Scalable Generation of Mesenchymal Stem Cells from Human Embryonic Stem Cells in 3D

Abstract

Human embryonic stem cell (hESC) derived mesenchymal stem cells (EMSC) are efficacious in treating a series of autoimmune, inflammatory, and degenerative diseases in animal models. However, all the EMSC derivation methods reported so far rely on two-dimensional (2D) culture systems, which are inefficient, costive and difficult for large-scale production. HESC, as an unlimited source, can be successively propagated in spheroids. Here, we demonstrate that hESC spheroids can directly differentiate into MSC spheroids (EMSC_Sp) within 20 days in one vessel without passaging and the system is scalable to any desired size. EMSC_Sp can further differentiate into osteocytes and chondrocytes in spheres or demineralized bone matrix. EMSC_Sp also retains immune-modulatory effects in vitro and therapeutic effects on two mouse models of colitis after dissociation. Compared to EMSC differentiated in monolayer, EMSC_Sp-derived cells have faster proliferation and higher yield and develop less apoptosis and slower senescence. Thus, the 3D differentiation system allows simple, cost-effective, and scalable production of high-quality EMSC and subsequently bone and cartilage tissues for therapeutic application.

Introduction

Mesenchymal stem cells (MSC) are a unique stem cell type with not only multipotency as other somatic stem cells, but also the capability of immunomodulation and migration, and low immunogenicity. They have been used in clinical trials for the treatment of many degenerative, autoimmune, and inflammatory diseases ¹. MSC can be isolated from a variety of fetal and adult tissues such as amniotic membrane, amniotic fluid, placenta, umbilical cord, umbilical blood, dental pulp, adipose, and bone marrow. However, MSC isolated from these somatic tissues are subject to great variations of the source tissues, limitation of donors, and risk of disease transmission from donors.

In contrast, human pluripotent stem cells (hPSC) including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are unlimited and stable sources with the potential to differentiate into any cell type in the body. MSC have been previously derived from hESC via co-culturing with mouse OP9 cells, sorting, scraping or handpicking of cells ^2-7. However, these methods are limited by low MSC yield and purity. We have recently reported two methods for MSC derivation from hESC (EMSC). The first uses a multi-step protocol including embryoid body formation followed by hemangioblast enrichment ⁸. Whereas, the second uses a simplified protocol with one intermediate step of trophoblast differentiation followed by multiple rounds of passaging in MSC medium ⁹. Like somatic tissue-derived MSC, EMSC as well as iPSC-derived MSC also have immunomodulatory effect in vitro and can reduce the severity of an increasing list of degenerative, autoimmune, and inflammatory diseases in animal models such as osteochondral defects ⁵, allergic airway inflammation ¹⁰, lupus nephritis, uveitis ¹¹, multiple sclerosis ^{8, 9}, and inflammatory bowel disease ⁹. Following intra-femoral transplantation into immunocompromised mice, EMSC can engraft and support hematopoiesis ¹². However, the above methods are all in two-dimensional (2D) systems, which demand tedious cell maintenance, passaging, and large culture spaces and supplies while having low EMSC yield.

It has been known that 3D spheroid formation grants somatic tissue-derived MSC with enhanced paracrine secretion of angiogenic, anti-tumorigenic, and pro- and anti-inflammatory factors, improved cell survival, increased differentiation potentials, and delayed replicative senescence ¹³. Genes involved in hypoxia ¹⁴, apoptosis ^{15, 16}, inflammation ^{15, 17}, and mechanophysical changes ^{18, 19} are differentially expressed in MSC cultured in 2D versus 3D. Based on these studies, we recently demonstrated that spheroid formation allows hESC and MSC to tolerate ambient conditions for up 4 and 10 days, respectively, without loss of viability and functions including the efficacy of the spheropreserved MSC on mouse colitis models ²⁰. This method may replace cryopreservation to ship cells worldwide.

We have previously demonstrated that hESC-formed spheroids (hESC_Sp) can be passaged in 3D culture with sustained pluripotency, normal karyotype, and capability to efficiently differentiate to functional cardiac spheres ^{21, 22}. In this study, we further show that hESC_Sp could directly differentiate into MSC in spheroids (EMSC_Sp). Unlike the 2D differentiation systems, our method allows all the multi-step differentiation procedures to be completed in one vessel in 3D without a need for cell passaging. EMSC_Sp could further differentiate to osteocytes, chondrocytes, and adipocytes all in spheroids and in demineralized bone matrix. EMSC_Sp could also be reattached and passaged in monolayer (EMSC_Sp-ML) without compromising their multipotency. EMSC_Sp-ML retained immune-modulatory effects in vitro and therapeutic effects on mouse colitis models. Compared to EMSC without spheroid formation, EMSC_Sp-ML had faster proliferation and underwent less apoptosis and slower senescence.

Results

EMSC_Sp generation and characterization

H9 and CT3 hESC were initially cultured in monolayer in the mTeSR1 medium (Fig. 1A and 1B). To generate hESC_Sp, we dislodged hESC colonies with dispase and passed cell clusters through a mesh (Fig. 1C) to form hESC_Sp (Fig. 1D), which were cultured in suspension and passaged via spheroid splitting as we reported recently ²¹. Immunostaining and flow cytometric analysis confirmed that hESC_Sp retained pluripotency markers (Figs. S1A and S1B). Measured with ImageJ, the size of hESC_Sp was 108.2 ± 22.0 μm in diameter on day one after spheroid passaging (Fig. S1C). On day 2, hESC_Sp were treated with 10-ng/ml BMP4 and 1-μM A83-01 in mTeSR1 Minus Select Factors to initiate trophoblast-like (TB) differentiation in spheres. At day 3 of the treatment, the expression of several typical trophoblast-associated genes remarkably increased in the cells differentiating in spheres (TB_Sp/d3) as well as in monolayer (TB_ML/d3), indicating that the spheroid differentiation was also through a TB stage (Figs. S2A and S2B) as we reported before on hESC differentiation in monolayer ⁹. On day 5, the consumed medium was replaced with MSC medium, followed by continuous culture for 15 days (Fig. 1E). To further determine the nature of the differentiated spheroids, we dissociated the spheroids, re-plated the single cells into a flask, and cultured them in 2D monolayer (EMSC_Sp-ML), which demonstrated spindle-like phenotype similar to that of typical MSC (Fig. 1F).

