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PLOS ONE logoLink to PLOS ONE
. 2020 Jun 2;15(6):e0225485. doi: 10.1371/journal.pone.0225485

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

Margaux Deynoux 1,#, Nicola Sunter 1,#, Elfi Ducrocq 1, Hassan Dakik 1, Roseline Guibon 2,3, Julien Burlaud-Gaillard 4,5, Lucie Brisson 3, Florence Rouleux-Bonnin 1, Louis-Romée le Nail 6, Olivier Hérault 1,7, Jorge Domenech 1,7, Philippe Roingeard 4,5, Gaëlle Fromont 2,3, Frédéric Mazurier 1,*
Editor: Atsushi Asakura8
PMCID: PMC7266346  PMID: 32484831

Abstract

Mesenchymal stem cells (MSC)-spheroid models favor maintenance of stemness, ex vivo expansion and transplantation efficacy. Spheroids may also be considered as useful surrogate models of the hematopoietic niche. However, accessibility to primary cells, from bone marrow (BM) or adipose tissues, may limit their experimental use and the lack of consistency in methods to form spheroids may affect data interpretation. In this study, we aimed to create a simple model by examining the ability of cell lines, from human (HS-27a and HS-5) and murine (MS-5) BM origins, to form spheroids, compared to primary human MSCs (hMSCs). Our protocol efficiently allowed the spheroid formation from all cell types within 24 hours. Whilst hMSC-spheroids began to shrink after 24 hours, the size of spheroids from cell lines remained constant during three weeks. The difference was partially explained by the balance between proliferation and cell death, which could be triggered by hypoxia and induced oxidative stress. Our results demonstrate that, like hMSCs, MSC cell lines make reproductible spheroids that are easily handled. Thus, this model could help in understanding mechanisms involved in MSC functions and may provide a simple model by which to study cell interactions in the BM niche.

Introduction

Over the last two decades, extensive studies have attempted to characterize mesenchymal stem cell (MSC). Initially described in the bone marrow (BM), MSCs were later found in almost all adult and fetal tissues [1]. Their classification rapidly suffered from a lack of clear phenotypical definition. Therefore, in 2006, the International Society for Cellular Therapy (ISCT) defined MSCs according to three minimal criteria: adherence to plastic, specific cell surface markers and multipotent potential. Indeed, MSCs are classically described as stem cells that are able to differentiate into osteoblasts, adipocytes and chondroblasts [2], making them an attractive source of cells in regenerative medicine. Subsequent studies have also established their ability to differentiate into cardiomyocytes [3], neurons [4], epithelial cells [5] and hepatocytes [6]. The discovery of the multiple functions of MSC, such as those involved in the anti-inflammatory response [7] and in injury repair [8,9] confirmed them as promising cellular tools in regenerative medicine.

Furthermore, MSCs represent a key component of the BM microenvironment supporting normal hematopoiesis through the regulation of stem cell renewal and differentiation processes, but also fueling malignant cells and protecting them from therapeutic agents [10]. As such, primary MSCs have often been used as feeder layers in long-term co-culture of hematopoietic cells in vitro in preclinical studies [11]. With the aim of standardization, the murine MS-5 cell line became a standard for both normal or malignant hematopoietic cell culture [12]. This robust co-culture model has been widely used and has contributed to the characterization of hematopoietic stem cells (HSC) [11]. This two-dimensional (2D) system, while closer to BM physiology than the culture of hematopoietic cells alone, still lacks the three-dimensional (3D) complexity of the BM niche. Thus, although widely used, it is certainly not sufficiently consistent at predicting in vivo responses [13]. Therefore, a 3D system might be a better alternative to mimic the BM microenvironment.

Critically, the culture leads to rapid loss of MSC pluripotency and supportive functions. Therefore, a wide range of techniques to form 3D MSCs aggregates, from the simplest spheroids to the more complex matrix-based structures, have been proposed [14]. Studies of spheroids, also called mesenspheres, were mostly dedicated to the examination of MSC stemness and differentiation abilities, such as osteogenesis, in order to improve their in vitro expansion and transplantation efficacy in regenerative medicine [15,16]. Furthermore, this model has also been tested as a surrogate niche for hematopoietic cells [1723]. Spheroids take advantage of the ability of MSCs to self-aggregate, which is improved by using various approaches such as low adhesion plates, natural and artificial (centrifugation) gravity, cell matrix or more complex scaffolds [13,14,24,25]. Classically, studies have used human primary MSCs, from BM, cord blood and lipoaspirate, or rodent sources [15,26].

Although immortalized MSCs, or well characterized cell lines, could bypass the lack of primary cells and avoid the variability involved with use of primary human MSCs (hMSCs) samples, they are rarely employed to make spheroids [27,28]. Cell lines would also allow better standardization of the spheroid formation protocol. In this study, we examined the spheroid-forming capacity of two human cell lines (HS-27a and HS-5) and the currently used murine MS-5 cell line, in comparison with primary hMSCs. We defined a simple and fast method using standard matrix to form spheroids and characterized them in terms of physical features, cell proliferation and death.

Materials and methods

Cell culture and reagents

The murine MS-5 bone marrow (BM) stromal cell line was kindly provided by Mori KJ (Niigata University, Japan) [29]. HS-27a and HS-5 human BM stromal cell lines were purchased from American Type Culture Collection (CRL-2496 and CRL-11882, respectively). Primary BM hMSCs were obtained by iliac crest aspiration from healthy donors (without hematological disorders) undergoing orthopedic surgery at the University Hospital of Tours, after informed consent, for cell banking according to the Declaration of Helsinki, as approved by the French Ministry of Education and Research (authorization number No. DC-2008-308). Samples from BM aspirates were diluted in MEM Alpha (Life Technologies, Villebon-sur-Yvette, France) and filtered through a cell-strainer prior to centrifugation (350 x g, 10 min). Cells were resuspended and seeded at 105 to 2.105 cells/cm2 in MEM Alpha supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (both from Life Technologies) and 1 ng/mL of recombinant human FGF basic (FGF-2, R&D Systems, Abingdon, United Kingdom). Medium was changed twice a week until cells reached confluency. In experiments, primary hMSCs were used at passage 2. HS-27a and HS-5 cell lines were cultured in RPMI 1640 (Life Technologies) and MS-5 in MEM Alpha. All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine (Life Technologies), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were maintained in a saturated humidified atmosphere at 37°C and 5% CO2.

Spheroids formation

For one spheroid, 30,000 cells were cultured in 100 μL of medium, supplemented by 0.25% to 1% of either MethocultTM H4100 or SF H4236 (StemCell, Grenoble, France), and seeded in U-bottomed 96-well plate (Sarstedt, Marnay, France). Both media contains methylcellulose in IMDM, but SF H4236 is supplemented with bovine serum albumin, recombinant human insulin, human transferrin (iron-saturated), 2-Mercaptoethanol and unknown supplements as described by the manufacturer. The medium was the same as that of the normal culture for each cell line but supplemented with heat inactivated FBS to reach 15%. At days as detailed, microscopic analysis was performed using a Leica DMIL microscope (Leica, Nanterre, France), coupled to a DXM1200F camera (Nikon, Champigny-sur-Marne, France). To determine the number of cells in each spheroid over time, only wells with a unique, fully-formed spheroid were selected. Twelve spheroids per experiment were pooled and dissociated with 2 mg/mL collagenase 1A (Sigma-Aldrich, Saint-Quentin-Fallavier, France), 10 min at 37°C, with agitation every two minutes, and then counted by the trypan blue exclusion assay.

Time-lapse video

Automatic acquisitions were performed on a Nikon Eclipse TI-S microscope, coupled to a DS Qi2 camera (Nikon). The system includes a cage incubator (Okolab, Pozzuoli, NA, Italy) controlling temperature and level of CO2. Analyses were performed using both NIS Element BR (Nikon) and Fiji/ImageJ softwares.

Scanning electron microscopy

Spheroids were fixed by incubation for 24 h in 4% paraformaldehyde, 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). Samples were then washed in phosphate-buffered saline (PBS) and post-fixed by incubation with 2% osmium tetroxide for 1 h. Spheroids were then fully dehydrated in a graded series of ethanol solutions, and dried in hexamethyldisilazane (HMDS, Sigma-Aldrich). Finally, samples were coated with 40 Å platinum, using a PECS 682 apparatus (Gatan, Evry, France), before observation under an Ultra plus FEG-SEM scanning electron microscope (Zeiss, Marly-le-Roi, France).

