Stromal cells in health and disease (2018)

Stromal cells are a heterogeneous class of cells and fulfill a variety of highly important roles in health and disease. They are important during development, tissue injury, regeneration, immune responses, cancer, and other pathologies by contributing to several biological processes in different tissues. Their phenotype and function are dependent on the specific tissue microenvironment, but stromal cells themselves also shape the organization, integrity, and the dynamic of their own microenvironment. The multidirectional role of stromal cells in tissues and organs is receiving much needed attention and due to the cutting-edge methods and elegant studies (1-12; and articles in this issue), we are discovering their key role in fundamental biology and their clinical importance. One of the most studied types of stromal cells is mesenchymal stromal cells (MSC) 12, 13. The same acronym is also used to define mesenchymal stem cells; however, it is debatable whether the “stem” nomenclature is appropriate, since postnatal MSC are not necessarily biologically functional stem cells 14. Furthermore, in vitro MSC differentiation does not necessarily recapitulate in vivo differentiation. Thus, the International Society for Cellular Therapy (ISCT) supports that the abbreviation “MSC” should stand for mesenchymal stromal cells 15, 16. MSC are known to be present in bone marrow (BM) 17 and adipose tissue (AT) 18 and can also be found in dental pulp 19-21, umbilical cord blood, placenta, and other tissues 22. Recently, it was found that MSC are also present in the heart 23 and other nonmesenchymal vascularized sources 24-26. In cell culture, the cells are plastic adherent and spindle- or star-shaped morphology and can be maintained under standardized culture conditions 12, 13, 27 (Fig. 1). Moreover, they are purported to be multipotent, which can be tested by osteogenic, adipogenic, and chondrogenic differentiation in vitro (Fig. 1). According to Horwitz et al., this feature provides evidence that the investigated cells are indeed MSC 16. MSC are therefore good candidates for cell therapies, as they can easily be isolated, expanded, and re-implanted, in the autologous and allogeneic setting. Their great therapeutic potential in tissue engineering is of utmost relevance, since they have the ability to differentiate into any mesenchymal cell type, such as chondrocytes, adipocytes, and osteocytes. On a molecular level, MSC should routinely express a set of surface markers (Fig. 1), “Clusters of Differentiation” (CD) on their cell membrane composed of CD73, CD90, and CD105, which were defined by the ISCT as the standard minimal required markers for MSC 28. In contrast, MSC should not express the hematopoietic antigens CD45, CD34, CD14, and CD19, which distinguish them from hematopoietic stem cells, often found in the same region (Fig. 1). Additionally, they are negative for CD11b, CD79a, and HLA-DR 23. These criteria, however, are not unique to MSC, and continued research is needed to identify surface markers that are more specific to this kind of stromal cell 14, 24, 29. As surface markers can be lost or even gained during culturing, the results do therefore not necessarily show the situation in vivo 23. Three aspects of MSC detection by cytometry that are critical to the widespread application of multicolor flow cytometry to both basic and clinical investigations are 30: the first is instrument standardization, which includes the determination of optimal PMT settings and the use of calibration beads and target channels to ensure consistent results from day to day and laboratory to laboratory. The second is the lower limit of detection. In flow cytometry, the limiting factor can either be signal to noise (where noise is the proportion of the background or “false positive” events that appear in a negative control) or by the number of events acquired. The American College of Pathologists mandates that at least 100 events of interest be counted in an analytical gate for a result to be reportable. This is based on Poisson statistics, where 100 counted events yield a coefficient of variation of 10% 31. This is an important consideration when deciding how many events to acquire, especially for rare event problems. Finally, the data analysis strategy should be as finely crafted as the staining panel. The International Society of Hematotherapy and Graft Engineering (ISHAGE) method for the analysis of CD34+ cells provides a model for rare event detection 32. Rather than starting with the conventional forward by side light scatter gating, the ISHAGE strategy begins with CD45 by side scatter, immediately eliminating debris that is difficult or impossible to “gate out” with light scatter alone. A second check is that identified CD34+ events have appropriate CD45 expression. Finally, light scatter (and in some applications a viability dye) is used to define the final population of interest. Although far from intuitive, this methodology (combined with lyse/no wash staining and spiked counting beads) has been almost universally adopted by clinical laboratories allowing unprecedented standardization of a rare events assay that is routinely used to make lifesaving decisions concerning hematopoietic progenitor cells (HPC) dose. An alternative analytical strategy for the multicolor analysis of MSC in heterogeneous samples such as BM, digested tissues, and blood might begin with the acquisition of 1 million or more events 33. After elimination of cell clusters and coincident events using doublet discrimination 34 on forward light scatter and nonviable cells using an exclusion dye (such as Sytox red and Draq7), a negative gate (e.g., CD14−/CD45−/CD34−) could be used to define the denominator population of nonhematopoietic cells. By the time all of this “cleaning” is done, the nonhematopoietic denominator among which MSC will