The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

Abstract Introduction Low back pain (LBP) is a global health concern. Increasing evidence implicates intervertebral disk (IVD) degeneration as a major contributor. In this respect, tissue‐specific progenitors may play a crucial role in tissue regeneration, as these cells are perfectly adapted to their niche. Recently, a novel progenitor cell population was described in the nucleus pulposus (NP) that is positive for Tie2 marker. These cells have self‐renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies. Objective Here, we studied the influence of the culture method and of the microenvironment on the proliferation rate and the differentiation potential of human NP progenitors in vitro. Method Cells were obtained from human NP tissue from trauma patients. Briefly, the NP tissue cells were cultured in two‐dimensional (2D) (monolayer) or three‐dimensional (3D) (alginate beads) conditions. After 1 week, cells from 2D or 3D culture were expanded on fibronectin‐coated flasks. Subsequently, expanded NP cells were then characterized by cytometry and tri‐lineage differentiation, which was analyzed by qPCR and histology. Moreover, experiments using Tie2+ and Tie2− NP cells were also performed. Results The present study aims to demonstrate that 3D expansion of NP cells better preserves the Tie2+ cell populations and increases the chondrogenic and osteogenic differentiation potential compared to 2D expansion. Moreover, the cell sorting experiments reveal that only Tie2+ cells were able to maintain the pluripotent gene expression if cultured in 3D within alginate beads. Therefore, our results highly suggest that the maintenance of the cell's multipotency is mainly, but not exclusively, due to the higher presence of Tie2+ cells due to 3D culture. Conclusion This project not only might have a scientific impact by evaluating the influence of a two‐step expansion protocol on the functionality of NP progenitors, but it could also lead to an innovative clinical approach.


| INTRODUCTION
The intervertebral disk (IVD) consists of superior and inferior cartilaginous endplates (CEP), outer annulus fibrosus (AF), and central gel-like nucleus pulposus (NP). IVD degeneration is a major cause of lower back pain, and the burdens caused by IVD degeneration-related morbidities extend to society and the economy as a whole. [1][2][3][4] Current therapies cannot cure IVD degeneration and are limited to conservative treatments (such as medication or physical therapy) or surgical interventions (such as spinal fusion). 5 Regeneration therapy for IVD degeneration mainly targets the NP tissue, as its degeneration is considered to be a major cause of low back pain. 5,6 Therefore, understanding the cell morphology and gene expression profile of NP cells (NPCs) is critical for developing new therapeutic strategies for IVD regeneration.
The recent discovery of a new cell candidate for cell therapy, the NP progenitor cell (NPPC), advances this research focus as this cell type might be involved in the innate maintenance of disk health. 7 These progenitor cells represent a rare subpopulation (between 1% and 5%) of the total NP cell population and were shown to decrease markedly with age and to be nearly absent in people >40 years of age. 7 However, NPPCs fulfilled formally progenitor criteria and possess the ability for multilineage differentiation. 7,8 Such NPPCs were identified and successfully isolated from human, bovine, murine, and canine NP tissues using fluorescence-activated cell sorting (FACS) through the angiopoietin-1 receptor (aka. Tie2). 9 Nonetheless, it is still to be clarified how this Tie2 positive (Tie2 + ) cells are related to mesenchymal stromal cells (MSCs) and the recently closer characterized notochordal cells. 10 Recently, a study reported the expression of two classical MSC markers, CD29 and CD105, in NPPCs compared to umbilical-cord-derived MSCs. 11 These results were in accordance with findings of niches for multipotent stem cells in the disk itself or possibly in the nearby bone marrow of the vertebrae close to CEP cells. [12][13][14] The ability to culture and expand NPCs (and especially NPPCs), while maintaining their gene expression profile and their stem potential is essential in experiments using these cells, as dysregulation of multiple intracellular signaling pathways may play a key role in IVD degeneration. 15,16 Several cell based or cytokine therapies have been investigated using rat, rabbit, bovine, mouse, and human NPC culture, 6,[17][18][19] but molecular pathways such as NPC intracellular signaling pathways are still not fully understood. 1,5 Cell therapy seems to be a more promising approach for disk regeneration. However, so far, there have been few reports using human NPCs caused by the difficulty associated with harvesting adequate quantities of these cells from available IVDs. Additionally, despite their early fading, the study of this marginal subpopulation within the NP might give rise to a better understanding of how these cells affect IVD health, and what conditions will be optimal to extend their number in vitro and to maintain them longer in their native stem state. Moreover, a deeper understanding might lead to new approaches for the regeneration of the IVD. Furthermore, the gene expression profile of human NPCs is easily lost with conventional two-dimensional (2D) monolayer culture, which does not replicate the three-dimensional (3D) hydrophilic aggrecan-rich gelatinous extracellular matrix (ECM) with unique avascular, hypoxic, nutrient-deficient, high osmotic pressure characteristics in which NPCs are naturally supported. Therefore, a novel culture method is required, which allows for the number of NPCs to be expanded while maintaining their gene expression profile. In this study, we aimed to establish an in vitro culture method for human NPCs to maintain and increase the amount of NPPCs over time. We also aimed to preserve the stem potential of those cells regarding their multipotent gene expression and phenotype but also their ability to differentiate into a chondrogenic, adipogenic, and osteogenic lineage. Moreover, experiments were performed in which NPCs were sorted into Tie2 + and Tie2 − subpopulations combined with our new culture method was also performed to better understand the metabolism and gene expression profile of those cells under those specific conditions.

