neurotrophic factors and their intracellular signal transduction on neurogenesis, fate determination and cell survival of NSCs are discussed. More knowledge on mechanisms of neural differentiation is essential for regeneration therapy of neurodegenerative diseases and brain injury by NSC transplantation and recruitment of endogenous repair mechanisms.

Oliveira S . B, Pillat M M, Cheffer A, Lameu C, Schwindt T T, Ulrich H. Functions of neurotrophins and growth factors in neurogenesis and brain repair. Cytometry A 2013; 83A: 76–89. DOI: 10.1002/cyto.a.22161

Do we need liver stem cells?

The answer is clearly: Yes! Each developmental or regenerative process initiated in the liver is followed by the emergence of progeny of putative liver stem cells. The only question is: Where is the stem cell and what is its nature? Christ and Pelz discuss the obvious dilemma with “the” liver stem cell. It must exist but has not been identified unequivocally so far. Novel flow cytometry approaches have been used to isolate subpopulations of liver cells tentatively representing the stem cell fraction. Yet, it remains unclear whether this is really an individual stem cell fraction or is representing a specific developmental stage of immature or mature hepatocytes activating expression of stem cell markers upon a specific challenge. It is also discussed in the article whether the chance to use non-hepatocyte stem cells for cell therapy of liver diseases is realistic. This is a highly interesting issue since the increasing shortage of donor organs for liver transplantation in end-stage liver diseases urgently demands alternatives.

Christ B and Pelz S. Implication of hepatic stem cells in functional liver repopulation. Cytometry A 2013; 83A: 90–102. DOI: 10.1002/cyto.a.22232

Hemo-Vascular Progeny of Human ESC and iPSC

Human induced pluripotent stem cells (hiPSC) represent an unlimited resource for cellular therapies. For example, generation of engraftable vascular and hematopoietic progenitors from patient-specific hiPSC may have great clinical utility for the effective, long-term treatment of hemato-vascular disorders. However, most hiPSC do not produce hemato-endothelial progeny with efficiencies comparable to bona fide human embryonic stem cells. In these studies, the authors developed an optimized hiPSC differentiation system that simultaneously generated multipotent hematopoietic CD34+CD45+ progenitors and vascular precursors from adherent embryoid body-derived cells with characteristics of hemogenic endothelium. Using intracellular flow cytometry analysis they demonstrated embryonic, fetal and adult hemoglobin expressions in hiPSC-derived erythroid cells generated with this system. Furthermore, FACS-purified CD31+CD146+ vascular progenitor cells were demonstrated to generate expandable populations possessing potent endothelial functionality. This two-dimensional differentiation system can be employed for direct time-lapse single cell videography, time-course studies of hematopoietic genesis events, or kinetic flow cytometry analyses of newly emerging hematopoietic and vascular progenitors in normal and diseased hiPSC model systems.

Park TS, Zimmerlin L,  Zambidi E T. Efficient and simultaneous generation of hematopoietic and vascular progenitors from human induced pluripotent stem cells. Cytometry A 2013; 83A: 114–126. DOI: 10.1002/cyto.a.22090

Flow cytometric determination of stem/progenitor content in epithelial tissues

Flow cytometry can be applied to solid tissues, with the advantages of multidimensional analysis and the ability to detect and sort rare populations.  In this issue, two companion papers illustrate multidimensional analysis of stem/progenitor and epithelial differentiation markers to compare the stem-cell content of non-small cell lung cancer and normal lung.  The first paper, by Donnenberg and coworkers, concentrates on technical issues of tissue disaggregation and staining, providing a working standard operating procedure and MIFlowCyt specifics as online supporting information. This article also examines the types of sample preparation and staining artifacts encountered with solid tissues, and illustrates ways to recognize and eliminate them during data analysis.  The second paper, by Normolle and coworkers takes up where the first manuscript left off, examining the use of multivariate classification techniques to discriminate between lung cancer and normal lung.  Starting with conventional region and gate type analysis performed on each data file, the authors illustrate how to make sense of the resulting 86 variables, tackling the p > n (variables greater than number of specimens) problem encountered in other high dimensional data sets, such as transcriptomics.  The paper compares several different statistical techniques including Elasticnet and Random Forests, and shows the importance of bootstrapping to determine the most informative variables and estimate the accuracy, sensitivity and specificity of the final classification scheme.  In online supporting information, Normolle explains the assumptions of alternative classification methods and supplies a tutorial on zero-filling, transformation and standardization, and implementation of the methods, including working code written for the robust statistical shareware R.  In addition to providing new information about the stem-cell content of lung cancers and normal lung, these papers provide a how-to guide that can be generalized to multidimensional flow cytometry on solid tissues.

Donnenberg V S, Landreneau R J, Pfeifer M E, Donnenberg A D. Flow cytometric determination of stem/progenitor content in epithelial tissues: An example from nonsmall lung cancer and normal lung. Cytometry A 2013; 83A: 141–149. DOI: 10.1002/cyto.a.22156

Normolle D P, Donnenberg V S, Donnenberg A D. Statistical classification of multivariate flow cytometry data analyzed by manual gating: Stem, progenitor, and epithelial marker expression in nonsmall cell lung cancer and normal lung. Cytometry A 2013; 83A: 150–160. DOI: 10.1002/cyto.a.22240

Mesenchymal markers on human adipose stem/progenitor cells

The discovery that mesenchymal stromal cells (MSC)—fibroblast-like cells expanded in culture from plastic-adherent bone marrow cells—are capable of multilineage differentiation has created much excitement, both for what they reveal about the biology of adult tissue stem cells, and for their potential as cellular agents of regenerative and anti-inflammatory therapy.  The fact that the initial characterization was performed on tissue-expanded cells has led to some ambiguity concerning the nature of their rare in vivo precursors.   In this issue, Zimmerlin and coworkers examine the properties of several distinct populations of cells derived from the stromal-vascular fraction of human fat, which are also capable of MSC-like multilineage differentiation.  The most prevalent of these populations, sometimes referred to as preadipocytes, was originally recognized by expression of the heme/endothelial marker CD34, which is absent in the classical definition of mesenchymal stem cells.  Here Zimmerlin and coworkers examine the coexpression of mesenchymal markers on four phenotypically distinct stem/progenitor populations present in human adipose tissue.  They conclude that unlike culture-expanded bone marrow derived MSC, MSC-like cells from adipose tissue gain expression of CD34 in addition to CD73, CD105 and CD90 as they differentiate in vivo from CD34 negative pericytes.

Zimmerlin L, Donnenberg V S, Rubin J P, Donnenberg A D. Mesenchymal markers on human adipose stem/progenitor cells. Cytometry A 2013; 83A: 134–140. DOI: 10.1002/cyto.a.22227