Transmission electron microscopy

Spheroids were fixed by incubation for 24 h in 4% paraformaldehyde, 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). Samples were then washed in phosphate-buffered saline (PBS) and post-fixed by incubation with 2% osmium tetroxide for 1 h. Spheroids were then fully dehydrated in a graded series of ethanol solutions and propylene oxide. Impregnation step was performed with a mixture of (1∶1) propylene oxide/Epon resin, and then left overnight in pure resin. Samples were then embedded in Epon resin, which was allowed to polymerize for 48 h at 60°C. Ultra-thin sections (90 nm) were obtained with an EM UC7 ultramicrotome (Leica). Sections were stained with 5% uranyl acetate (Agar Scientific, Stansted, United Kingdom), 5% lead citrate (Sigma-Aldrich) and observations were made with a transmission electron microscope (Jeol, JEM 1011, Croissy-sur-Seine, France).

Immunohistochemistry

At least five spheroids per condition were pooled, fixed in formalin, embedded in paraffin and cut in 3–4 μm sections on Superfrost Plus slides. Slides were deparaffinized, rehydrated and heated in citrate buffer pH 6 for antigenic retrieval. After blocking for endogenous peroxidase with 3% hydrogen peroxide, the primary antibodies were incubated. The panel of primary antibodies included anti-HIF-1α (Abcam ab51608, Paris, France) (dilution 1/200, incubation 1 h), anti-VEGF-A (Abcam ab1316, dilution 1/200, incubation 1 h), anti-HO-1 (Abcam ab52947, dilution 1/1 000, incubation 1 h), anti-CA-IX (Novocastra clone TH22, Nanterre, France) (dilution 1/100, incubation 20 min), anti-Ki-67 (DakoCytomation clone 39–9, Glostrup, Denmark) (dilution 1/50, incubation 30 min), anti-caspase 3 (Novocostra clone JHM62, Nanterre, France) (dilution 1/100, incubation 1 h) and anti-LC3B (Novus Biological NB 600–1384, Cambridge, UK) (dilution 1/200, incubation 1 h). Immunohistochemistry was performed with either the automated BenchMark XT slide stainer (Ventana Medical System Inc.) using OptiView Detection Kit (Ventana Medical System Inc.) (for CA-IX and Ki-67), or manually using the streptavidin-biotin-peroxidase method with diaminobenzidine as the chromogen (Kit LSAB, DakoCytomation). Slides were finally counterstained with haematoxylin. Negative controls were obtained after omission of the primary antibody or incubation with a non-specific antibody.

Quantitative real-time PCR

Total RNAs were extracted using TRIzol reagent (Life Technologies) and reverse transcription was performed with the SuperScriptTM VILOTM cDNA Synthesis Kit (Invitrogen, Villebon-sur-Yvette, France), both according to the manufacturer’s procedures. qRT-PCR was performed on a LightCycler® 480 (Roche, Switzerland) with the LightCycler® 480 Probes Master (Roche). GAPDH, ACTB, RPL13A and EF1A genes were used as endogenous genes for normalization. Primer sequences (S1 Table) were designed with the ProbeFinder software (Roche), and all reactions were run in triplicate.

Cell cycle analysis

Spheroids were dissociated with 2 mg/mL collagenase 1A (Sigma-Aldrich), 10 min, at 37°C, with agitation every two minutes. Cells were fixed with 2% paraformaldehyde/0.03% saponin for 15 min at room temperature (RT), and washed three times for 5 min with 10% FBS/0.03% saponin. Cells were then stained with 7-Aminoactinomycin D (7-AAD, Sigma-Aldrich) and an AF488-conjugated anti-KI-67 antibody (BD Biosciences, Le Pont de Claix, France) or the AF488-conjugated IgG1 isotype control (BD Biosciences). Experiments were performed on an AccuriTM C6 flow cytometer (BD Biosciences) and data were analyzed with the FlowJo V10.4.1 software (Tree Star Inc.).

Statistical analysis

All statistical analyses were performed using R software. Since our sampling never exceeds n = 6, we used nonparametric tests. The Mann-Whitney test was used to compare two conditions and Kruskal-Wallis for multiple comparisons, followed by a Dunn’s post hoc test. The threshold for significance was set up to a p-value of 0.05.

Results

Establishment of primary hMSC-spheroids by cell aggregation method

From the different methods to form MSC-spheroids, we followed an approach based on cell aggregation in methylcellulose-based medium [27]. To establish a protocol that is simple, reproducible and compatible with hematopoietic cell culture, two commercial methylcelluloses (MethoCult H4100 and SF H4236) developed for hematopoietic progenitors assays were tested (Fig 1A). A range from 0.01 to 1% of methylcellulose has been previously used [27,3033], so we tested three different concentrations (0.25, 0.5 and 1%). We also tested the hanging drop technique [31,3335] and the previously described U-bottomed 96-well plates methods [27,30,32,33,36]. Both techniques worked well for primary hMSCs but the second was more appropriate for further analyses since the handling is easier and the volume of medium higher, which could prevent starvation and dehydration in long-term cultures. The SF H4236 methylcellulose at a concentration of 0.5% was adopted because it generated only one spheroid in most of the wells with lower condensation aspect for primary hMSCs (Fig 1B). Under these culture conditions, MSCs were able to form spheroids rapidly, in as little as five hours of culture (S1 Video), which is consistent with previous studies [27,32,37].

Fig 1. Spheroids formation from primary hMSCs.

Fig 1

(A) Schematic representation of experimental plan. (B) 30,000 primary hMSCs per well were seeded into U-bottomed 96-well in medium containing 0.25%, 0.5% or 1% of methylcellulose (MethocultTM H4100 or SF H4236). Microscopy analysis was performed after 24 h (scale bars = 500 μm).

Formation of spheroids from MSC lines

The spheroid-forming capacity was followed for two human cell lines, HS-27a and HS-5, and compared to that of primary hMSCs. The two cell lines were obtained by immortalization of hMSCs from the same BM sample with the papilloma virus E6/E7 genes [38,39]. HS-27a cells support hematopoietic stem cell maintenance (self-renewal, formation of cobblestone areas), whereas HS-5 cells mainly sustain proliferation and differentiation [3840]. Like primary hMSCs, they both retained the ability to form spheres but required about 10 hours to make rounded spheroids (S2 and S3 Videos). Although cells of various origins formed spheroids of equivalent sizes (about 300 μm of diameter) after 24 hours, primary hMSC-spheroids rapidly condensed and reached half of their initial perimeter after 14 days of culture (Fig 2A and 2B). In contrast to primary hMSCs, the perimeter of spheroids resulting from both cell lines remained constant during three weeks. Knowing that primary hMSCs and cell lines may differ in their growth properties, we used the murine MS-5 cell line that has contact inhibition [29]. This cell line was able to quickly form spheroids similarly to the other cell lines (S4 Video). It is noteworthy that MS-5 cells initially formed a flat multilayer disk of cells prior to contracting into spheres. Similarly to the spheroids from human cell lines, spheroids from MS-5 cells kept the same size over time (Fig 2A and 2B). This suggests that shrinking might be an intrinsic property or extracellular matrix (ECM) composition of primary cells rather than related to cell proliferation control. We thus examined whether the difference in the size maintenance between various MSCs might be attributed to the cell number per spheroid. In order to quantify the viable cells, spheroids were dissociated at different timepoints after seeding. In accordance with the decrease in circumference, the number of cells per spheroid from primary hMSCs dramatically dropped within seven days (Fig 2C), in agreement with other studies [31,35]. Remarkably, although keeping the same size, HS-27a-spheroids, as well as the MS-5 ones, had lost viable cells similarly to primary hMSCs (Fig 2C). In contrast, HS-5-spheroids had less obvious decrease in cell number with time (Fig 2C). Overall, the size reduction does not seem to be strictly attributable to reduced cell number in spheroids and could be possibly attributed to other factors such as the ECM composition.

Fig 2. Follow up of the spheroids from various MSCs.

Fig 2

(A) Microscopy analysis of primary hMSC-, HS-27a-, HS-5- and MS-5-spheroids over 21 days in culture (scale bars = 100 μm). (B) Perimeter was measured with an arbitrary unit; each experiment is the mean of at least 10 spheroids from n = 3 experiments. Data are shown as mean ± SD; * compared to day 1; * p ≤ 0.01. (C) Number of living cells per spheroid over 21 days in culture (primary hMCSs and MS-5 n = 3; HS-27a and HS-5 n = 4). Data are shown as mean ± SD; *, **, *** compared to day 0; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Electron microscopy observation of the MSC-spheroids

Scanning electron microscopy (SEM) confirmed the shrinking of primary hMSC-spheroids (Fig 3A and S1A Fig). SEM also revealed, at higher magnification, that spheroids from primary hMSCs are highly cohesive, showing tight intercellular connections forming a flat surface, whereas HS-27a- and HS-5-spheroids, and to a lesser extend MS5-spheroids, exhibited more rounded cells at their surface (Fig 3B and S1B Fig). This phenomenon intensified over time and may explain the size reduction of hMSC-spheroids compared to the cell lines. Absence of ECM is not involved, since ECM deposition is visible for all cell types (Fig 3B). From day 7 for cell lines and day 14 for primaries, spheroid structure began to change, showing loss of cell-cell adhesion, and cell death at the surface.