| Cell isolation
Human IVD tissue was obtained from 10 patients ranging from 20 to Gibco, Life Technologies, Zug, Switzerland), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/glutamine (#10378016; 10 000 units of penicillin, 10 000 μg of streptomycin, and 29.2 mg/mL of L-glutamine in a 10 mM citrate buffer; Merck, Darmstadt, Germany). The AF and CEP cells were directly frozen and stored right after isolation.
Concerning human bone marrow mesenchymal stromal cells (hBMSCs), human bone marrow aspirates (20 mL) were obtained during routine orthopedic surgical procedures on the same patients described above undergoing spinal surgery after experiencing trauma to their disks. After surgery, the bone marrow aspirate was immediately transferred into plastic tubes containing heparin, S-Monovette (#01.1634; Sarstedt, Nümbrecht, Germany). After diluting the marrow aspirates with phosphate-buffered saline (PBS) at a ratio of 1:4, nucleated cells were isolated using a density gradient solution (Histopaque, Sigma). The complete medium consisted of α-minimum essential medium (αMEM) with 10% fetal bovine serum, 1% of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (aka. HEPES) at 1 M, 1% sodium pyruvate (100 mM), and 1% of penicillinstreptomycine-glutamine (100×) solution (P/S/G) (from Gibco and Merck, respectively). Nucleated cells were plated at a density of 3.10 6 cells/cm 2 in complete medium supplemented with 5 ng/mL of fibroblast growth factor-2 (FGF-2; R&D Systems, Zug, Switzerland) and cultured in a humidified incubator at 37 C and 5% CO 2 . The medium was changed twice a week. Here, hBMSCs were selected based on adhesion and proliferation on the plastic substrate 1 week after seeding and were used at passage 3. HEPES (1 M), 1% sodium pyruvate (100 mM), and 1% of penicillinstreptomycin-glutamine (100×) solution (all from Gibco) and 10 ng/mL of FGF-2 (R&D Systems) until confluence. After reaching confluence, cells were directly used for the differentiation assays. Microscopic pictures were taken for each condition at the end of the first and second phase of expansion using the Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan).

| FACS analysis
The phenotype of NPCs was determined by cytofluorimetric analysis
The cells were fixed in 4% formaldehyde, and the calcium deposition of the cell layers was evaluated with 2% Alizarin red staining (ALZR) solution (Sigma-Aldrich) for 45 minutes. The ALZR was released from the cell layers by the addition of a 10% cetylpyridinium chloride solution (Sigma-Aldrich) for 1 hour under agitation. Optical density was measured at 570 nm with the microplate reader SpectraMax M5.
The sample sections were stained with 0.02% Fast Green solution (Merck) for 10 minutes. Then they were rinsed with 1% acetic acid and then immersed in 0.1% Safranin-O (Merck) at pH 2.5 for 15 minutes to stain sulfated glycosaminoglycans (GAGs). Additionally, sections were stained for sulfated GAGs with 1% Alcian blue 8GX (Sigma-Aldrich) in 3% acetic acid at pH 1.0. 25 Once dehydrated and mounted, slides were imaged using an inverted microscope (Eclipse E800; Nikon), and single images were taken at ×4 magnification and stitched together using NIS Elements microscope imaging software (Nikon) and edited for easier comparison with ImageJ 1.51j8 (Rasband WS, 2020, ImageJ, National Institutes of Health, Bethesda, Maryland, http://rsb.info.nih.gov/ij/).