Figure 1.

Derivation of EMSC_Sp from hESC_Sp (A-D) At day 1, H9 hESC colonies (A) were dislodged and passed through a 50-μm strainer using mechanical pressure (B) to generate hESC_Sp of similar sizes (C). At day 2, the spheroid sizes were measured using ImageJ (D). (B) Scale bar is 100 μm. (D) Scale bar is 400 μm. (E) hESC_Sp were treated with 10-ng/ml BMP4 and 1-μM A83-01 in mTeSR1 Minus Select Factors for 3 days to induce differentiation into trophoblast-like cells also in spheroids. Scale bar is 400 μm. (F) The spent medium was replaced with the MSC medium to culture the trophoblast spheroids for 15 days with the medium refreshed every 5 days to form MSC_Sp, which were then dissociated and attached as a monolayer in a new dish. The resultant cells are termed EMSC_Sp-ML. Scale bar is 100 μm.

To track the multi-step differentiation, we analyzed cell surface markers TROP2 for trophoblast-like cells and CD73 for MSC on cells dissociated from spheroids at days 0, 5, 10, and 20 of the differentiation. On day 0, hESC (99.1%) were negative for either TROP2 or CD73. By day 5 of the differentiation, 98.2% of the cells became positive for TROP2. On day 10, 99.4% of the cells turned out positive for both markers. By day 20, 97.9% of the cells were positive for only CD73 (Fig. 2A). These results indicate a transition from hESC to trophoblast-like cells and then to MSC all in 3D, similar to that in 2D observed in our previous study ⁹. Immunostaining of the day-20 spheroids shows that cells in the spheroids expressed typical MSC markers CD44, CD105, and CD90, but negative for the apoptotic marker activated caspase-3 (Fig. 2B), indicating that the 3D method allows generation of EMSC_Sp without noticeable apoptosis.

Figure 2.

Characterization of EMSC_Sp and EMSC_Sp-ML (A) Stepwise differentiation from H9 hESC to trophoblasts and then MSC (EMSC_Sp) all in spheroids, reflected by transitional occurrence of markers of trophoblasts (TROP2) and MSC (CD73) via flow cytometry analysis. Blue dots stand for isotype control and red for tested markers. (B) Frozen sections of EMSC_Sp were immunostained positive for MSC markers CD90, CD105, and CD44, and negative for the apoptotic marker active caspase-3. Scale bars: 100 μm. (C) EMSC_Sp-ML were dissociated and quantified for the percentage of cells positive for four MSC markers and a mixture of negative control markers. Blue peaks stand for isotype control and red for tested markers. (D) Tri-lineage differentiation of EMSC_Sp-ML into chondrocytes, osteocytes, and adipocytes. Scale bars: 100 μm.

Flow cytometry demonstrates that EMSC_Sp-ML also expressed CD44, CD73, CD90, and CD105, but negative for a cocktail of hemangioblast and hematopoietic markers (Fig. 2C). Similar results were observed with EMSC_Sp-ML derived from three other hESC lines (CT2, CT3, and Envy) and one iPSC line derived from human peripheral blood mononucleocytes (PBY4) ²³. Human bone marrow MSC (BMSC) from two donors were tested as positive controls (Table S1). The ratio of CD105⁺ cells was lower among the cells that differentiated from PBY4 than that from hESC, which may be associated with different origins or epigenetic memory ²⁴. EMSC_Sp-ML also possessed the capability of differentiation into the three lineages of chondro-, osteo-, and adipocytes (Fig. 2D). These results suggest that EMSC_Sp and the subsequently derived EMSC_Sp-ML both possess typical features of MSC. In addition, G banding analysis shows that EMSC_Sp-ML derived from H9 and CT3 hESC lines both maintained normal keryotypes after prolonged culture (at p5 and p6, respectively) (Figs. S2C and S2D).

Scale-up production of EMSC_Sp and further differentiation in the demineralized bone matrix

Given the potential value of EMSC_Sp in clinical application, it is important to determine if our new method allows scale-up production of EMSC_Sp. Using a 100-ml plastic bag with an input for medium change and a filter for air exchange, we generated (7.0 ± 0.5) x 10⁷ EMSC in spheres at (7.0 ± 0.3) x 10⁵ cells/ml) from 3 x 10⁶ hESC within 20 days (Fig. 3A and 3B). In contrast, using 21 T75 flasks we obtained (4.2 ± 0.3) x 10⁷ EMSC in 2D monolayer (EMSC_ML), which took 5 passages and 39 days and consumed 555-ml MSC medium (Fig. 3B). Thus, compared to the 2D method, the 3D method produced 67% more EMSC, using half amount of medium in almost halftime in a single container without cell passaging. Moreover, following medium change EMSC_Sp in the same vessel could directly differentiate into spheroids of chondrocytes or adipocytes in 20 days, depending on the differentiation medium used. Staining shows that chondrocyte spheroids were positive for Alcian Blue, and adipocyte spheroids positive for Oil Red O with clear oil droplets (Fig. 3C).

Figure 3.