Fig 3. Scanning electron microscopy (SEM) observation of MSC-spheroids.

Fig 3

(A) Spheroids from primary hMSCs are round with a smooth surface and show progressive shrinking. HS-27a- and HS-5-spheroids are more irregular and granular. (B) Higher magnification show that cells are cohesive at the surface of primary hMSC-spheroids and more chaotic with distinguishable cells of different shapes for human cell lines. ECM deposition (arrow heads) is visible on all spheroids. Scale bars = 100 μm (A) and 20 μm (B).

Further analysis by transmission electron microscopy (TEM) was performed to investigate the ultrastructure of the cells within the spheroids. Between day 1 and day 7, cells showed the swelling of the cell cytoplasm and the presence of an increasing number of necrotic cells, thus suggesting particularly rapid induction of cell death for primary hMSCs compared to cell lines (S2A–S2L Fig). Strong induction of autophagy, indicative of cell stress, was demonstrated by appearance of numerous cytoplasmic vacuoles and autophagosomes for primary hMSCs, as soon as day 1 (S2A Fig), and could certainly explain the shrinking. Remarkably, despite a higher apparent loss of cell adhesion by TEM, induction of cell death (necrosis) appeared delayed for cell lines. In addition, although the number of cytoplasmic vacuoles increased over time, no autophagosomes were noted in cell lines. Consistent with this observation, although LC3B expression can be triggered in HS-27a cells (S3 Fig), its subcellular expression remains uniformly cytoplasmic rather than as dot-like staining patterns, which may thus indicate a block in the autophagic mechanism as previously described [41,42].

Cell death and proliferation of the MSC-spheroids

To explain why spheroids showed decreased cell number over time, we proposed an imbalance between cell death and cell proliferation. Cell death has already been noted by electronic microscopy observation though autophagy and cell lysis. Apoptosis and cell cycle were first determined by flow cytometry using 7-AAD/Ki-67 staining (Fig 4A). Increasing sub-G0/G1 cell population revealed a strong induction of necrosis and/or late apoptosis after 14 days in hMSC-spheroids, whereas none or moderate cell death was observed for the two human cell lines (Fig 4B). In accordance with these data, caspase-3 staining showed few apoptotic cells until day 7 for primary hMSCs and HS-5 cells (Fig 4C). In contrast, apoptosis in HS-27a-spheroids was observed as soon as day 1, and increased with time, which is consistent with earlier detection of death cells by flow cytometry (Fig 4B). Regarding the proliferation, although harvested at the same confluency, primary hMSCs appeared already much more quiescent than HS-27a or HS-5 cells at day 0 (Fig 4D). A significant proportion of cells remained proliferating in spheroids until day 3 for HS-27a and day 7 for HS-5 cells. Remarkably, while closer to HS-27a cells in terms of perimeter and number of cells, MS-5 cells had a massive increase in cell death and almost no proliferation (S1C and S1D Fig), similarly to primary hMSCs. Ki-67 detection by immunochemistry, in primary hMSCs and human cell lines, revealed homogeneous staining at day 1 indicating proliferation in the whole spheroid (Fig 4E) in agreement with a previous study [43]. Staining confirmed a lower proliferation rate of primary hMSCs compared to cell lines and a rapid proliferation arrest with only few Ki-67-positive cells remaining at the periphery of the spheroid at day 3. A progressive decrease in proliferation for the human cell lines supported the results obtained by flow cytometry. Remarkably, decreased proliferation appears in the entire spheroid and is not restricted to in-depth localizations. These data showed that spheroids are characterized by imbalance between cell death and proliferation, which may explain the highest loss of cells over time.

Fig 4. Determination of proliferation and apoptosis of MSC-spheroids.

Fig 4

(A, B and D) Cell cycle analysis of spheroids over 21 days in culture. (A) Representative gating strategy from primary hMSCs at day 0, (B) sub-G1 apoptosis quantification (primary hMSCs n = 6; HS-27a and HS-5 n = 3) and (D) cell cycle quantification (primary hMSCs n = 6; HS-27a and HS-5 n = 5; * for G0; ǂ for G1; # for S/G2/M) (data are mean ± SD; *, ǂ, # compared to day 0; *, ǂ p ≤ 0.05; **, ǂǂ, ## p ≤ 0.01). (C and E) Immunohistochemistry of (C) caspase-3 and (E) Ki-67 at days 1, 3 and 7 for primary hMSC-, HS-27a- and HS-5-spheroids (scale bars = 50 μm (C) and 100 μm (E)). Arrow heads indicate Ki-67-positive cells.

Hypoxia and oxidative stress in MSC-spheroids

Like in tumor spheres [4446], the appearance of an oxygen gradient and hypoxia in MSC-spheroids has been demonstrated [47,48]. Carbonic anhydrase IX (CA-IX), a mediator of hypoxia-induced stress response, is commonly used as marker in tumors [49]. Increased CA-IX has been observed in MSC-spheroids, particularly for HS-27a cells (Fig 5A). The pro-survival adaptation to hypoxia occurs mainly through the stabilization of the hypoxia-inducible factors (HIFs). HIFs are key regulators of multiple cell processes, including cell cycle, metabolism, pH control and autophagy. Increasing expression of HIF-1α protein expression has been observed in spheroids over time, as well as at the mRNA level (Fig 5B). Finally, we examined the expression of VEGFA, a typical HIF transcriptionally regulated gene [50]. Its expression in hMSC- and HS-27a-spheroids was already elevated at day 1, but strongly increased at both protein and mRNA levels over time (Fig 5C).

Fig 5. Hypoxia detection of primary hMSC- and HS-27a-spheroids over 7 days in culture.

Fig 5

(A) Immunohistochemistry of CA-IX. (B) Immunohistochemistry and mRNA of HIF-1α. (C) Immunohistochemistry and mRNA expression of VEGF-A. (primary hMSCs n = 5; HS-27a n = 3; * p ≤ 0.05; ** p ≤ 0.01; scale bars = 100 μm).

In certain circumstances, very low level of oxygen (anoxia) or long exposure to hypoxia may provoke DNA damage and oxidative stress that trigger apoptosis [44,46]. Besides hypoxia appearance in spheroids, cell aggregation may also stress the cells by itself and increase reactive oxygen species (ROS). Heme oxygenase 1 (HO-1) is induced by a variety of stressors, and is therefore a marker of hypoxia and oxidative stress [48,51]. Indeed, oxidative stress triggers nuclear relocation of NRF-2, a HO-1 transcription factor, which then leads to antioxidant response through induced expression of antioxidants by HO-1. In the spheroids, we observed a high expression of HO-1 at day 1, which increased over time (Fig 6A). Conversely, among the 24 antioxidant genes [52], we found a total of seven genes upregulated in spheroids from the primary hMSCs and the HS-27a cell line (Fig 6B). Remarkably, of these genes, four (GPX1, PRDX2, SOD1 and SOD2) were commonly upregulated in both cell types irrespective of their initial expression level.

Fig 6. Oxidative stress detection in primary hMSC- and HS-27a-spheroids.

Fig 6

(A) Immunohistochemistry of HO-1 (scale bars = 100 μm). (B) Expression of antioxidant genes (n = 3; data are mean; * compared to 2D control (CTL); * p ≤ 0.05; ** p ≤ 0.01).

Together, these data indicate concomitant appearance of hypoxia and oxidative stress in both primary hMSC- and HS-27a-spheroids, which could therefore explain initial cell cycle arrest and further apoptosis in prolonged hypoxia.