| Cell sorting
Tie2 + cells were sorted using FACS as previously described. 8,9,26 Freshly isolated NPCs (N = 3) were incubated with an anti-human reported. 26 The Mouse IgG 1 PE-conjugated Antibody (#IC002P, clone 11 711, R&D systems) was used as isotype control to set the gate for sorting. RNA isolation was performed as previously described. 27 DNase (DNase 1 Kit, Sigma-Aldrich) was used to degrade residual DNA, and iScript cDNA Synthesis Kit (Bio-Rad Inc., Switzerland) was used for reverse transcription. Thereafter, the cDNA was mixed with iTaq universal SYBR Green Supermix (Bio-Rad) and with forward and reverse primers (Microsynth, Switzerland) for each gene (Table 1). qPCR was performed in duplicates (CFX96 Touch, Bio-Rad) using 18S and GAPDH as reference genes. The relative gene expression was determined using the 2 −ΔΔCt method, 28 and data were normalized to the control condition (hBMSCs in complete medium or NP cells in 2D culture).

| Cell activity
Mitochondrial activity was determined with the Alamar Blue assay

| GAG content
The same papain digested samples were used to determine the amount of GAG and proteoglycans. For this, 1,9-dimethyl-methylene blue (Sigma-Aldrich) was used, and absorbance was read at 600 nm with a SpectraMax M5 plate reader. 29 GAG content was calculated from a standard curve obtained from chondroitin sulfate (Sigma-Aldrich). Data were normalized to the amount of cells that were seeded in each culture condition.

| Statistical analysis
Data are presented as mean ± SD (SD). For all data, a nonparametric distribution was assumed. The percentage of cells positive after cyto-

| Conventional 2D monolayer culture vs 3D culture into alginate beads
To find a better way to culture NPCs, we were following a two-step protocol. Microscopic pictures of the NPCs were taken at each round of this two-step protocol ( Figure 1). Briefly, during the first phase of expansion, we observed that NPCs formed a monolayer when culturing them in 2D with ascorbic acid supplemented medium ( Figure 1A,E, I). However, if cells were within alginate beads with ascorbic acid supplemented medium NPCs presented a round shape in the hydrogel ( Figure 2B-D). In the same way, NPCs in 2D or in 3D were both expressing CD45 (50 ± 5%) ( Figure 2E) and CD146 (25 ± 3%) ( Figure 2G). In contrast, only cells cultured in 3D (alginate beads) were significantly positive for CD34 (10 ± 5%) ( Figure 2F) and the Tie2 marker (45 ± 5%) ( Figure 2H).
Concerning the relative expression of pluripotent genes and Tie2 3.2 | NPCs maintained their gene expression profile and NPPCs proliferated when cultured back in 2D For the second step (second expansion phase), following the first phase of expansion, NPCs cultured in 2D monolayer or 3D alginate beads were put back in 2D fibronectin-coated flasks ( Figure 2M).
To further increase the number of human NPCs, after the first phase of expansion (in 2D or 3D), we performed the second phase of expansion in 2D using fibronectin-coated dishes with FGF-2 supplemented medium. The analysis performed was the same as the one used for the first expansion phase, meaning flow cytometry ( Figure 2N-S) and qPCR ( Figure 2T-W).
Concerning the relative expression of pluripotent genes and Tie2 ( Figure 2T-W), the expression of NANOG ( Figure 2T) and OCT4 After quantifying the Oil-Red-O stain ( Figure 3M), we could observe that a higher amount was stained with hBMSCs and 3D NPCs compared to 2D NPCs (1.7-fold increase for both).

| Osteogenic differentiation of NPCs
Afterwards, we attempted to differentiate the expanded NPCs (at the end of the second expansion phase) into an osteogenic lineage compared to hBMSCs. After 21 days of culture, ALZR staining was performed and a representation of this assay is shown ( Figure 4A-F).
We could observe a coloration of the mineralized extracellular matrix   Figure 5D-F). Thereafter, we also quantified the GAG content in the supernatant ( Figure 5G). We showed that the amount of GAG was significantly higher (10 μg/mL compared to 0.5 μg/mL) in the NP (3D) cells cultured with chondrogenic medium compared to the control medium ( Figure 5G).

| Chondrogenic differentiation of NPCs
To continue to analyze those samples, two histological colora- chondrogenic medium compared to ones cultured with the control medium ( Figure 6C).

