A Method For Providing A Cartilage Implant With Chondrocytes

Abstract

Disclosed is a method for differentiating induced pluripotent stem cells (iPSCs) into chondrocytes and integrating them into a matrix/scaffold to provide a cartilage implant. The method comprises the steps of seeding a surface of a substrate with iPSCs. The surface is coated with nanoparticles in a particle density of at least 500 particles/μm2, and parts of the surface in between said nanoparticles are coated with a coating agent. Growth differentiation factor 5 (GDF5) molecules are attached to the nanoparticles. The method further comprises the steps of adding a first differentiation medium to the seeded iPSCs and allowing the seeded iPSCs to differentiate at least into chondrocyte progenitor cells on the surface in the presence of the first differentiation medium. The obtained differentiated cells are integrated into a matrix/scaffold.

Claims

1. A method for differentiating induced pluripotent stem cells (iPSCs) into chondrocytes and integrating the differentiated cells into a matrix/scaffold to provide a cartilage implant, said method comprising the steps of: seeding a surface of a substrate with iPSCs to produce seeded iPSCs, wherein the surface is coated with nanoparticles in a particle density of at least 500 particles/μm.sup.2 and less than 1500 particles/μm.sup.2, and parts of the surface in between said nanoparticles are coated with a coating agent, wherein growth differentiation factor 5 (GDF5) molecules are attached to said nanoparticles; adding a first differentiation medium to the seeded iPSCs; allowing the seeded iPSCs to differentiate at least into chondrocyte progenitor cells on the surface of the substrate in the presence of the first differentiation medium; and integrating the differentiated cells into the matrix/scaffold.

2. The method according to claim 1, wherein said iPSCs are differentiated for between 3 and 10 days on said substrate surface.

3. The method according to claim 1, wherein the method further comprises: removing formed condensed chondrocyte progenitor cell aggregates from the substrate surface with GDF5 attached thereto; and further differentiating the removed condensed chondrocyte progenitor cell aggregates into chondrocytes, in a second differentiation medium.

4. The method according to claim 3, wherein said removed condensed cell aggregates are formed into a three-dimensional pellet structure before being further differentiated.

5. The method according to claim 3, wherein the step of further differentiating the removed condensed chondrocyte progenitor cell aggregates is performed for 4 to 10 weeks.

6. The method according to claim 3, wherein said first and second differentiation medium both comprise Dulbecco's modified Eagle's medium (DMEM), Insulin-Transferrin-Selenium, Ascorbic acid, Dexamethasone, Linoleic acid, Sodium Pyruvate, transforming growth factor beta 1 (TGFβ-1), transforming growth factor beta 3 (TGFβ-3), or a combination thereof.

7. The method according to claim 1, wherein a maintenance medium is added to the substrate surface after the iPSCs have been seeded to the surface and before the differentiation medium is added.

8. The method according to claim 1, wherein said iPSCs are chondrocyte derived iPSCs.

9. The method according to claim 1, wherein said coating agent is a proliferative agent; and/or wherein the nanoparticles are gold particles coated with thiolated streptavidin, and wherein the GDF5 molecules are biotinylated and attached to the nanoparticles through biotin/streptavidin interaction.

10. The method according to claim 1, wherein 20,000 to 100,000 iPSCs/cm.sup.2 are seeded on the substrate surface.

11. A cartilage implant, wherein said cartilage implant is obtained by differentiating induced pluripotent stem cells (iPSCs) into chondrocytes and integrating them into a matrix/scaffold in accordance with claim 1.

12. A chondrocyte for use in chondrocyte implantation, said chondrocyte being integrated into a matrix/scaffold, wherein said chondrocyte is obtained by a method for differentiating induced pluripotent stem cells (iPSCs) into chondrocytes, said method comprising the steps of: seeding a surface of a substrate with iPSCs to produce seeded iPSCs, wherein the surface is coated with nanoparticles in a particle density of at least 500 particles/μm.sup.2 and less than 1500 particles/μm.sup.2, and parts of the surface in between said nanoparticles are coated with a coating agent, wherein growth differentiation factor 5 (GDF5) molecules are attached to said nanoparticles; adding a first differentiation medium to the seeded iPSCs; allowing the seeded iPSCs to differentiate at least into chondrocyte progenitor cells on the surface of the substrate in the presence of the first differentiation medium; removing formed condensed chondrocyte progenitor cell aggregates from the substrate surface with GDF5 attached thereto; further differentiating the removed condensed chondrocyte progenitor cell aggregates into chondrocytes in a second differentiation medium; and integrating the obtained chondrocyte into the matrix/scaffold.

13. The chondrocyte according to claim 12, wherein said iPSCs are differentiated for between 3 and 10 days on said substrate surface.

14. The chondrocyte according to claim 12, wherein said removed condensed cell aggregates are formed into a three dimensional pellet structure; before being further differentiated.

15. The chondrocyte according to claim 12, wherein the step of further differentiating the removed condensed chondrocyte progenitor cell aggregates is performed for 4 to 10 weeks.

16. The chondrocyte according to claim 12, wherein said first and second differentiation medium both comprise Dulbecco's modified Eagle's medium, Insulin-Transferrin-Selenium, Ascorbic acid, Dexamethasone, Linoleic acid, Sodium Pyruvate, transforming growth factor beta 1 (TGFβ-1), transforming growth factor beta 3 (TGFβ-3), or a combination thereof.