Dedifferentiation in MSC-spheroids

The 2D culture of MSCs critically leads to rapid loss of pluripotency after few passages, whereas MSC-spheroids can induce dedifferentiation, demonstrated by the expression of three pluripotent transcription factors (OCT-4, SOX-2 and NANOG) [32,53]. Furthermore, it has been described that hypoxia transcriptionally regulates these factors in a HIFs-dependent manner [54]. Therefore, in order to validate whether HS-27a behave similarly to primary hMSCs, we examined the expression of OCT-4, SOX-2 and NANOG, over time. Results showed that hMSC-spheroid formation was accompanied by upregulation of OCT4 and SOX2, in agreement with previous studies, but surprisingly showed no upregulation of NANOG (Fig 7A). HS-27a had similar expression level of the three genes to hMSCs in 2D culture and had progressive increased expression of all markers, suggesting that the HS-27a retains dedifferentiation capacity like primary hMSCs (Fig 7B).

Fig 7. Dedifferentiation detection of hMSC- and HS-27a-spheroids over 7 days in culture.

Fig 7

(A and B) Gene expression of OCT4, NANOG and SOX2 for (A) primary hMSC- and (B) HS-27a-spheroids (primary hMSCs n = 5; HS-27a n = 3; * p ≤ 0.05; ** p ≤ 0.01).

Discussion

MSC-spheroids have mainly been designed to study biological processes such as those related to ex vivo stem cell maintenance, differentiation capacity, immunity or anti-inflammatory, with the aim to improve their use in regenerative medicine [25,55]. For instance, studies have demonstrated the benefit of using MSC-spheroids in treating cardiac, cerebral, kidney and hindlimb ischemia [5659] or in tissue cartilage and bone repair [6063]. MSCs are also highly involved in hematopoietic homeostasis [1,10]. However, until recently, they were viewed as rare heterogeneous populations lining vessels in the BM. Using multiscale 3D quantitative microscopy, Gomariz et al. have elegantly revealed that mesenchymal reticular subsets are remarkably more abundant than previously estimated [64]. This reinforces the interest of studying MSCs in the hematopoietic context and investigating them from a 3D angle.

Many studies have examined complex matrix-based 3D models, while ultimately, only few have approached MSC-spheroids as in vitro surrogate of the hematopoietic niche. In brief, HSCs can migrate and settle into MSC-spheroids that provide higher potential to promote HSCs expansion and stemness maintenance, compared to 2D co-cultures [18,19]. Direct intrabone delivery of MSC-spheroids containing HSCs also sustained higher engraftment and retention of HSCs compared to injected HSCs [17]. However, Schmal et al. drew our attention on the fact that hanging drop method led to lower proliferation of hematopoietic cells and decreased generation of progenitors compared to 2D co-culture, although the stem cell capability has not been yet tested [35]. These contrary data suggest that spheroid colonization by HSCs may certainly depend on the method used to create spheroids. Spheroids are also attractive tools to uncover mechanisms by which malignant cells remodel their microenvironment and MSCs participate in chemoresistance and relapse [65]. As a proof of concept, Reagan et al. showed that MSC-spheroids help to unravel mechanisms of regulation of osteoblasts in multiple myeloma [66], while Aljitawi et al. demonstrated that chemotherapy is linked to the expression of N-cadherin in AML [23].

Although MSC-spheroids appear to be a promising tool, studies might have been limited by the availability of primary hMSCs and the reproducibility due to different sources. Strangely, 2D co-cultures with hematopoietic cells have long been established with cell lines, mostly murine, such as MS-5 or M2-10B4 [11], but they have almost never been used to create 3D aggregates. Our study provides an evaluation of the ability of three cell lines to form spheroids. We chose HS-27a and HS-5 cell lines for their human origin and their known capacity to sustain hematopoiesis [38]. The HS-5 cells are described as fibroblastoid cells, secreting high amount of growth factors that support hematopoietic progenitors proliferation. The HS-27a cell line has epithelioid morphology, producing low level of growth factors but supporting hemapoietic stem cells [38]. Unlike MS-5 cells, HS-5 and HS-27a cells do not retain contact inhibition that certainly, although of human origin, have limited their use for long-term cultures. Independently of the contact inhibition capacity, the three cell lines were able to provide quick and reproducible spheroids. The delay to achieve a complete spheroid, compared to primary hMSCs, could certainly be attributed to sedimentation speed, cell lines being much smaller than primaries that could hence sediment faster. In agreement with previous studies [18], we found that primary hMSCs provide highly cohesive spheroids with a smooth surface. MS-5 had a similar appearance, whereas spheroids from human cell lines were more disorganized with distinct cells at the surface. ECM deposition was visible for all types of spheroids but ECM composition could differ, and the higher proliferation of human cell lines, which certainly induces ECM remodeling, may explain the differences.

Contrary to primary hMSC-spheroids, all spheroids from cell lines kept a constant size over time. This could be partially explained for human cells by the fact that cell lines continue to proliferate in spheroid culture, whereas primary MSCs are mostly quiescent, and may compensate cell death. However, HS-27a showed a decrease in viable cells similarly to primary hMSCs and MS-5 are quiescent but do not shrink. Shrinking has already been reported for primary hMSCs [31,32,35,37,6769] and has been attributed to autophagy [32]. Reduced cell growth or arrest is known to trigger autophagy [70]. Since, HS-27a and HS-5 cell lines proliferate until 7 days in spheroids, one could hypothesize that transformed cell lines may have lower autophagy, contrary to quiescent primary hMSCs. Consistent with this assumption, TEM revealed high amounts of cytoplasmic vacuoles and autophagosomes in primary hMSC-spheroids, as early as day 1. Although cytoplasmic vacuoles progressively appeared in spheroid from cell lines, almost no autophagosomes were detected. It is worth noting that HS-27a and HS-5 were obtained from primary hMSCs transformed with Human papillomavirus 16E6/E7, which activates autophagy via Atg9B and LAMP1 in cervical cancer cells [71]. However, neither TEM, nor LC3B staining showed autophagy in HS-27a cells. Indeed, LC3B expression was strongly induced in HS-27a cells, which indicates response to stress and induction of the autophagic process, but staining remains diffuse in the cytoplasm, suggesting a blockage of the autophagy process. However, diffuse LC3B staining may hamper the interpretation in IHC, while a dot-like staining patterns is more indicative of autophagy [41]. Dots may also reflect the accumulation of autophagosomes due to induction of autophagy, or due to inhibition of autophagy resulting from a lack of autophagosome degradation upon fusion with lysosomes [72]. In the absence of obvious autophagy in cell lines, cell death could therefore be explained by necrosis, as shown by SEM, and apoptosis observed by caspase-3 staining in IHC (early apoptosis) and 7-AAD staining in flow cytometry (late apoptosis and necrosis). Low apoptosis is detected for primary hMSC-spheroids, but massive lysed and necrotic cells are seen by SEM as early as day 1, in addition to autophagy. In agreement with our results, others studies have also demonstrated induction of apoptosis after several days [35,67,73]. While MS-5 cells resemble primary cells with low proliferation and strongly increased cell death, probably due to their contact inhibition, they conversely did not shrink like human cell lines, probably because of low or no autophagy.

Strong hypoxia and oxidative stress are among the stressors that could have induced autophagy and/or apoptosis in spheroids. Oxygen gradients have been frequently reported in tumor-spheres, with deep hypoxia surrounding a necrotic core [4446], as well as in MSC-spheroids [47]. The hypoxia response mainly occurs through the stabilization of hypoxia-inducible factors (HIFs), which are regulators of multiple biological processes, such as angiogenesis or energetic metabolism. HIFs have an essential pro-survival role by promoting genes, such as those involved in metabolism and autophagy [50]. However, acute and prolonged hypoxia may also trigger cell death through blocking DNA replication and induced oxidative stress [44,46]. In MSC-spheroids, we found increased expression of hypoxia markers, including HIF-1α and its target CA-IX, VEGF-A, concomitant to induced oxidative stress, as revealed by increased expression of HO-1 and the antioxidant response. SOD2 and GPX1 were the two genes with the greatest upregulation, which indicates strong oxidative stress. Interestingly, although HO-1 and antioxidant genes are typically NRF-2 targets, they could also be regulated by HIFs. Altogether, these data indicate appearance of hypoxia and oxidative stress in primary hMSC- and HS-27a-spheroids, which could therefore explain cell cycle arrest, induction of autophagy and further apoptosis in prolonged hypoxia.

The 2D culture of MSCs critically leads to rapid loss of pluripotency after few passages, whereas 3D culture has showed greater MSC stemness maintenance, and induced dedifferentiation, demonstrated by the expression of three pluripotent transcription factors (OCT-4, SOX-2 and NANOG) and multipotent differentiation capacity [32,43,53,74]. It is worth noting that hypoxia favors stemness and dedifferentiation and induces expression of pluripotent transcription factors through HIFs [75,76]. Like primary hMSC-spheroids, HS-27a-spheroids had increased expression of pluripotent markers. This confirmed that HS-27a behave similarly to primary hMSCs and had preserved dedifferentiation capacity that could also be (re)activated during spheroid formation.

