Drzeniek, Norman

Geissler, Sven

Kamhieh‐Milz, Julian

Moll, Guido

Reinke, Petra

English

Institutional Repository of the Freie Universität Berlin

Editorial comment: variables affecting the presence ofmesenchymal stromal cells in the peripheral blood and theirrelationship with apheresis productGuido Moll,1,2 Norman Drzeniek,1,2 Julian Kamhieh-Milz,3 Sven Geissler1,4 and Petra Reinke1,5,61BIH Center for Regenerative Therapies (BCRT), 2Berlin-Brandenburg School for Regenerative Therapies (BSRT), 3Department ofTransfusion Medicine, 4Julius Wolff Institute (JWI), 5Department of Nephrology and Internal Intensive Care Medicine and 6BerlinCenter for Advanced Therapies (BECAT), all Charite Universit€atsmedizin Berlin, corporate member of Freie Universit€at Berlin, Hum-boldt-Universit€at zu Berlin, and Berlin Institute of Health (BIH), Berlin, GermanyKeywords: haematopoietic stem cell transplantation,granulocyte colony-stimulating factor, stem cell mobi-lization, mesenchymal stromal cells, blood circulation,peripheral blood, apheresis product, flow cytometry.In this issue of the British Journal of Haematology, Jain et al.identified circulating mesenchymal stromal cells (MSCs) inperipheral blood (PB) and apheresis product (AP) collectedin the context of haematopoietic stem cell (HSC) mobiliza-tion for HSC transplantation (HSCT). As pointed out by theauthors themselves and others (Fox et al., 2007; Jones &McGonagle, 2008; Moll et al., 2019), the presence of circulat-ing MSCs in PB is debatable and their identification may behampered, among others, by: (i) their low frequency inperipheral blood, (ii) their unclear phenotypic characteriza-tion, and (iii) the large biological variations related to donorpathology, disease status, and corresponding treatment regi-mens.Within the light of these challenges, it is still not cleartoday if MSCs naturally extravasate and circulate withinblood, or if the presence of MSCs in the circulation is a mereartefact resulting from the blood sampling procedure (e.g.vessel puncture), major micro- or macrovascular injury (e.g.severe fractures or surgery), or non-physiological mobiliza-tion strategies (e.g. mobilization drug toxicity) (Marquez-Curtis et al., 2011). Either point could likely lead to anartificial presence of perivascular MSCs in the blood stream,resulting from the breakdown of vascular barrier integrity(Alm et al., 2010; Hoogduijn et al., 2014; Moll et al., 2019).Jain et al. investigated if MSCs can be mobilized into theblood stream in response to drug regimens alreadyapproved/applied for HSC collection in HSCT. Two reviewsgive an elaborate outline on the strategies used for progenitorcell mobilization (Marquez-Curtis et al., 2011; To et al.,2011). One prime example comprises the targetting of cyto-kine/chemokine and growth factor signalling with granulo-cyte colony-stimulating factor (G-CSF) and the CXCR4-antagonist plerixafor (AMD3100) for the modulation of thestromal-derived factor-1a (SDF-1a)/CXCR4-signalling axisthat controls the retention of HSCs in the bone marrow(BM) (Fig 1).Intriguingly, Larsen et al. noted in a baboon model thatMSC mobilization and colony-forming unit fibroblast (CFU-F) in PB in response to G-CSF did only occur when addingstem cell factor (To et al., 2011). Rankin et al. found in mice,that MSCs/CFU-F were not found in PB post-mobilizationwith G-CSF, but when adding vascular endothelial growthfactor and CXCR4-antagonist (Pitchford et al., 2009).Nonetheless, first reports exist that documented smallquantities of MSCs/CFU-F in PB of G-CSF-mobilizedpatients (Wexler et al., 2003; Kassis et al., 2006). This is inline with reports implicating the SDF-1a/CXCR4-axis in BMretention of various progenitor cell linages, but also inchemoattraction of CXCR4-expressing BM progenitors tosites of tissue damage and ischaemia through hypoxia-in-ducible factor-1 (HIF-1)-mediated induction of SDF-1a(Petit et al., 2002; Ceradini et al., 2004; Kucia et al., 2005;Otsuru et al., 2008; Yin et al., 2010).Jain et al. here report for the first time the detailed kineticsof: (i) circulating MSCs in PB of HSCT patients before HSCmobilization, (ii) in AP obtained at day 5 post start of mobi-lization with G-CSF (Grafeel, 10 µg/kg/day for 4 days), and(iii) in PB and AP at day 6 upon mobilization with G-CSF(10 µg/kg on day 6) and plerixafor (024 mg/kg given 12 hoursprior), obtained in a second round of apheresis for those withinadequate CD34+ counts in the first apheresis (<2 9 106/kg).To identify CD34+ HSCs and CD34dim MSCs, and to cal-culate their frequency in blood relative to white blood cells(WBCs), Jain et al. employed a dual platform strategy basedon multiparameter flow cytometry and total leukocyteCorrespondence: Guido Moll, BIH Center for Regenerative Therapies(BCRT), Charite Universit€atsmedizin Berlin, Campus VirchowKlinikum, Augustenburger Platze 1, D-13353 Berlin, Germany.E-mail: guido.moll@charite.decommentaryª 2020 The Authors. British Journal of Haematology published by British Society for Haematologyand John Wiley & Sons Ltd. British Journal of Haematology, 2020, 189, 593–596This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution andreproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.First published online 10 February 2020doi: 10.1111/bjh.16389counts. The MSCs were identified as CD34dim/CD73+/CD90+/CD105+ cells, with dual positivity for either CD73+/CD105+ or CD73+/CD90+ or CD90+/CD105+. The flowcytometry gating strategy and controls are shown in the pri-mary article and the MSC frequency was calculated as fol-lows: MSC (%) = (MSC Number 9 100)/Viable WBCs.The flow cytometry was performed according to Hasset al. (2011) in line with the current guidelines for the use offlow cytometry and cell sorting. In