Large-scale production of EMSC_Sp and continuous differentiation into chondrocytes and adipocytes all in spheroids. (A) Large-scale production of EMSC_Sp in a 100-ml plastic bag and EMSC_ML in multiple T75 flasks. Both were from 3 x 10⁶ CT3 hESC. (B) Comparison of EMSC_Sp and EMSC_ML derivation for the time of production, cell yield (mean ± SE, n = 4), and consumption of culture supplies. *Medium change every 5 days. (C) EMSC_Sp were directed to differentiate into chondrocytes and adipocytes both in spheroids. Scale bars: 50 μm. (D) Sections of DBM alone or recellularized with EMSC were subjected to H&E staining. (E) Osteo- and chondrocyte differentiation of EMSC in spheres and single cells (at 2:1 in cell number ratio) loaded in two neighboring pieces of DBM. (F) H&E, Alizarin Red S, and Alcian Blue staining of the EMSC-loaded DBM cultured in the osteo- and chondrogenic differentiation media for 20 days. Scale bars: 100 μm.

Demineralized bone matrix (DBM) is a widely accepted scaffold for bone and cartilage repair in clinic. Low attachment and viability of single cells filled into scaffolds are key problems that affect the outcomes of the cell-filled scaffolds in tissue engineering. Since spheroid formation of MSC enhances the cell viability and preserve the cell functions ²⁰, we decided to fill DBM with EMSC_Sp and dissociated EMSC to enhance the cell survival and differentiation in the scaffold. We found that DBM could be recellularized with EMSC (Fig. 3D). Two cubes of DBM were aligned side by side, rinsed in osteo- or chondrogenic differentiation medium, covered with a mixture of EMSC_Sp and dissociated EMSC at a ratio of 2:1 in cell number (Fig. 3E), and cultured in a 37^oC incubator for 20 days.

Both DBM cubes in each medium gradually fused with each other and became a whole piece of hard tissue (Fig. 3E). H&E staining shows that DBM alone contained lots of empty spaces, whereas the spaces inside EMSC-loaded DBM were filled with cells. Lineage-specific staining indicates that the cells in the spaces of the DBM cultured in the osteo- and chondrogenic differentiation media were positive for Alizarin Red S and Alician Blue, respectively. The Alcian Blue-stained cells were mostly aligned on the surface of the DBM (Fig. 3F). Together these data suggest that EMSC_Sp can differentiate into the skeletal tissues in DBM, which might be implanted for repair of bone fracture and cartilage injury.

Immune-responsive and -modulatory effects of EMSC_Sp-ML in comparison with authentic MSC from bone marrow

To further verify the therapeutic values of EMSC_Sp-ML, we tested the response of EMSC_Sp-ML to the inflammatory cytokine IFNγ, in comparison with BMSC cultured in monolayer (BMSC_ML) as a positive control. Following IFNγ treatment for 24 h, a series of anti- and pro-inflammatory genes including IDO, PD-L1, CCL2, and CXCL-10 were up-regulated in EMSC_ML, EMSC_Sp-ML and BMSC_ML compared to the untreated controls, whereas expression of the inflammatory cytokines IL6 changed little and IL8 decreased in all the three IFNγ treated samples (Fig. 4A).

Figure 4.

Immune-responsive and -suppressive effects of EMSC_Sp-ML(A) EMSC_ML and EMSC_Sp-ML and BMSC_ML, pre-treated with or without 20 ng/ml IFNγ for 24 h, were subjected to RT-PCR assay for inflammation-related genes. GAPDH was detected for internal control. A mixture of all the samples was processed for the assay without reverse transcriptase to serve as a negative control (NC). (B and C) IFNγ-treated EMSC_Sp-ML as well as BMSC_ML were mixed at the cell ratio of 1:40 or 1:80 with CFSE-labeled mouse lymph node lymphocytes (2×10⁵ cells/well) in a 96-well plate. The lymphocytes in the mixture were stimulated with anti-CD3 and -CD28 antibodies for 5 days. Lymphocyte proliferation was assayed via flow cytometry for CSFE-diluted cells (hollow peaks) against original CSFE-stained cells (the solid dark peak) as displayed in the histograms (C). The percentages of inhibition are shown displayed in D as mean ± SE (n = 3) (D) Correlation coefficients for comparisons of EMSC_ML and EMSC_Sp-ML with BMSC_ML based on microarray assay.

Furthermore, the IFNγ-treated EMSC_Sp-ML and BMSC_ML cells dose-dependently inhibited proliferation of lymphocytes isolated from mouse lymph nodes and stimulated by antibodies against CD3 and CD28 (Fig. 4B). The percentages of inhibition are shown in Fig. 4C. These data suggest that EMSC_Sp-ML, like BMSC_ML, cells can respond to the inflammatory cytokine and repress lymphocyte proliferation.

Microarray assays show that the global gene expression profiles of both EMSC_ML (which had not experienced spheroid formation) and EMSC_Sp-ML are similar to that of BMSC_ML with the correlation coefficient as 0.89 and 0.93, respectively (Fig. 4D). The fact that EMSC_Sp-ML are more similar to BMSC_ML than EMSC_ML indicates that derivation via spheroidal formation may make EMSC to resemble the authentic BMSC more than derivation in monolayer culture.