Conclusions

Overall our data indicate that, like hMSCs, MSC cell lines can be used to make reproductible and easily handled spheroids. HS-27a cells resemble primary cells, and are of a particular interest for further studies, since they provide better support to HSCs compared to HS-5 cells [3840]. Thus, this model could help in understanding mechanisms involved in MSC physiology and may be a simple model to study cell interactions in the hematopoietic niche. The model could also be extended to research metastatic process as previously described for breast cancer [28].

Supporting information

S1 Fig. Spheroids formation of mouse MS-5 cell line.

(A and B) Scanning electron microscopy (SEM) analysis over 14 days (scale bars = 100 μm (A) and 20 μm (B). (C) Sub-G1 apoptosis quantification (n = 3) and (D) cell cycle quantification over 21 days in culture (n = 3; data are mean ± SD).

(TIF)

S2 Fig. Transmission electron microscopy (TEM) observation of MSC-spheroids.

TEM analysis of primary hMSC-spheroids at day 1 (A), day 3 (B) and day 7 (C); Higher magnification is also shown to highlight autophagosomes. HS-27a-spheroids at day 1 (D), day 3 (E) and day 7 (F); HS-5-spheroids at day 1 (G), day 3 (H) and day 7 (I) and MS-5-spheroids at day 1 (J), day 3 (K) and day 7 (L). Scale bars = 20 μm.

(PPTX)

S3 Fig. LC3B expression in HS-27a-spheroids.

Immunohistochemistry of LC3B is shown at days 1, 3 and 7 for HS-27a-spheroids (scale bars = 50 μm).

(TIF)

S1 Video. A representative time-lapse video of spheroid formation.

30 000 primary MSCs seeded into U-bottomed 96-well, in medium containing 0.5% of methylcellulose (MethocultTM SF H4236) were followed via a Nikon Eclipse TI-S microscope for 24 hours.

(MP4)

S2 Video. A representative time-lapse video of spheroid formation.

30 000 HS-27a cells seeded into U-bottomed 96-well, in medium containing 0.5% of methylcellulose (MethocultTM SF H4236) were followed via a Nikon Eclipse TI-S microscope for 24 hours.

(MP4)

S3 Video. A representative time-lapse video of spheroid formation.

30,000 HS-5 cells seeded into U-bottomed 96-well, in medium containing 0.5% of methylcellulose (MethocultTM SF H4236) were followed via a Nikon Eclipse TI-S microscope for 24 hours.

(MP4)

S4 Video. A representative time-lapse video of spheroid formation.

30,000 MS-5 cells seeded into U-bottomed 96-well, in medium containing 0.5% of methylcellulose (MethocultTM SF H4236) were followed via a Nikon Eclipse TI-S microscope for 24 hours.

(MP4)

S1 Table. List of primers and probes sequences.

(DOCX)

Acknowledgments

We would like to thank P. G. Genever (University of York, UK) for providing valuable support, providing protocol and recommendations to establish spheroids. We also thank Sophie Hamard (University of Tours, France) for her technical assistance.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors acknowledge the Ministry of Research (MD), the ARC Foundation (MD, HD), the French Society of Hematology (MD), the “Ligue contre le Cancer (NS)”, and the Lebanese south governate (HD) for their funding. This work was supported by the French Committees of the “Ligue Contre le Cancer Grand-Ouest” [16 (Charente), 36 (Indre), 37 (Indre-et-Loire), 41 (Loire et Cher), and 86 (Vendée)] and the Région Centre Val de Loire (FM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Atsushi Asakura

14 Jan 2020

PONE-D-19-30255

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

PLOS ONE

Dear Dr Mazurier,

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"Primary normal BM-MSCs were isolated from healthy donors (without any hematological disorder) undergoing orthopedic surgery (University Hospital, Tours,

France) after informed consent and following a procedure approved by the local ethical committee."

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Reviewer #2: Partly

Reviewer #3: Partly

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Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors used spheroids to study differences of primary human mesenchymal stem cells (hMSCs) and immortalized mesenchymal stem cell (MSC) lines. Overall, it seems that the study properly designed to achieve their research goal. However, the current manuscript still has a number of major and minor concerns and needs better clarifications in several places.

Major concerns:

1. The rationale behind the study is a bit unclear. The authors should describe more how MSC-based spheroids are specifically available to study the hematopoietic niche. The sentences in the first paragraph of the Discussion section are vague or not specific for MSC-based spheroids.

2. It is expected that the cells in each well gradually formed a single spheroid at the end. However, in Figures1B, 2A, and Supplementary Videos, small cell aggregates are identified near a large spheroid. Please clarify how these aggregates might influence the analysis in this study. For instance, were these small aggregates included (or excluded) when the number of cells was analyzed in Figure 2C?

3. Figure 2B: As this is 3D culture, the spheroid volume may sound more reasonable compared to the perimeter.

4. Figure 3: Additional indications or labeling should be used to point out specific histological features. The figure legend should also be expanded more, not only describing the results in the text. In Figure 3C, where is the appearance of a progressive cell injury?. By the way, what does “a progressive cell injury” mean?

5. Figure 4: Like Ki67 staining, it is worth to have additional immunohistochemical results showing cell death in the spheroids.

6. Figure 6B: Although the results are summarized as a heat map without specific values, statistical differences are presented there. Is this appropriate?

7. Page 16, the section named “Stemness in MSCs-derived spheroids”: The word “stemness” sounds too definitive here, as the conclusion was only supported by the results of gene expression for specific stem cell markers.

8. The Discussion section may be short a bit, when compared to the amount of experiments and results.

9. The abbreviations “MSCs-“ or “MSCs-derived” are used in many places. They should be “MSC-“ and “MSC-derived”.

Minor concerns:

1. Page 3, Line 54 from the bottom: Please spell out “2D” as this abbreviation comes at the first place in the main text.

2. An abbreviation “3D” should be defined with “three-dimensional” in Page 3, Line 55.

3. Page 5, Line 90: Please describe the dose of FGF-2 with nanogram or microgram/mL. A use of % is not common and not helpful for readers.

4. Page 5, Line 93: The passage number of established MSC lines (“between passage 5 and 20”) would not be accurate, as these cells were already expanded in culture before the authors had received them from the repositories.

5. Page 8, Line 151-158, Quantitative real-time PCR: Why does only this section have the catalogue numbers for the products?

6. Page 9, Line 170-173, Statistical analysis: Nonparametric statistics are commonly used. I understand that in general nonparametric methods are not easy to achieve a statistical difference when the number of subjects is relatively small like n=3 or 4. This may be still acceptable, but please justify why these statistical methods were specifically applied.

7. Page 9, Line 180-181: Please add “(MethoCult H4100 and SF H4236)” after “two commercial methylcelluloses.” How are these two products different in terms of components?

8. Page 14, Line 281, Figure 4D legend: Please revise “Arrows” to “Arrow heads.”

9. Page 15, Line 309: What does “(Patent WO2016083742)” mean?

10. Page 15, Line 320: The word “established MSCs” is vague. Be specific.

11. Page 16, the word “stemness”: What retains stemness is the cells cultured in spheroids, not spheroids themselves.

12. These words should be reconsidered in use: 3D MSC structures (Page 4, Line 60), gold-standard (Page 4, Line 74), a stemness capacity (Page 16, Line 334-335), stemness detection (Page 16, Line 337).

Reviewer #2: In this study, the authors examined the sphere-forming capacity of two human bone marrow stromal cell lines, morphologically resembling epithelial cell or fibroblast, and a murine bone marrow stromal cell line, comparing to primary human bone marrow-derived MSCs. An approach based on cell aggregation in methylcellulose-based medium was used. The size of the sphere, the number of cells constituting the sphere, morphology, cell cycle, and gene expression related to hypoxia-induced stress response were examined chronologically. Although it is interesting to know them, honestly, it is not clear to me what model the authors tried to create. A simple model to evaluate sphere-forming capacity of cells or searching cell line exhibiting MSC-like cellularity in terms of sphere formation capacity? It has been reported that in comparison with dissociated cells or cells expanded on adhesion culture, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining or enhancing their differentiation capacity. The reviewer somehow thinks that there is something missing.

Major concern

If the authors are trying to find a cell line exhibiting MSC-like cellularity in terms of sphere formation capacity, a single cell culture may be needed to assess sphere forming capacity. Proof of MSC-like cellularity such as self-renewability, tri-linage differentiation capacity is, of course, necessary.