| Comparison of Tie2 + and Tie2 − NP cells population
To look at the effect of Tie2 + cells on the expression of pluripotent genes and other factors, we followed the two-step protocol previously described with the addition of a sorting step (FACS) after digesting the NP tissue ( Figure 7A). Briefly, after dissecting the human IVD and digesting the NP tissue, NPCs were sorted for the Tie2 marker. Next, Tie2 + and Tie2 − NP cells were seeded in 2D as a monolayer or 3D into alginate beads ( Figure 7A).
We initially looked at the cell metabolism using the Alamar Blue assay on day 4 ( Figure 7B) and day 7 ( Figure 7C), normalized to the amount of DNA. We noticed that the cell metabolism (proportional to the amount of DNA) was lower in cells (either Tie2 + or Tie2 − ) cultured in 3D compared to cells cultured in 2D on day 4 and day 7 (twofold and 3.5-fold decrease, respectively). Moreover, looking at the DNA content at day 7 ( Figure 7D), in each condition, we can observe a lower amount of DNA measured in 3D culture (either Tie2 + or Tie2 − ) compared to 2D culture (2.25-fold decrease). Concerning the GAG load of each sample at day 7 ( Figure 7E), we can note no statistical differences between each condition (either Tie2 + or Tie2 − and 2D or 3D culture).
Regarding the relative expression of pluripotent genes and Tie2 ( Figure 7F-I), the expression of NANOG ( Figure 7F) and SOX2 ( Figure 7H) was showing a significant up-regulation for Tie2 + NPCs that were cultured in 3D compared to NPCs cultured in 2D (either Tie2 + or Tie2 − ). However, OCT4 ( Figure 7G) was not showing any statistical differences in NPCs for both conditions, so for cells cultured in 2D or 3D (either Tie2 + or Tie2 − ). Finally, looking at the relative gene