Efficacy of EMSC_Sp-ML on colitis model in mice

Given the immune responsive and modulatory effects of EMSC_Sp-ML cells, we decided to test whether the cells can treat mouse models of inflammatory colitis induced by DSS or TNBS. DSS involves immune responses driven by both Th1 and Th2, whereas TNBS involves only Th1-driven immune response ²⁵. DSS addition into the drinking water for 7 days caused dramatic decline of the body weight of C57BL/6 mice within 10 days or so. EMSC_Sp-ML treated with IFNγ, re-suspended in PBS at 1×10⁶ cells/100 μl, and injected i.p. at days 1 and 2 post-DSS treatment reduced the loss of the body weight, compared to the control group that had been exposed to DSS water and injected with PBS (Fig. 5A). The length of the intestine was shortened the most in the DSS/PBS mice but moderately in the DSS/EMSC_Sp-ML mice, compared to the untreated mice (Figs. 5B and S3A). Histological analysis also supports the efficacy of EMSC_Sp-ML as the intestinal epithelium was less disrupted in the DSS/EMSC_Sp-ML group than the DSS/PBS group (Figs. 5C and S3C).

Figure 5.

Alleviation of DSS-induced mouse colitis by IFNγ-treated EMSC_Sp-ML(A) Colitis was induced with DSS in C57BL/6 mice as described in Methods. IFNγ-treated EMSC_Sp-ML cells were injected i.p. on days 1 and 2 of the experiment. Body weight (mean ± SE) change of the mice was monitored everyday on days 0 through 15. The mice were sacrificed on day 15 and their colons were isolated. Control, n = 4; DSS+PBS, n = 6; DSS+EMSC_Sp-ML, n = 7. (B and C) The colon length (B) and histological scores (C) were measured on day 15. Data were analyzed via Student's t-test and presented as mean ± SE. (D) Colitis was induced with TNBS in C57BL/6 mice as described in Methods. IFNγ-treated EMSC_Sp-ML cells were injected i.p. on days 0 and 1 of the experiment. Body weight (mean ± SE) change of the mice was monitored every day on days 0 through 5. The mice were sacrificed on day 5 and their colons were isolated. Control, n = 4; TNBS+PBS, n = 7; TNBS+EMSC, n = 7. (E and F) The colon length (E) and histological scores (F) were measured on day 5. Data were analyzed via Student's t-test and presented as mean ± SE.

For the TNBS-induced colitis model, TNBS was administered into the anus at 2 mg/mouse twice at days 0 and 1. EMSC_Sp-ML cells were injected i.p. at 1x10⁶ cells/100 μl/mouse also twice on days 0 and 1, respectively. These cells had even more obvious efficacy to prevent body weight loss than they did in the DSS-induced colitis model (Fig. 5D). Although EMSC_Sp-ML reduced colon length shortening in the TNBS-treated mice (Figs. 5E and S3B), it is much less obvious than in DSS-treated mice. EMSC_Sp-ML also reduced colon histology scores in the TNBS-treated mice compared to the vehicle control (Figs. 5F and S3D). These data together suggest that EMSC_Sp-ML have therapeutic effects on both colitis models.

Differential gene expression profiles in 2D- and 3D-derived EMSC

To elucidate differences between 2D- and 3D-derived EMSC at the molecular level, we analyzed the profiles of global gene expression using microarray in EMSC_ML and EMSC_Sp-ML at early (p3) and late (p8) passages. We also tested the response of the p3 EMSC samples to IFNγ treatment. First, we determined the quality of the dataset by comparing the probe intensity of all genes between the samples. As expected, the overall gene expression profiles of all the EMSC samples (no matter whether they were from 2D or 3D, and p3 or p8) are quite similar for comparisons between each other (R² ≥ 0.8), whereas their profiles are very different from that of the parental hESC sample (R² = 0.68-0.71) (Fig. S4A).

Next, the microarray dataset was normalized via Quantile Adjustment method, and filtered to remove probes with detection P value > 0.01 in at least 50% of the samples ²⁶. The rest of the data are shown in a heat-map to display expression of about 17,000 genes in each sample (Fig. S4B). Then, we compared the gene expression profiles of the samples in volcano plots. 190 (p3) and 115 (p8) genes were expressed 2-fold or higher, and 168 (p3) and 123 (p8) genes expressed 1/2-fold or lower in EMSC_Sp-ML compared to EMSC_ML (P < 0.001) (Figs. S4C and S4D).

These genes were analyzed based on gene ontology using DAVID bioinformatics resources. First, pluripotency genes such as POU5F1 (OCT4), SOX2, and NANOG were down-regulated, and MSC marker genes including CD44, CD73, CD90, and CD105 up-regulated in all the MSC samples, which further increased at the later passage (Fig. 6A).

Figure 6.

Transcriptomic analysis of EMSC_ML and EMSC_Sp-MLHeat-maps are presented for differentially expressed genes associated to pluripotency and MSC (A), proliferation (B), inflammation (C), apoptosis and senescence (D).

In addition, many of the differentially expressed genes are related to cell proliferation, inflammation, apoptosis, and senescence (Figs. 6B-D). For example, genes that positively regulate cell proliferation including CENPO, CEP152, CEP55, etc., were expressed higher in EMSC_Sp-ML than EMSC_ML, whereas genes that repress cell proliferation including CDK6, CAV2, and GADD45 expressed lower in EMSC_Sp-ML than EMSC_ML, which is more obvious at p8 than p3 (Fig. 6B). These data are consistent with the cell proliferation data in Figs. 3A-B. On the other hand, pro-inflammatory genes, e.g., CD72 and CD46 were expressed higher in EMSC_ML (p8) than EMSC_ML (p3) and EMSC_Sp-ML (both p3 and p8), and anti-inflammatory genes, e.g., TSG6, PTGS2, etc., expressed higher in EMSC_Sp-ML (p3) than EMSC_ML (p3) and in MSCs (either ML or Sp-ML) at p3 than p8 (Fig. 6C). Moreover, genes that promote apoptosis and senescence such as BAX, DAPK2, CDKN2A, etc., were expressed lower in EMSC_Sp-ML than EMSC_ML. In contrast, genes that repress apoptosis and senescence such as BCL2, AVEN, STAT4, etc., were expressed higher in EMSC_Sp-ML than EMSC_ML. Together, these results suggest that EMSC_Sp-ML, like MSC in spheroids derived from somatic issues ¹³, express higher levels of proliferative genes, and lower levels of inflammatory, apoptotic, and senescent genes than EMSC_ML, which is more obvious at the early stage than the late stage.