Reviewer #3: Although the manuscript is interesting in the comparison of the capacity in the spheroid culture from three types of hMSCs there are some serious problems. The authors should be addressing them.

Special comments

1) It totally is the serious problem that the authors did not examine the difference in the differentiation ability into osteoblasts, adipocytes, and chondrocytes from spheroid with primary or cell line hMSCs in the present experiments because it is an important role of regeneration ability for cellular therapy using hMSCs.

2) Materials and Methods: There is no ethics statement (permission number) and preparation and condition of primary hMSCs. Did the authors conducted in compliance with Declaration of Helsinki and get the informed consent from patients? Furthermore, did the authors separate and collect the primary hMSCs with stemness makers (CD29, CD44, and CD105 etc.) or not (heterogeneity) in present experiments?

3) Results: Data interpretation involved in Results section. The authors should describe them in Discussion section.

4) Results: The authors stated the primary hMSCs was less optimal for spheroid culture. It is well known that the proliferation and maintenance of spheroid culture is dependent on the number of spread cells. Did the authors should examine and confirm the less number of spread cells or diameter less than 300 micrometer without shrinking spheroid in primary hMSCs? Furthermore, the authors should examine whether the smaller spheroid derived from primary hMSCs induced activation of HIF-1alpha and apoptosis or not.

5) Figure1: Why did the authors use the two types of methylcelluloses? What is the difference, such as components, viscosity, and moisturization etc.? The authors should explain them.

6) Figure 2: It confuses the murine or human hMSCs. In Fgure2, the authors should add the murine MS-5 data with perimeter and cell number/spheroid, and culture days, but not its supplemental video.

7) Discussion: The authors explain the induction of autophagy in primary hMSCs. However, the authors did not check it. The authors should check the expression of autophagy related molecules, such as LCII etc.

Minor comments

8) Results p10, line 204: It confuses the primary or immortalization hMSCs. The authors should add the word primary MSCs.

9) Discussion p17, line 352: Was it inserted the reference, not referenced number?

10) Fig3C and Fig.5 were low density images, then it is difficult to interpret them.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2020 Jun 2;15(6):e0225485. doi: 10.1371/journal.pone.0225485.r002

Author response to Decision Letter 0


16 Apr 2020

Rebuttal letter [PONE-D-19-30255]

April 6th, 2020

Dear Editor,

First, we would like to thank the editor and reviewers for their remarks and hope that we have now answered most of their concerns and improved the manuscript to fully meet PLOS ONE’s publication criteria. To make it easier, our answers are in blue.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

PLOS ONE’s requirements have been carefully rechecked, and errors are corrected.

2. Thank you for including your ethics statement:

"Primary normal BM-MSCs were isolated from healthy donors (without any hematological disorder) undergoing orthopedic surgery (University Hospital, Tours, France) after informed consent and following a procedure approved by the local ethical committee."

Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

In the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

We apologize for the lack of information and now provide a more detailed sentence (page 5, line 82): “Primary BM hMSCs were obtained by iliac crest aspiration from healthy donors (without hematological disorders) undergoing orthopedic surgery at the University Hospital of Tours, after informed consent, for cell banking according to the Declaration of Helsinki, as approved by the French Ministry of Education and Research (authorization number No. DC-2008-308)”. Please note that the local ethical committee does not provide permission number.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

The phrase was « Both techniques worked well for primary hMSCs but the second was more appropriated for further analyses and offered lesser dehydration (data not shown) ». This was a simple observation that we though informative for readers. We understand the general requirement and removed the statement. The sentence (page 11, line 193) is now « Both techniques worked well for primary hMSCs but the second was more appropriate for further analyses since the handling is easier and the volume of medium higher, which could prevent starvation and dehydration in long-term cultures ».

______________________________

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors used spheroids to study differences of primary human mesenchymal stem cells (hMSCs) and immortalized mesenchymal stem cell (MSC) lines. Overall, it seems that the study properly designed to achieve their research goal. However, the current manuscript still has a number of major and minor concerns and needs better clarifications in several places.

Major concerns:

1. The rationale behind the study is a bit unclear. The authors should describe more how MSC-based spheroids are specifically available to study the hematopoietic niche. The sentences in the first paragraph of the Discussion section are vague or not specific for MSC-based spheroids.

We understand the criticism of the reviewer and the need of clarification. Our work is not limited to hematopoietic niche research, although we are convinced about the real need, but more widely of interest for any kind of specific issue including those in regenerative medicine that require spheroids.

Since the Reviewer had also found the discussion to short (concern #8), we decided to fully reformat the discussion and develop the use of MSC-derived spheroids in hematological research (Page 20, Discussion). We believe that would answer both criticisms.

2. It is expected that the cells in each well gradually formed a single spheroid at the end. However, in Figures1B, 2A, and Supplementary Videos, small cell aggregates are identified near a large spheroid. Please clarify how these aggregates might influence the analysis in this study. For instance, were these small aggregates included (or excluded) when the number of cells was analyzed in Figure 2C?

In Figure 1B, SF H4236 with 0.5 and 1 % provided obviously a unique sphere. This is why for further experiments only the SF H4236 condition was retained. This was mentioned in the text (page 11, line 195) “The SF H4236 methylcellulose at a concentration of 0.5 % was adopted because it generated one spheroid per well with lower condensation aspect for primary hMSCs (Fig 1B)”. In this figure, cells were not enumerated.

In figure 2A, very few aggregates are seen for MS-5 and a bit of spreading for HS-5 only after 14 days. Small aggregates occurred from time to time, certainly due to remaining debris (medium, plastic), but it was very marginal. That appears, however, when we acquired videos. Although the low number of cells in tiny aggregates may account for a low percentage and in all conditions, we decided at the beginning to take into consideration only wells with unique spheroids and no spreading. Therefore, the count in Figure 2C is from fully formed spheroid. We do understand the concern of the reviewer and thus modified our sentence in Materials and Methods section (page 6, line 107) “To determine the number of cells in each spheroid over time, only wells with unique, fully-formed spheroid were selected ”.

3. Figure 2B: As this is 3D culture, the spheroid volume may sound more reasonable compared to the perimeter.

Since spheroids are not perfectly rounded and may sometime be slightly ovoid (see figures 2A and 3A), we believed that giving the perimeters, even as arbitrary unit values, will be more exact and rigorous than any other parameter. Volume will require diameter measurement and be a rough estimation by applying the same formula “4/3 x π x R3” on all spheroids considering they are round, otherwise we will have to measured length and width, with unknown height. Thus, the difference between spheres will be almost the same at the end but with increased technical and calculation errors. So, we think that this is useless and certainly not reasonable to do. In the literature, volume is not frequently provided, whereas authors mostly show diameter (Pennock et al., Schmal et al., Tsai et al., De Barros et al., etc.), which may introduce a bias as abovementioned.

4. Figure 3: Additional indications or labeling should be used to point out specific histological features. The figure legend should also be expanded more, not only describing the results in the text. In Figure 3C, where is the appearance of a progressive cell injury? By the way, what does “a progressive cell injury” mean?

We agree with the reviewer. We kept only SEM as main figure, pointed out specific histological features and consequently modified the legend. To get a better resolution, we had to transfer TEM in supplemental material, as S2 Fig, with individual photos for each spheroid type from day 1 to day 7. “Progressive cell injury” is illustrated by loss of cell-cell adhesion, swelling of the cytoplasm, increased lysed cells and cell death. This has been changed in the text/legend due to figure modifications (page 14, line 253).

5. Figure 4: Like Ki67 staining, it is worth to have additional immunohistochemical results showing cell death in the spheroids.

We initially though that death shown by electronic microscopy and flow cytometry would be enough. We, however, agree with the reviewer that it is worth showing IHC. We therefore included caspase 3 staining, which could detect much earlier induction of apoptosis than 7-AAD staining. Results were added as figure 4C and reported in the text (page 15, line 281).

6. Figure 6B: Although the results are summarized as a heat map without specific values, statistical differences are presented there. Is this appropriate?

To summarize the high amount of transcriptomic data, we believe that heatmap might be better representation than multiple histograms, but thus lost the possibility of showing statistics. Possibly not classically, we believed we had found a way that might be the most informative to the reader and rigorously important to prove which of the gene is statistically different by adding a star. So, as it does not seem appropriate, we do not mind removing stars.

7. Page 16, the section named “Stemness in MSCs-derived spheroids”: The word “stemness” sounds too definitive here, as the conclusion was only supported by the results of gene expression for specific stem cell markers.