be detected will be a small fraction of the millions of events originally collected. MSC may then be defined on the nonhematopoietic fraction using the ISCT criteria (CD90+/CD73+/CD105+). Only then should a forward by side light scatter gate be used as a final “clean up” of events with inappropriate scatter (very low forward scatter, low forward scatter with high side scatter). It should also be pointed out that “dump parameters” (i.e., markers for which the population of interest is assumed a priori to be negative) could be combined in a single fluorescence channel, freeing up channels for the detection of other rare nonhematopoietic populations such as circulating endothelial cells or epithelial cells. Another aspect that may be exploited to quantify MSC in tissues such as BM is the ability to measure the percent of HPC according to the ISHAGE strategy 35. In heterogeneous samples, the ratio of MSC to HPC, in conjunction with an additional sample assayed for absolute CD34 count by a single platform assay, can be used to approximate an absolute MSC count 33. Very rare events such as native BM MSC cannot be assayed directly by a single platform assay because of the sample dilution (at least 1:10) required by the lyse/no wash protocol and the large number of events which must be acquired. Multicolor panels for MSC identification extend a standardized approach for MSC identification in other tissues such as adipose 36-38, perivascular tissues 25, 39, and the dermis 26. This special issue on cytometric analysis of stromal cells illustrates the challenges and opportunities for stromal cell research. Here, we selected a variety of manuscripts dealing with stromal cells in a wide variety of conditions and diseases but with a major focus resting on the diversity and complexity of stromal cells in vitro versus in vivo as well as how their fate varies in regular homeostasis, cancer, or fibrosis. Holzwarth and colleagues (this issue, page 876) combine multiplexed multi-epitope-ligand-cartography (MELC) histology in the BM with image segmentation to elucidate cell-object based heterogeneity among stromal cell subsets. The authors used a MELC system, which automatically delivers washing and staining solutions to the sample, thereby allowing the detection of up to 100 parameters on one histological section 40. The combination of image segmentation with various approaches for the quantification of preferential object contact allowed the analysis of stromal cell networks, as well as neighboring cells of interest. This study not only confirms MELC as a robust histological method but also reveals a heterogeneous expression of leptin receptor (LpR), BP-1, and VCAM-1 in the stromal network, as well as LpR enrichment in stromal somata. Additionally, the authors have shown that not only did B cell’ subsets preferentially contact stromal processes in comparison to stromal stomata, but that plasma cells were also in intimate contact with the stromal compartment. In summary, this study introduces a nice and versatile pipeline for the spatial analysis of complex tissue structures and underlines the importance of studying stromal cells in intact in vivo niches. In this issue, Consentius and colleagues (this issue, page 889) also investigated the in vivo phenotype of stromal cells. The authors addressed the general question whether MSC express the same set of surface markers in vivo that is used for their identification after in vitro expansion by using multiplex-immunohistology (Chipcytometry), a technique that allows staining of more than 100 biomarkers consecutively on the same cell 41. Chipcytometry demands that the interrogated cells be fixed on coated glass surfaces, thereby allowing a long-term stability of surface and intracellular biomarkers. This is of significant interest to biobanking of irreplaceable or rare samples. The authors first report that CD73+CD90+CD105+CD45−CD34−CD31−CD14−CD19− MSC can be found in human early and full-term placenta as well as in human BM aspirates. Future studies will show how similar or diverse in situ versus ex vivo expanded MSC are. Particularly, technologies such as MELC and Chipcytometry will significantly aid in improving our knowledge of stromal cells in vivo. The paper by Moravcikova and colleagues (this issue, page 894) provides, for the first time, a detailed characterization of native unpassaged BM MSC as compared to phenotypic changes that occur during the initial and late expansions in vitro. Using the surface proteomic platform FACSCAP Lyoplate of 242 surface antigens (BD Biosciences, La Jolla, CA), this study addresses some of the limitations with MSC identification. Although a large number of MSC can be propagated from a very small initial sample, several lines of evidence indicate that MSC lose their potency after multiple passages. Therefore, there is an unmet need to discover new biomarkers that are associated and can independently predict MSC function in vivo. Using a high-throughput cell surface proteomic approach, the authors characterized the phenotypic profile of unpassaged BM MSC and determined the modulation in cell surface protein expression of stromal cells from unpassaged BM through 10 serial culture passages. As predicted by the changes in MSC function with an increase in passage, several functional classes of proteins were found to be affected. This included those influencing cell death, immune regulation, membrane transport, cell adhesion, motility, and proliferation; all are molecular changes, which have significant consequences on MSC function. These data not only suggest potential markers of native MSC but also identify potential new biomarkers, which could serve as quality control in the manufacture of optimal MSC products. Lekishvili and colleagues (this issue, page 905) also analyzed MSC from different passages and found similar results to Moravcikova et al., by using fluorescent