Upon IFNγ treatment, pro-inflammatory genes such as IL6, CXCL10, CXCL11, etc., were highly expressed in IFNγ-treated EMSC_ML but not EMSC_Sp-ML, whereas anti-inflammatory genes such as IDO1, IL10RB, and IFNγ-inducible genes (IFITM1, IFI35, IFI6, and IFIT2) were highly expressed in both IFNγ-treated EMSC_ML and EMSC_Sp-ML (Fig. 6C). These gene expression changes suggest that both EMSC_ML and EMSC_Sp-ML can respond to IFNγ, a feature important for their immune-modulatory and therapeutic effects. Together, the global gene expression profile of EMSC_Sp-ML is generally consistent with the features identified for MSC spheroids derived from other sources ^13-19.

EMSC_Sp-ML have higher proliferation, less apoptosis, and delayed senescence than EMSC_ML

To obtain cellular evidence for the differences in the gene expression profile between the 2D- and 3D-derived EMSC, we compared the cells for some biological functions below. First, we monitored the proliferation of both EMSC_ML and EMSC_Sp-ML by tracking their confluence after being seeded in a 6-well plate in the IncuCyte Live-Cell Analysis System. As shown in Fig. 7A, while all groups starting from similar confluence at around 2.5%, p8 and p10 EMSC_Sp-ML reached 38.5% and 20.4% confluence, respectively, after being cultured for 110 h. In contrast, p8 and p9 EMSC_ML only reached 5.9% and 9.3% confluence, respectively, within the same period of time. This indicates that EMSC_Sp-ML proliferate faster than EMSC_ML, which was supported by the recording of cell numbers during the first five passages (derived from two hESC lines H9 and CT3). Higher cell numbers (about 50-100 fold) were obtained from EMSC_Sp-ML than EMSC_ML (Fig. 7B).

Figure 7.

Comparison of proliferation, apoptosis, and senescence between EMSC_Sp-ML and EMSC_ML (A) EMSC_ML and EMSC_Sp-ML at various passages were seeded at 3×10⁴/well in a 12-well plate. Percentage of cell confluency in each well was monitored in the IncuCyte ZOOM Live-cell Analysis System. Results are presented as mean ± SD (n = 3). (B) EMSC_ML and EMSC_Sp-ML from two lines of hESC (H9 and CT3) were cultured for five consecutive passages and accumulated cell number was counted at each passage. (C) The concentrations of dissociated EMSC_ML and EMSC_Sp-ML were adjusted to 1×10⁶ cells/ml. An Eppendorf tube containing 1 ml of the cell suspension was stored at 4^oC for 24 h, and the cells were then subjected to flow cytometry analysis with Annexin V staining for apoptotic cells and PI staining for dead cells. The ratios of live and dead cells in the same samples from (C) was indicated in the histogram. Results are displayed as mean ± SD (n = 3). *P < 0.05 for comparison of EMSC_Sp-ML with EMSC_ML. (D) EMSC_ML and EMSC_Sp-ML were cultured at 37^oC in a 12-well plate until about 50% confluency and stained for β-gal positive cells (dark blue). Cell nuclei were counterstained with DAPI (light blue). Results are displayed as mean ± SD (n = 3) in a bar chart, and representative images are shown beside. *P < 0.05. **P < 0.01. (E) Schematic to accommodate all the findings in this study.

Since refrigeration is often used to protect viability of stored cells, we dissociated EMSC_ML and EMSC_Sp-ML and stored them at 4^oC for 24 h followed by analysis of apoptosis. A higher ratio of apoptotic cells (about 2-fold) was found in culture of EMSC_ML (10.2%) than EMSC_Sp-ML (5.1%), based on staining for annexin V and propidium iodide (Fig. 7C). Again, higher viability was found in EMSC_Sp-ML than EMSC_ML. In addition, we tested cell senescence using staining for senescence-associated (SA) β-gal and found that up to 37.5% and 50.4% EMSC_ML were SA-β-gal⁺ at p8 and p9, respectively, where only 25.9% and 27.0% EMSC_Sp-ML were SA-β-gal⁺ at p8 and p10, respectively (Fig.7D). These results suggest that EMSC_Sp-ML have higher proliferation, less apoptosis, and delayed senescence than EMSC_ML, consistent with the gene expression profiles revealed via the microarray analysis (Fig. 6).

Discussion

Human MSC are a highly promising therapy for degenerative, inflammatory, and autoimmune diseases, which have been in preclinical studies and clinical trials worldwide. However, the great demand for a large number of MSC in clinic has proved to be a top challenge due to the limited number of MSC isolated from donated tissues and expanded in 2D monolayer cultures ²⁷. As alternative sources, hESC and iPSC can generate MSC theoretically for an unlimited time. Recently, substantial progress has been achieved to directly differentiate hESC spheroids into 3D structures such as cardiomyocytes ²¹, neural progenitors and hepatocytes ^{28, 29}. Without the need for culture splits (which are associated with repeat cell detachment, dissociation, and reattachment), these 3D direct differentiation methods have fewer cell loss, save space, time, supplies, and labor, thus having great advantages over 2D differentiation systems and favoring large-scale production of the therapeutic cells in a bioreactor. Although micro-beads, scaffold, and matrix have been used to generate MSC spheroids from adherent MSC culture ^30-32, they demand trypsin treatment and cell dissociation. It has not yet been reported whether hESC spheroids can be directly differentiated into MSC spheroids, i.e., EMSC_Sp.