| DISCUSSION
In this study, we established and studied a novel and efficient twophase expansion culture method for human NPCs, using a series of primary 3D alginate bead culture followed by 2D monolayer culture on fibronectin-coated dishes with FGF-2.
In the first phase of expansion, NPCs cultured in alginate beads (3D) showed the most relevant results. First, NPCs cultured in 3D were showing the same phenotype as hBMSCs by expressing CD90, CD73, CD105, and CD45 as surface markers. 30 Moreover, cells cultured in 3D presented a higher percentage of cells positive for CD34 (10 ± 6.2%) compared to NPCs cultured in 2D (2.5 ± 2.3%) showing a higher potential of differentiation of this population (3D) compared to the 2D as CD34 is a common marker for diverse progenitors. 31 Moreover, the expression of the CD34 surface marker by a small percentage of NPCs could also suppose that those cells are closely related to quiescent hematopoietic stem cells/progenitor cells that were identified to express both CD34 and Tie2. [32][33][34] Only CD146 did not reveal any differences in our analysis. This could be explained by the fact that disks were coming from a healthy donor and that this marker can distinguish stem cell subpopulations with distinct migration and regenerative potential in degenerative IVDs. 35  Regarding the second phase of expansion, the addition of FGF-2 accelerated the proliferation of human NPCs (data not shown), as already described. 37 This result is consistent with previous studies, which have reported enhanced cell proliferation following FGF-2 administration. 16,38,39 However, in agreement with previous reports, 40,41 the culture of NPCs in monolayer (2D) with or without FGF-2 leads to a loss of the phenotype. Moreover, fibronectin has been shown to be a key component in IVDs and more precisely NP tissue. [42][43][44] In this study, the combination of fibronectin coating with the addition of FGF-2 into the culture media, as previously described with the culture of murine NPCs, should overcome this limitation of "stemness" potential loss. 45 This expansion protocol, by still keeping the cells in a mimicking microenvironment after previously cultured in 3D (alginate beads), will maintain the cells with a phenotype and gene expression close to cells in their native tissue. Briefly, even at the end of the second phase of expansion, NPCs were still showing a phenotype similar to hBMSCs, meaning CD90 + , CD73 + , CD105 + , and CD45 + . Moreover, only cells previously cultured in 3D (in the first phase) were still positive for CD34 and Tie2, two characteristic markers of NPPCs of the IVD across species. 9 However, the fibronectin coating combined with the treatment with FGF-2 was not as efficient as the 3D culture in alginate, which can be seen with a lower expression of two pluripotent genes, NANOG and OCT4, but still a higher relative gene expression of SOX2 and TEK for the cells previously cultured into alginate beads.
All those genes are well described for the regulation of stem cell pluripotency and differentiation 46  To verify the multipotent potential of the NPCs that we were expanding, we decided to perform a tri-lineage differentiation of those cells into adipogenic, osteogenic, and chondrogenic differentiation as previously described. 26,48 Osteogenic and adipogenic differentiation of NPCs were performed at 20% oxygen as difficulties to obtain mineralization and adipogenesis at low oxygen levels are well documented in the literature. [49][50][51][52][53][54] Culturing in adipogenic medium following a second phase expansion culture with FGF-2 was showing a great formation of lipid droplets in our control condition (hBMSCs) but also in both NPCs samples (2D and 3D). This was already described in the literature 55 and confirmed the stem potential of our expanded cells. Concerning the osteogenic potential after 21 days of culture, the NPCs (2D and 3D) also showed a strong production of extracellular mineralized matrix, comparable to the hBMSCs. This osteogenic potential was already described, 56  This study has several limitations. First, we did not distinguish between "notochordal cells" and "chondrocyte-like cells" among our mix of NP cells after dissection and digestion of the NP tissue. 6,61 However, morphological differences between "notochordal" cells and "chondrocyte-like"/"nucleopulpocyte-like" cells have recently been distinguished in the stages of cellular differentiation. 6,[62][63][64][65][66][67] In this study, both large vacuolated cells and small round cells were observed during the early stages of 3D alginate bead cultures (Figure 1). However, the small cells dominated the cell population as cell proliferation increased. Accordingly, NP "chondrocyte-like" and NP progenitor cells identified as small cells were mainly used in this study.
Regarding the Tie2 + and Tie2 − experiment, the first phase of expansion with 3D culture (alginate beads) seems to keep the NPCs (either Tie2 + or Tie2 − ) at a lower metabolism, compared to 2D culture, by having them in a microenvironment close to the native NP tissue. 68,69 Increasing evidence highlights a pivotal role for metabolism in stem cell physiology and lineage specification. 70,71 Metabolism, indeed, is no longer considered merely an energy source nor an endpoint of gene regulation. Instead, metabolites and the nutrient environment are active players in determining intracellular signaling and enzymatic activities and consequently modulators of the stem cell's fate. Moreover, metabolic intermediates of cellular metabolism regulate epigenetic mechanisms, including histone modifications, DNA methylation, and noncoding RNAs, thereby modulating the global epigenome landscape and stemness. 72 Concerning GAG production by NPCs (either Tie2 + or Tie2 − ), it was impossible to detect any statistical difference probably due to the early time point of measurement (day 7), knowing that it was early shown that ECM mimicking scaffold (including GAG) promote stemness maintenance of mesenchymal stem cells via spheroid formation. 73 Recent research showed that the enrichment of the culture environment, either by chemically defined medium, growth factor supplementation, or modification of the culture surface stiffness, helped to preserve progenitor cells. 74,75 Concerning the pluripotent gene expression for NP Tie2 + and Tie2 − cells, the higher expression of NANOG and SOX2 only found in NPCs positive for Tie2 and cultured in 3D reveals the central role of 3D mimicking microenvironment for NPPCs culture. Even if in 2D, during the first phase of expansion Tie2 + cells were still expressing the TEK gene (aka. Tie2), they were however not able to maintain any of their pluripotent gene expression, as described with hBMSCs. 76 Our method has the potential to become a useful tool for basic research and clinical research (cell therapy) aiming at elucidating molecular mechanisms in human NPCs and in developing cell-based regenerative therapies for IVD degeneration using expanded NPCs population enriched in NPPCs (Tie2 + ), which cannot be obtained using a conventional monolayer culture method. 77 Furthermore, it will be useful for research related to tissue engineering or regenerative medicine in which the expansion of progenitor cells plays an important role.

| CONCLUSIONS
We established a novel and efficient primary culture and expansion culture method for human NPCs consisting of sequential primary 3D alginate culture with ascorbic acid supplemented medium followed by 2D monolayer culture on fibronectin-coated dishes containing FGF-2.
This protocol shows that heterogenic NP cell populations are closer to a multipotent phenotype if cultured in 3D within alginate beads compared to 2D culture. Moreover, NPCs were able to better differentiate into osteogenic, chondrogenic, and to a lesser extent adipogenic lineage even after in vitro expansion than 2D monolayer expanded NPCs.
As we experimented with pure Tie2 + and Tie2 − cell populations, it is highly suggested that the maintenance of multipotent capacity was mainly but not exclusively due to the higher presence of Tie2 + cells in the 3D culture. This project not only has a scientific impact by evaluating the influence of a two-step expansion protocol on the functionality of NP progenitors but could also lead to an innovative clinical approach with cell therapy for IVD regeneration and repair.