We agree with the reviewer. The reviewer 3 also mentioned that in his concern #11/12. We, therefore, modified the paragraph (page 18, line 345), excluding the word stemness and dedifferentiation, which seems more suitable.

8. The Discussion section may be short a bit, when compared to the amount of experiments and results.

We agree. We reformatted and increased the Discussion section (See also concern #1).

9. The abbreviations “MSCs-“ or “MSCs-derived” are used in many places. They should be “MSC-“ and “MSC-derived”.

We thank the reviewer for drawing our attention to this error. This is now corrected.

Minor concerns:

1. Page 3, Line 54 from the bottom: Please spell out “2D” as this abbreviation comes at the first place in the main text.

We thank the reviewer for drawing our attention on this error. It is now modified (page 3, line 54).

2. An abbreviation “3D” should be defined with “three-dimensional” in Page 3, Line 55.

It has been also modified (page 3, line 55).

3. Page 5, Line 90: Please describe the dose of FGF-2 with nanogram or microgram/mL. A use of % is not common and not helpful for readers.

This is true and has been changed for “….1 ng/mL of recombinant human FGF basic” (page 5, line 90).

4. Page 5, Line 93: The passage number of established MSC lines (“between passage 5 and 20”) would not be accurate, as these cells were already expanded in culture before the authors had received them from the repositories.

The reviewer is right. We thus removed this description and only kept the information for primary “homemade” cells (page 5, line 92).

5. Page 8, Line 151-158, Quantitative real-time PCR: Why does only this section have the catalogue numbers for the products?

This was an error. We removed numbers (page 9, line 159).

6. Page 9, Line 170-173, Statistical analysis: Nonparametric statistics are commonly used. I understand that in general nonparametric methods are not easy to achieve a statistical difference when the number of subjects is relatively small like n=3 or 4. This may be still acceptable, but please justify why these statistical methods were specifically applied.

Our sampling was always lower than n = 6, which does not allow us to make parametric statistics. Therefore, we used nonparametric tests. We added this notion in the ‘Materials and methods” (page 10, line 178).

7. Page 9, Line 180-181: Please add “(MethoCult H4100 and SF H4236)” after “two commercial methylcelluloses.” How are these two products different in terms of components?

All information is available online but to clarify we added a sentence in the “Materials and Methods” section: “Both medium contains methylcellulose in IMDM, but SF H4236 is supplemented with bovine serum albumin, recombinant human insulin, human transferrin (iron-saturated), 2-Mercaptoethanol and unknown supplements as described by the manufacturer”. (page 6, line 101).

8. Page 14, Line 281, Figure 4D legend: Please revise “Arrows” to “Arrow heads.”

We agree. This has been changed (page 16, line 304).

9. Page 15, Line 309: What does “(Patent WO2016083742)” mean?

The list of genes was included in a patent from our laboratory, it is now published in a paper as indicated by the following reference [52]: “Picou et al., Bone marrow oxidative stress and specific antioxidant signatures in myelodysplastic syndromes. Blood Adv. 2019 Dec 23;3(24):4271-4279. doi: 10.1182/bloodadvances.2019000677”. This is now cited changed (page 18 line 333).

10. Page 15, Line 320: The word “established MSCs” is vague. Be specific.

We agree. The words “established MSCs” has been changed to “both primary hMSC- and HS-27a-spheroids” (page 18 line 342).

11. Page 16, the word “stemness”: What retains stemness is the cells cultured in spheroids, not spheroids themselves.

We agree and made modifications in the title and in the text of this paragraph. We wrote dedifferentiation instead of stemness (See major concern #7) (pages 18, line 345 and following).

12. These words should be reconsidered in use: 3D MSC structures (Page 4, Line 60), gold-standard (Page 4, Line 74), a stemness capacity (Page 16, Line 334-335), stemness detection (Page 16, Line 337).

The terms condensates or aggregates are indeed more often used in the literature. We therefore changed (page 4, line 59). We are not sure about what the reviewer means by reconsidering gold-standard but changed it for “the currently used murine MS-5 cell line” instead (page 4, line 73). As mentioned above, the text including stemness was replaced by dedifferentiation.

______________________________

Reviewer #2: In this study, the authors examined the sphere-forming capacity of two human bone marrow stromal cell lines, morphologically resembling epithelial cell or fibroblast, and a murine bone marrow stromal cell line, comparing to primary human bone marrow-derived MSCs. An approach based on cell aggregation in methylcellulose-based medium was used. The size of the sphere, the number of cells constituting the sphere, morphology, cell cycle, and gene expression related to hypoxia-induced stress response were examined chronologically. Although it is interesting to know them, honestly, it is not clear to me what model the authors tried to create. A simple model to evaluate sphere-forming capacity of cells or searching cell line exhibiting MSC-like cellularity in terms of sphere formation capacity? It has been reported that in comparison with dissociated cells or cells expanded on adhesion culture, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining or enhancing their differentiation capacity. The reviewer somehow thinks that there is something missing.

Major concern

If the authors are trying to find a cell line exhibiting MSC-like cellularity in terms of sphere formation capacity, a single cell culture may be needed to assess sphere forming capacity. Proof of MSC-like cellularity such as self-renewability, tri-linage differentiation capacity is, of course, necessary.

It is not clear for us what the reviewer means by “MSC-like cellularity in terms of sphere formation capacity?”. Our purpose never aimed to identify/compare stem cell or differentiation capacities of cell lines, but rather to determine whether, like primary hMSCs, they can form spheroids. HS-27a and HS-5 human are transformed cells. What will be the purpose of testing for their self-renewability ability? Both are capable of tri-lineage differentiation in 2D (1. Ischac et al., submitted manuscript Figure 1; 2. Liu et al., 2015; 3. Vallet et al., 2011). We could certainly expect them to do the same in 3D as figure 2, but in modified medium.

[For figures please check Cover Letter file]

1. Figure 1. Differentiation potential of HS-27a and HS-5 cells into osteoblasts, adipocytes, chondrocytes and vascular smooth muscle. Scale bars indicate 100 µm. Images from Ischac et al., submitted manuscript.

2. Liu, B., Wu, S., Han, L., Zhang, C., 2015. Β-Catenin Signaling Induces the Osteoblastogenic Differentiation of Human Pre-Osteoblastic and Bone Marrow Stromal Cells Mainly Through the Upregulation of Osterix Expression. Int. J. Mol. Med. 36, 1572–82. doi:10.3892/ijmm.2015.2382

3. Vallet, S., Pozzi, S., Patel, K., Vaghela, N., Fulciniti, M.T., Veiby, P., Hideshima, T., Santo, L., Cirstea, D., Scadden, D.T., Anderson, K.C., Raje, N., 2011. A Novel Role for CCL3 (MIP-1α) in Myeloma-induced Bone Disease via Osteocalcin Downregulation and Inhibition of Osteoblast Function. Leukemia 25, 1174–81. doi:10.1038/leu.2011.43.

MSC-spheroids are mainly developed in a context of bioengineering, to study impact on survival, differentiation capacity, etc. They were also suggested as in vitro surrogate models for the hematopoietic bone marrow microenvironment. Therefore, using cell lines may help in investigating mechanisms in a simple system whatever is the research domain, bioengineering or hematopoiesis, in the absence of primary hMSCs. Nothing has been previously published on cell lines in 3D, whereas cells lines are often used in 2D to support hematopoiesis, for instance. To our knowledge, all studies were based on MSC-aggregates, using different matrix and methods, and not on single cells. Probably few data such as those from Isern et al. used human single cells in order to study their stem cell capacity, and others on murine cells, possibly. We fit our work on methods reported before.

The authors are very sorry and apologize if the reviewer did not pick up the message and hope that the manuscript is more comprehensible now.

______________________________

Reviewer #3: Although the manuscript is interesting in the comparison of the capacity in the spheroid culture from three types of hMSCs there are some serious problems. The authors should be addressing them.

Special comments

1) It totally is the serious problem that the authors did not examine the difference in the differentiation ability into osteoblasts, adipocytes, and chondrocytes from spheroid with primary or cell line hMSCs in the present experiments because it is an important role of regeneration ability for cellular therapy using hMSCs.

As mentioned above, HS27a and HS5 are known to be able to differentiate into the tri-lineages. We agree that might be worth to test in 3D, but this would require specific media, different than the one we used. In addition, we wondered at which timepoint the reviewer think it will be relevant to perform differentiation. Alternately, based on protocol from the literature, direct differentiation and staining could be performed directly on spheroids (Ref 74 - Cheng et al. 2012), by adding specific growth factors in our medium in order to obtain osteoblasts, chondrocytes and adipocyte differentiation. Before closing our laboratory, due to Covid-19, we were only able to show adipocytic differentiation of the HS-27a cell line (see figure 2). However, we thought that would, first, change the purpose of our main message of our actual manuscript and, second, require to set up much more methods, particularly to get osteogenic and chondrogenic differentiation as for 2D (Figure 1, reviewer#2 concerns). Thus, this would certainly be of interest for a further story.