In this study, we derived EMSC completely in 3D with much higher efficiency and quality than EMSC derived in 2D, and show that EMSC_Sp could further differentiate into spheroids of chondrocytes and adipocytes in 3D in the same vessel. In addition, our method for EMSC_Sp generation does not require enzymatic treatment and culture split, thus it remarkably reduces cell damage and loss, and saves labor of experimenters. It also saves reagents and other culture consumables and enhances the differentiation efficiency and MSC yield. Moreover, we have shown that up to 70-million EMSC in spheroids could be generated in a 100-ml plastic bag. EMSC_Sp loaded into DBM differentiated into osteocytes or chondrocyte in the matrix, depending on the corresponding differentiation medium (Fig. 3).

Following re-plating of EMSC_Sp, the resultant EMSC_Sp-ML retained typical phenotypes, cell surface markers, and tri-lineage differentiation capability of MSCs. Interestingly, the spherical culture history enabled EMSC_Sp-ML to retain some unique features including faster proliferation, less apoptosis, and slower senescence than EMSC_ML. Consistently, gene expression profiles show that EMSC_Sp-ML had higher expression of pro-proliferative, anti-inflammatory, anti-apoptotic, and anti-senescent genes, and lower expression of pro-apoptotic, and pro-senescent genes than EMSC_ML. These features are also similar to those of MSC spheroids derived from somatic tissues ^{17, 33, 34}, which can be retained even after 10 passages in monolayer culture ¹⁶.

It has been known that IFNγ-treated BMSC can enhance immunosuppressive factors, which then repress T cell activation, differentiation, and functions, and also have increased migratory and reparative capacities, which may aid tissue repair and prolong allograft survival ³⁵. We also found that IFNγ-treated EMSC_Sp-ML inhibited lymphocyte proliferation and reduced the severity of DSS-induced colitis in mice, suggesting that EMSC_Sp-ML are therapeutically effective similar to EMSC_ML ³⁶ and MSC from other sources ¹.

In spite of the remarkable functional changes in MSC following spherical culture, underlying molecular mechanisms and signaling pathways that trigger and sustain these changes are largely unknown. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. It has been known that the self-aggregation process of BMSC initiates caspase-dependent IL1 autocrine signaling. IL1 then stimulates expression of early growth response gene-2 (EGR2), a zinc finger transcription factor that promotes synthesis of PGE2 an inhibitor of innate immunity via up-regulation of PTGS2/COX2 in MSCs ^{34, 35}. The IL1 autocrine signaling subsequently induces expression of chemokine receptors, such as CXCR4, or immunomodulatory mediators, such as TNFAIP6/TSG6 and IL6 ^{18, 20}. Although EGR2 was expressed low in all the MSC samples (data not shown), we found that transcripts for TGS6 and PTGS2 two anti-inflammatory genes were higher in EMSC_Sp-ML (p3) and EMSC_ML (p8), respectively, than the corresponding EMSC_ML samples (Fig. 6C), indicating that the IL1 autocrine signaling might be more active in EMSC_Sp-ML than EMSC_ML. Nevertheless, further investigations are warranted to illustrate the molecular mechanisms for how EMSC_Sp retain featured MSC capacity while gaining properties in cell functionalities different from EMSC_ML.

In summary, we have established a simple 3D method to efficiently differentiate hESC into MSC and then into chondrocytes and adipocytes, all in spheroids and in a single vessel. EMSC_Sp can recellularize DBM and differentiate in situ into osteocytes and chondrocytes. Following re-plating of EMSC_Sp, the resultant EMSC_Sp-ML share similarities in phenotypes and functionalities with BMSC, while possessing higher growth speed, lower apoptosis, and slower senescence than EMSC_ML (Fig. 7E). Since EMSC_Sp can be stored under ambient conditions for up to 10 days ²⁰, EMSC_Sp generated using our method at any scale can be shipped worldwide without cryopreservation for research and therapeutic applications.

Methods

hESC culture and MSC generation from hESC spheroids and human bone marrow

All the hESC lines H9 ³⁷, CT2, CT3 ³⁸, and Envy (GFP⁺) derived from hES3 ³⁹ used in this study had been registered with the National Institutes of Health, USA. For all these registered hESC lines, informed consents were taken from all the donors of leftover early-stage human embryos. hESC were cultured on Matrigel in E8 medium (StemCell Technologies) ⁴⁰. The PBY4 iPSC line was derived from donated human peripheral blood via Millipore ²³. For EMSC_Sp-ML generation, we first made hESC spheroids of homogenous sizes as described ^{21, 22}. Briefly, typical hESC colonies were digested for 5-10 min with 1 mg/ml dispase (StemCell Technologies). Once hESC colonies began to roll up, dispase was removed and cells were rinsed with Ca²- and Mg²-free PBS twice. The colonies were gently washed off the plate with mTeSR1 and collected with a 5-ml pipette. We then cut the colonies by passing them through a strainer with pore size at 50 μm in diameter (CellTrics) under pipette pressure to generate cell clusters of about 50-μm. The clusters were collected, re-suspended in 3 ml/well pre-warmed mTeSR1 medium (StemCell Technologies) supplemented with 10-μM Y-27632 (Sigma) to inhibit apoptosis, and cultured in suspension on Petri dish (Corning).