cell barcoding (FCB). In this study, the authors have modified an FCB assay for multiplexed analysis of human MSC to evaluate the quality of these cells during bioprocessing. An antibody panel was used to target 15 ubiquitously expressed or stage-specific markers together with a fixable viability dye acting as the cell barcoding agent. Using this system, the authors found that inter-analyst expression patterns between MSC cultures were similar at discrete passages but that diverse marker expression was evident between passages. The application of FCB in flow cytometry has been effective for the discrimination of intracellular protein targets and has yielded a number of high-content screening applications 42, 43. The assesments of surface marker expression, however, have been limited when using this methodology 44. The current study represents a proof of principle where a modified FCB method for the rapid characterization of BM-derived hMSC. The authors provide compelling evidence using side-by-side analysis of barcoded and nonbarcoded cells that this technique is suitable for the rapid phenotypic characterization of cells exposed to different bioprocessing conditions. Additionally, the method incorporates fewer subjective factors such as sample preparation and instrument day-to-day variations and is flexible across a wide range of diverse cell types. MSC also have an important role in many diseases. In their review article, Aanei and Campos Catafal (this issue, page 916) discuss the relevance of the BM microenvironment for myelodysplastic syndromes (MDS) with a particular focus on stromal cell contribution to the MDS pathology. MDS are a heterogeneous group of clonal hematopoietic disorders, and the therapies available have limited efficacy. This review analyzes the recent findings regarding the role of the BM microenvironment in MDS pathogenesis and opines how this information can improve diagnosis and therapy. Podszywalow-Bartnicka and colleagues (this issue, page 929) present an experimental setup based on the hypoxic co-culture of stromal cells with different cell lines derived from leukemia patients. This work describes several assays how to control and investigate the cross-talk between leukemic cells and stromal cells. The authors demonstrate that stromal cells protect leukemia cells from imatinib-induced cell death contributing to leukemia progression and perhaps to the development of therapy resistance. This report underscores the findings that stromal cells are not just passive supportive cell type found in the connective tissue but that MSC actively participate in shaping the tissues micro- and macroenvironment and that validated stromal cell-specific mulltiparametric assays need to be incorporated into discovery-based scientific research Despite the vast characterization efforts of MSC, the stromal cells remain a rather undefined population. While MSC seem relatively well characterized, our knowledge of fibroblasts, myofibroblasts, or pericytes is still quite limited. Even with new sequencing technologies readily available, such as single cell RNAseq, the cell phenotype and definition is extremely critical to the success of the transcriptomic data. One such cell type is cancer-associated fibroblasts (CAF), especially because the phenotypic markers used currently are not unique to this population, and the identification of fibroblast-specific surface molecules is still a very active area. In their paper, Kahounová and colleagues (this issue, page 941) aimed to define a CAF-specific marker that would help to identify fibroblasts and distinguish them from epithelial cancer cells that have undergone an epithelail-to-mesenchymal transition (EMT) 45, 46. To this end, the authors verified the specificity of several commercially available antibodies that detect surface epitopes of fibroblasts and CAF. Unfortunately, none of the tested markers was expressed exclusively by fibroblasts, suggesting that the identification of fibroblasts and CAF should not rely on the detection of a single marker, indicating that combinations of multiple markers for both the positive (fibroblast markers) and the negative (epithelial cells, endothelial cells, and mesenchymal cells) must be utilized for the identification of CAF. The final paper of this special Issue on Stromal Cells, by Reichard and colleagues (this issue, page 952), expands on a pathology driven by stromal cells that can be seen in different situations such as cancer, inflammation, and asthma: fibrosis. This manuscript focuses on airway fibrosis, which is a prominent feature of chronic asthma and is the result of collagen deposition by myofibroblasts. As established methods of collagen deposition quantification are far from reliable, especially in airway myofibroblasts, the authors present a novel flow cytometric-based method for CD45−αSMA+ myofibroblast identification 47, to analyze collagen-I biosynthesis and deposition. Because of the quantitative aspect of this technology, intracellular collagen synthesis can be determined very early on in the disease process, enabling a significantly earlier evaluation of therapeutic effects on extracellular remodelling. Taken together, stromal cells are a very heterogeneous cell population, which is still quite undefined, not only phenotypically but also functionally. While in the past, stromal cells were considered to be passive supportive cells of the connective tissue, we now understand that these cells play an active role in both health and disease. The current remaining challenges are to select cell-specific and validated markers to define various subsets of stromal cells, in vitro, in vivo, and ex vivo, especially after an expansion. Therefore, there is a need for high-throughput, sensitive yet easy to use multiplex technologies, that will drive the discovery of novel markers and ways to dissect the complex biology of stromal cells.