[For figures please check Cover Letter file]

Figure 2. Adipogenic differentiation of HS-27A cells revealed after 2 weeks by neutral lipid vacuoles stained with Nile Red (Yellow).

2) Materials and Methods: There is no ethics statement (permission number) and preparation and condition of primary hMSCs. Did the authors conducted in compliance with Declaration of Helsinki and get the informed consent from patients?

We agree with the reviewer, the original sentence already mentioned “informed consent” but was not complete. This has been corrected. Please refer to above Journal requirements, concern #2.

Furthermore, did the authors separate and collect the primary hMSCs with stemness makers (CD29, CD44, and CD105 etc.) or not (heterogeneity) in present experiments?

MSCs from BM aspirates were seeded and cultured in current culture conditions with no additional cell staining and sorting. The protocol has been expanded in “material and methods” (page 5, line 86): “Samples from BM aspirates were diluted in MEM Alpha (Life Technologies, Villebon-sur-Yvette, France) and filtered through a cell-strainer prior centrifugation (350 x g, 10 min). Cells were resuspended and seeded at 105 to 2.105 cells/cm2 in MEM Alpha supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (both from Life Technologies) and 1 ng/mL of recombinant human FGF basic (FGF-2, R&D Systems, Abingdon, United Kingdom). Medium was changed twice a week until cells reached confluency”.

3) Results: Data interpretation involved in Results section. The authors should describe them in Discussion section.

We do not understand the concern. Could you please precise which data interpretation? Should we remove some from results or discuss in discussion section? We did not find much interpretation in results and describe mostly everything in the discussion. The discussion has been, however, rewritten to answer the concerns of reviewer 1.

4) Results: The authors stated the primary hMSCs was less optimal for spheroid culture.

It is not clear for us what the reviewer means by that. Less optimal than what? Cell lines? If so, it is not what we wished to state, or we were misunderstood. We seek for a cell line that should replace, somehow, the primary hMSCs with similar properties.

It is well known that the proliferation and maintenance of spheroid culture is dependent on the number of spread cells. Did the authors should examine and confirm the less number of spread cells or diameter less than 300 micrometer without shrinking spheroid in primary hMSCs? Furthermore, the authors should examine whether the smaller spheroid derived from primary hMSCs induced activation of HIF-1alpha and apoptosis or not.

We do not see spreading, except from time to time with HS-5 after 14 days, and were not interested in this aspect. Since this phenomenon is not observed, it does not seem relevant to look at smaller spheroids. Our method is based on a defined number of cells, which provided spheroids with an average diameter ≥ 300 �m, why should we start with lower number of cells to get smaller spheroids? We might not have completely understood the real meanings of this remark.

5) Figure1: Why did the authors use the two types of methylcelluloses? What is the difference, such as components, viscosity, and moisturization etc.? The authors should explain them.

We were looking for commercial methylcelluloses without cytokines that could be further used for hematopoietic cells. StemCell Technologies provides 3 products without cytokines: SF H4236, H4100 and H4230. After checking their composition, and based on other works, we excluded the H4230 because it contains fetal bovine serum (FBS), and we wished to use our own FBS selected for optimal growth of MSCs. SF H4236 has the same base composition as H4100, but also contains BSA, Insulin, transferrin and other unknown components. A sentence has been added in the Material and Methods (page 6, line 101): “Both media contains methylcellulose in IMDM, but SF H4236 is supplemented with bovine serum albumin, recombinant human insulin, human transferrin (iron-saturated), 2-Mercaptoethanol and unknown supplements as described by the manufacturer ”.

6) Figure 2: It confuses the murine or human hMSCs. In Figure2, the authors should add the murine MS-5 data with perimeter and cell number/spheroid, and culture days, but not its supplemental video.

Following the reviewer recommendations, this has been changed. Figure 2 now includes MS-5 data.

7) Discussion: The authors explain the induction of autophagy in primary hMSCs. However, the authors did not check it. The authors should check the expression of autophagy related molecules, such as LCII etc.

We agree with the reviewer and modified figures and text. As mentioned above (reviewer 1, concern #4) TEM images with higher magnification are chronologically shown in S2 fig. This figure shows autophagosome formation only in primary hMSC-spheroids, as soon as day1. We also stained HS-27a-spheroids for LC3B, as recommended by the reviewer, and found, although triggered, only diffuse cytoplasmic expression that do not reflect autophagosome formation as shown in S3 fig. LC3B detected by IHC, still seems under debate. This was underlined now in the “Electronic microscopy” chapter as well as in the discussion (page 22, line 422): “…diffuse LC3B staining may hamper the interpretation in IHC, while a dot-like staining patterns is more indicative of autophagy [41]. Dots may also reflect the accumulation of autophagosomes due to induction of autophagy, or due to inhibition of autophagy resulting from a lack of autophagosome degradation upon fusion with lysosomes [72]”.

Minor comments

8) Results p10, line 204: It confuses the primary or immortalization hMSCs. The authors should add the word primary MSCs.

We agree with the reviewer and changed the sentence for (page 12, line 207): “The spheroid-forming capacity was followed for two human cell lines, HS-27a and HS-5, and compared to that of primary hMSCs. The two cell lines have been obtained by immortalization of hMSCs from the same BM sample with the papilloma virus E6/E7 genes”.

9) Discussion p17, line 352: Was it inserted the reference, not referenced number?

We thank the reviewer for drawing our attention on this error. This has been changed.

10) Fig3C and Fig.5 were low density images, then it is difficult to interpret them.

We understand but the original has better quality. Due to the limited size required by the journal, we had to make it as low density. We now move fig 3C to supplemental data with its original size and intend to keep a better quality for fig 5.

Decision Letter 1

Atsushi Asakura

18 May 2020

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

PONE-D-19-30255R1

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Acceptance letter

Atsushi Asakura

22 May 2020

PONE-D-19-30255R1

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

Dear Dr. Mazurier:

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If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Spheroids formation of mouse MS-5 cell line.

    (A and B) Scanning electron microscopy (SEM) analysis over 14 days (scale bars = 100 μm (A) and 20 μm (B). (C) Sub-G1 apoptosis quantification (n = 3) and (D) cell cycle quantification over 21 days in culture (n = 3; data are mean ± SD).

    (TIF)

    S2 Fig. Transmission electron microscopy (TEM) observation of MSC-spheroids.

    TEM analysis of primary hMSC-spheroids at day 1 (A), day 3 (B) and day 7 (C); Higher magnification is also shown to highlight autophagosomes. HS-27a-spheroids at day 1 (D), day 3 (E) and day 7 (F); HS-5-spheroids at day 1 (G), day 3 (H) and day 7 (I) and MS-5-spheroids at day 1 (J), day 3 (K) and day 7 (L). Scale bars = 20 μm.

    (PPTX)

    S3 Fig. LC3B expression in HS-27a-spheroids.

    Immunohistochemistry of LC3B is shown at days 1, 3 and 7 for HS-27a-spheroids (scale bars = 50 μm).

    (TIF)

    S1 Video. A representative time-lapse video of spheroid formation.

    30 000 primary MSCs seeded into U-bottomed 96-well, in medium containing 0.5% of methylcellulose (MethocultTM SF H4236) were followed via a Nikon Eclipse TI-S microscope for 24 hours.

    (MP4)

    S2 Video. A representative time-lapse video of spheroid formation.

    30 000 HS-27a cells seeded into U-bottomed 96-well, in medium containing 0.5% of methylcellulose (MethocultTM SF H4236) were followed via a Nikon Eclipse TI-S microscope for 24 hours.

    (MP4)

    S3 Video. A representative time-lapse video of spheroid formation.

    30,000 HS-5 cells seeded into U-bottomed 96-well, in medium containing 0.5% of methylcellulose (MethocultTM SF H4236) were followed via a Nikon Eclipse TI-S microscope for 24 hours.

    (MP4)

    S4 Video. A representative time-lapse video of spheroid formation.

    30,000 MS-5 cells seeded into U-bottomed 96-well, in medium containing 0.5% of methylcellulose (MethocultTM SF H4236) were followed via a Nikon Eclipse TI-S microscope for 24 hours.

    (MP4)

    S1 Table. List of primers and probes sequences.

    (DOCX)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.


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