On day 2, hESC spheroids were treated with 10-ng/ml BMP4 and 1-μM A83-01 in mTeSR1 Minus Select Factors (StemCell Technologies) to induce differentiation into trophoblast-like cells, similar as we derived MSC from hESC cultured in monolayer via trophoblast differentiation in 2D ⁹. On day 5, the spent medium was removed and replaced with the MSC medium, i.e., MEM basal medium supplemented with 20% FBS, 1% NEAA, and 1% L-Glutamine. The culture medium was refreshed with the MSC medium every 5 days. Initial analyses were conducted on cell spheroids collected at various days during the differentiation. For continuous analyses, some spheroids were dissociated at day 20 with 0.25% trypsin for 5 min. and then plated into a T75 flask for expansion and passaging in 2D in the MSC medium.

BMSC were derived from bone marrow aspirates donated by two healthy male adults under the approval of the University of Macau Panel of Research Ethics #APP004-FHS. The aspirates were washed with PBS twice followed by isolation of the mononucleocyte (MNC) fraction via centrifugation over Ficoll (Sigma) at 800 RCF. BMSCs were plated in T75 flasks in the MSC medium and fed every 3 days. When cells reached 80% confluence, they were detached with 0.05% trypsin and re-plated at 30 cells/cm² (as passage 1 or p1) for further expansion. Resultant BMSC were passaged every 5-7 days.

Detection of cell surface markers for MSC

Cell surface markers were detected using BD Stemflow^TM Human MSC Analysis Kit (BD Biosciences). Briefly, cells were dissociated with 0.05% trypsin, washed and re-suspended in a staining buffer at 1×10⁷ cells/ml. Cell suspensions were stained for typical MSC markers (CD44, CD73, CD90, and CD105) and a cocktail of negative markers (CD45, CD34, CD11b, CD19, and HLA-DR). After staining, cells were analyzed on a BD Accuri^TM 6 flow cytometer (BD Biosciences).

Tri-lineage differentiation of MSC

MSC dissociated with 0.05% trypsin were seeded in a 12-well plate at 5×10⁴ cells/well. After two days, the cells were induced to differentiate into osteogenic, adipogenic, and chondrogenic lineages, using the StemPro differentiation kits (Life Technologies), according to the manufacturer's instructions. Media were changed every three days. Resultant osteocytes and chondrocytes were fixed on day 21 post-differentiation, and stained with Alizarin Red S and Alcian Blue, respectively. Due to the slower adipogenic differentiation from EMSC than bone marrow derived MSC ⁴¹, we prolonged the differentiation time to 40 days. Resultant adipocytes were fixed on day 40 and stained with Oil Red O. Cells were photographed following the staining.

EMSC spheroidal differentiation into chondrocytes and adipocytes in 3D

Following precipitation of EMSC_Sp in an ultra-low attachment plate, the consumed medium was gently aspirated. The spheroids were rinsed with PBS twice and then re-suspended in the Cartilage or Adipocyte Differentiation Medium (StemPro differentiation kits, Life Technologies) for differentiation into chondrocytes and adipocytes, respectively. The medium was refreshed every four days. On day 20 the spheroids were collected and fixed in 4% paraformaldehyde for two days. Frozen sections of those spheroids were used for identification of chondrocytes and adipocytes using Alcian Blue and Oil Red O, respectively, followed by photography. EMSC_Sp without chondro- or adipogenic differentiation were used as a negative control. For EMSC differentiation to osteocytes and chondrocytes in DBM (Anjiu, BioTech), multiple cubes of DBM were placed in a well of a 6-well plate, aligned side by side, and rinsed in the MSC medium overnight. Next day, the medium was removed, a mixture of EMSC_Sp and dissociated EMSC at the ratio of 2:1 in cell number was loaded into the DBM overnight to fill all the spaces between the cell spheres and scaffold. The recellularized DBM was then rinsed in osteo- or chondrocyte differentiation medium that was added to the well until 75% full, and the plate was carefully placed in an incubator for differentiation with the medium refreshed every 4 days until day 20. The cell-loaded DBM blocks were fixed in 4% paraformaldehyde and sectioned for further analyses.

Detection of apoptotic and live/dead cells

EMSC_Sp were fixed in 4% paraformaldehyde for two days followed by immunostaining for the apoptotic marker active caspase-3 using corresponding antibody (SantaCruz, USA). To quantify live/dead cells, EMSC in spheroids or monolayer were dissociated with 0.25% trypsin and stained with Annexin V (for apoptotic cells), and acridine orange (AO, which labels live cells green), and propidium iodide (PI, which labels dead cells orange) (Cellometer), according the manufacturers' instructions. AO/PI-stained cells were photographed and quantified on Cellometer, and Annexin V-stained cells quantified on BD Accuri^TM 6 flow cytometry.

Tracking of live cell proliferation

Cell growth was tracked using the IncuCyte ZOOM^TM Live-Cell Analysis System (Essen BioScience). MSC with different treatments were plated on a 6-well plate at the same initial density, and the plate was placed in the IncuCyte ZOOM™ system. Phase-contrast images were captured at 2 hours intervals for a total duration of 110 hours. Percentage of confluence in each well, which is proportional to cell proliferation rate, was calculated using the IncuCyte ZOOM™ software.

Detection of cell senescence

Cell senescence was analyzed using the Senescence β-Galactosidase Staining Kit (Beyotime). Cells cultured as a monolayer until ~70% confluence were fixed and stained with the kit overnight, rinsed with 1× PBS, and then penetrated with 0.2% Triton X-100 for 10 min. Nuclei were counterstained with DAPI for the total cell number. Five different microscopic views of the stained cells were randomly photographed. The ratios of β-gal⁺ cells among the total cells in all the views were analyzed for statistics.