addition, another methodto enrich MSCs from blood is elutriation based on theirphysical properties (e.g. size/density) in a liquid centrifuge(Zvaifler et al., 2000). Furthermore, adhesion to fibrinmicro-beads may also be used for MSC enrichment beforefurther phenotyping with flow cytometry and other methods(Kassis et al., 2006).Considering both patient background and sampling timepoints, the levels of MSCs detected by Jain et al. in PB andAP relative to viable WBCs were very similar in all threegroups: healthy controls (00013–00033% MSCs in >50% ofthe n = 8 patients tested positive), multiple myeloma group(00011–00041% MSCs in >35% of the n = 20 patients),and lymphoma group (00016–00077% MSCs in >60% ofthe n = 5 patients). The typical MSC-like fibroblasticphenotype of the cells was confirmed upon plating of thesorted cells in vitro onto standard cell and tissue culture plas-tic.Although the authors found small amounts of circulatingMSCs in all three groups of donors, their relative number,expressed as percentages of MSCs relative to viable WBCs,did not appear to be increased in PB post G-CSF treatment.Here, the mathematical expression chosen by Jain et al. maycarry a risk of misinterpretation, since WBC numbers in PBhave been shown to be increased in response to G-CSF (DeFelice et al., 2016; Melve et al., 2018). This may suggest thatthe absolute numbers of circulating MSCs may have actuallyincreased in response to G-CSF.Noteworthy, HSCs, endothelial progenitor cells (EPCs)and MSCs are not the only stem cell niches mobilized uponG-CSF treatment (Melve et al., 2018). Immune memory cells,like memory B cells, T central memory and T stem cellmemory cells are also affected. Thus, a closer look at subsetsof bone marrow-resident cells may yield great potential formore targeted mobilisation strategies as a new research direc-tive.In analogy to identification of MSCs within tissues (Men-dez-Ferrer et al., 2010; Consentius et al., 2018), a crucialFig 1. Different types of progenitor/stem cells (e.g. endothelial, haematopoietic, or mesenchymal lineages, EPCs, HSCs and MSCs, respectively)are lodged in extravascular bone marrow (BM) niches, lining the stromal tissue network and the BM microvascular sinusoids. The HSCs (andEPCs/MSCs?) are kept in the BM niche due to the chemokine gradient of locally produced stromal-derived factor 1a (SDF-1a/CXCL12) detectedby their expression of the corresponding receptor CXCR4. It is already well established that the HSCs can be mobilized into the bloodstream withgranulocyte colony-stimulating factor (G-CSF) and CXCR4-antagonist plerixafor. The injection of G-CSF leads to a strong expansion of myeloidcells within the BM and concomitant release of proteases, which can disrupt the SDF-1a gradient and local integrin tethering of progenitor cellsand thus release them into the blood stream (Marquez-Curtis et al., 2011). The MSCs are typically found in a perivascular location surroundingthe microvasculature, but on occasion small amounts of circulating MSCs can also be found in blood. Jain et al. studied whether apart fromHSCs, MSCs can also be mobilized into blood and apheresis product by the typical HSC mobilization regimen. A crucial aspect is the phenotyp-ing of circulating and in vitro cultured MSCs. [Colour figure can be viewed at wileyonlinelibrary.com]Commentary594 ª 2020 The Authors. British Journal of Haematology published by British Society for Haematologyand John Wiley & Sons Ltd. British Journal of Haematology, 2020, 189, 593–596aspect for flow cytometry-based identification of circulatingendothelial, haematopoietic, or mesenchymal progenitor lin-eages is their clear phenotypic distinction with appropriatemarker panels (Pitchford et al., 2009). Of importance is theirdistinction from circulating HSCs (Mendez-Ferrer et al.,2008), circulating EPCs and also circulating mature endothelialcells (CEC) (Asahara et al., 1997; Lanuti et al., 2018; Farinacci,et al., 2019). In particular, the latter two are phenotypicallyclosely related to MSCs, also adhere to culture plastic, and maytherefore potentially easily be mistaken for MSCs.A typical marker for identification of EPCs is the endothe-lial marker CD31, commonly employed to distinguish MSCsfrom endothelial cells (ECs) (Dominici et al., 2006), whichwas not included in the current panel of Jain et al. However,a major contamination with EPCs is unlikely, due to theexclusion of CD34+ cells (Farinacci et al., 2019). In line withthe confirmation of the MSC identity as plastic-adherentfibroblastic cells, another concern is the potential phenotypictransformation of circulating progenitor cells once plated ontissue culture plastic, akin to the processes of endothelial orepithelial to mesenchymal transition (EndMT/EMT) (Kren-ning et al., 2010).In conclusion, the studies of circulating MSCs in PB ofhumans are more than two decades old (Levesque et al., 2007).Most investigators agree that their frequency in blood is verylow in healthy individuals, but that the amounts of circulatingMSCs may increase under challenging conditions, thus support-ing the notion that MSCs can be transiently found ‘circulating’in blood. Jain et al. here provide further evidence that MSCscan be found in PB and AP of patients treated with a typicalG-CSF-based HSC mobilization regimen. 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Editorial comment: variables affecting the presence of mesenchymal stromal cells in the peripheral blood and their relationship with apheresis product

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Editorial comment: variables affecting the presence of mesenchymal stromal cells in the peripheral blood and their relationship with apheresis product

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