Reverse transcription- and quantitative polymerase chain reaction (RT-PCR and RT-qPCR)

Total RNA was extracted from cultured cells by using Trizol reagent (Life Technology) and cDNA was generated using PrimeScript RT Reagent kit (Clontech). PCR reactions were carried out by Quick-Load® Taq 2×Master Mix (NEB). PCR was performed under following condition: an initial denaturation at 95 °C for 30 sec.; followed by 28 cycles of 30 sec., denaturation at 95 °C, 20 sec., annealing at 53 °C, and 30 sec. extension at 68 °C; and with a final extension at 68 °C for 10 min.

qPCR was performed using iTaq^TM Universal SYBR Green Supermix (Bio-rad), and detected with the CFX96TM Real-Time PCR Detection System (Bio-rad). qPCR cycling protocol was as follows: an initial denaturation and enzymatic activation at 95 °C for 5 sec; followed by 39 cycles of 5-sec. denaturation at 95 °C, 30-sec. annealing at 60 °C, and melt curve analysis by 0.5 increments at 5 sec./step from 65 °C to 95 °C; and the final incubation was at 95 °C for 30 sec. for polymerase activation and DNA denaturation. Data are normalized to the expressional level of GAPDH and represented as fold changes. Primers used to detect expression of specific genes are listed in Table S2.

Microarray

RNA was isolated from EMSC_ML and EMSC_Sp-ML treated with or without IFNγ for 24 h, using RNAeasy mini kit (Qiagen), and cDNA libraries were synthesized and processed by using HumanHT-12 v4 Expression BeadChip. Further analysis was performed using the BeadStudio Data Analysis software (Illumina) and Excel.

The microarray dataset was normalized and filtered to remove probes with detection P value > 0.01 in at least 50% of the samples ²⁶. Two normalization methods were utilized to analyze the dataset on the basis of their biological relevance. First, average probe intensity of all biological and technical replicates was used to make comparison of gene expression profile between test and control groups. Second, the probe intensity of analyzed genes in hESC was used as a baseline to identify the gene expression differences between EMSC_ML and EMSC_Sp-ML. The microarray data has been deposited to Gene Expression Omnibus, and its accession number is GSE93218.

Inhibition of T lymphocyte proliferation in vitro

Lymphocytes isolated from peripheral lymph nodes of 8 to 10 weeks-old C57BL/6 mice were labeled with 5-μM CFSE (Beyotime) for 10 min, at 37°C, seeded at 2×10⁵ cells/100 μl/well in 1640 complete medium (RPMI-1640 + 10% FBS). MSC were seeded in a 6-well plate, cultured in the MSC medium for 7 days, dissociated with Trypsin-EDTA (StemCell Technologies), and added at the ratio of 1:40 or 1:80 (for cell number of MSC versus lymphocytes) per well into a 96-well plate containing lymphocytes at 100 μl/well. The lymphocytes in the mixture were stimulated with or without pre-coated 10-μg/ml anti-CD3 antibodies (BD Bioscience) and 2-μg/ml anti-CD28 antibodies (BD Bioscience). Suspended lymphocytes were collected 5 days after the stimulation and then subjected to flow cytometry analysis.

Mouse model of DSS/TNBS-induced colitis

Experiments were carried out on 8-week-old C57BL/6 mice, based on the animal use protocol #UMAEC-06-2015 approved by the University of Macau Panel of Research Ethics. The model was induced in mice as we recently reported ⁹ with minor modifications. Briefly, mice were administered with 2% (w/v) DSS (molecular weight 36,000-50,000, MP Biomedical) in the drinking water for 7 days. On days 1 and 2 after the start of the DSS treatment, mice were intraperitoneally (i.p.) injected with 1×10⁶ MSC/mouse. MSC were suspended in 100 μl PBS. Some mice were injected with 100 μl PBS alone as a negative control. Body weight loss, rectal bleeding, and stool consistency were monitored daily.

All mice were sacrificed on day 15 after the start of the experiment. The colon was dissected from each mouse and measured for its length. The colons were flushed with sterile 1× DPBS and fixed in 4% paraformaldehyde at 4^oC for 24 h. The distal part of the colon was embedded in paraffin wax and sectioned at 5 µm in thickness, mounted to glass slides for hematoxylin and eosin (H&E) staining. Images of the H&E stained sections were acquired on an Olympus CKX41 microscope. Histological score of the colon sections is calculated based on the percentage of area involved and crypt loss, erosions of the epithelium, and the severity of the other parameters ⁴².

The TNBS-induced colitis model was conducted on male BALB/C mice following the reported method ⁴³ with some modifications. 100 mg/kg TNBS in 100 μl 45% ethanol was intra-rectally administrated to mice on days 0 and 1. 6 h after the administration, mice were i.p. injected with 1x10⁶ MSC/mouse in 100-μl PBS or PBS alone. All mice were sacrificed on day 5 after the start of the experiment. The body weight loss, colon length, and histological score were documented as above.

Data analysis

Statistical analysis was performed with SPSS. Two-tailed Student's t-test was used to determine P values. Statistical significance was defined as *P < 0.05 and **P <0.01. Data obtained from flow cytometry were analyzed using FlowJo 7.6.

Supplementary Material

Supplementary figures and tables.

Abbreviations

hESC

human embryonic stem cell

hPSC

human pluripotent stem cell

iPSC

induced pluripotent sten cell

MSC

mesenchymal stem cell

BMSC

human bone marraw MSC

EMSC

hESC derived MSC

hESC_Sp

hESC spheroids

EMSC_Sp

EMSC spheroids

EMSC_ML

EMSC cultured in monolayer

EMSC_Sp-ML

EMSC_Sp reattached and passaged in monolayer

TB

trophlast

TB_Sp

TB spheroids

TB_ML

trophoblast cultured in monolayer

DBM

demineralized bone matrix

DSS

dextran sulfate sodium

TNBS

2,4,6-trinitrobenzenesulfonic acid.