17. The chondrocyte according to claim 12, wherein a maintenance medium is added to the substrate surface after the iPSCs have been seeded to the surface and before the differentiation medium is added.

18. The chondrocyte according to claim 12, wherein said iPSCs are chondrocyte derived PSCs.

19. The chondrocyte according to claim 12, wherein said coating agent is a proliferative agent; and/or wherein the nanoparticles are gold particles coated with thiolated streptavidin, and wherein the GDF5 molecules are biotinylated and attached to the nanoparticles through biotin/streptavidin interaction.

20. The chondrocyte according to claim 12, wherein 20,000 to 100,000 iPS cells/cm.sup.2 are seeded on the substrate surface.

21. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] By way of example, embodiments of the present invention will now be described with reference to the accompanying drawings, in which:

[0063] FIG. 1a shows a montage of SEM pictures taken at different positions with 1 mm interval along a gold nanoparticle gradient surface, the scale bar indicates 200 μm. The gradient is verified up to 8 mm on a 18×18 mm silica surface;

[0064] FIG. 1b shows a diagram of the gradient profile for a gold nanoparticle gradient functionalized with GDF5. The x-axis indicates gradient distance in mm and the y-axis shows GDF5 particles/μm.sup.2 as a function of the gradient distance;

[0065] FIG. 2a shows a schematic glass surface being 8 mm wide and provided with gold nanoparticles attached in a gradient pattern on a glass substrate;

[0066] FIG. 2b shows the schematic glass surface of FIG. 2a, having Laminin molecules attached to the glass surface between the gold nanoparticles, to avoid unwanted adherence of other molecules to the glass surface between each gold nanoparticle and aid the cell adherence to the surface and the differentiation of the cells;

[0067] FIG. 2c shows the schematic glass surface of FIG. 2b having Streptavidin attached to the gold nanoparticles. The Streptavidin will act as a linker to biotinylated biomolecules;

[0068] FIG. 2d shows a perspective view of the schematic glass surface of FIG. 2c. The gradient of Streptavidin coated gold nanoparticles is ready to be functionalized by biotinylated morphogens;

[0069] FIG. 2e shows a functionalised glass substrate surface, where biotinylated biomolecules have been attached to the Streptavidin. In the present disclosure, said biomolecules are GDF5, TGFβ-1 and TGFβ-3;

[0070] FIG. 3 shows laminin control gradient surfaces (A1, A2). The gradient surfaces were visualized with IN CELL Analyzer 6000 (IN Cell 6000, GE Healthcare, United Kingdom). Cavities with no cells in the centre were formed, mainly at higher laminin densities. The bars on each surface A1, A2 indicate the direction of the nanoparticle density gradient with laminin coupled to the surface;

[0071] FIG. 4 shows the budding zone in (A) a GDF5 gradient surface, 18×18 mm, seeded with c-iPSCs visualized using high throughput confocal IN CELL Analyzer 6000. The bar on the left upper side indicates the extent of the gradient where the continuous density increase is shown with a marker on the left-hand side, with low GDF5 density at the bottom of the image and high GDF5 density at the top, (B) focusing on the budding cell clusters identified at particle density of 500-1500 gold nanoparticles/μm.sup.2. Two white lines indicate the budding zone, and (C-D) show close up pictures of buds identified in the budding zone shown in (B), the scale bars in (C) and (D) are 10 μm;

[0072] FIG. 5A-F all show comparative pictures on cell behavior on GDF5, TGFβ-1 and TGFβ-3 h.d. surfaces, 18×18 mm, visualized using high throughput confocal IN CELL Analyzer 6000;

[0073] FIG. 5A shows GDF5 surfaces with a low particle density at 400 particles/μm.sup.2. No budding zones are identified;

[0074] FIG. 5B shows a GDF5 surface with a particle density in the range of the budding zone at 900 particles/μm.sup.2. Buds are clearly identified as brighter colored clusters;

[0075] FIG. 5C shows TGFβ-1 surfaces with a low particle density at 600 particles/μm.sup.2. Cell-free cavities were formed, a response comparable to cells on laminin only (FIG. 3);

[0076] FIG. 5D shows evenly distributed cells on TGFβ-1 surfaces with a high particle density of 2000 particles/μm.sup.2;

[0077] FIG. 5E shows TGFβ-3 surfaces with low density, 600 particles/μm.sup.2, and;

[0078] FIG. 5F shows TGFβ-3 surfaces with high density, 1900 particles/μm.sup.2;

[0079] FIG. 6 shows immunostaining and a marked expression of SOX9 in c-iPSCs after five days of differentiation on a GDF5 gradient. (A1-A3) Images were acquired in the budding zone using florescence microscopy, 20× objective. Scale bars denote 100 μm. (B1-B3) Images were acquired in the budding zone with a confocal microscope, 40× objective. Scale bars denote 10 μm;

[0080] FIG. 7 shows that TBX3 are localized in the nuclei and in vesicles in the budding zone. TBX3 expression is visualized after five days of differentiation on a GDF5 gradient. (A) shows a budding zone on a GDF5 gradient. Scale bar 100 μm. (B1-B3) and (C1-C3) show two different budding clusters. Images were acquired with a confocal microscope, 40× objective. Scale bars 20 μm. (D1-D3) is a closeup of a budding cluster. Images were acquired using a 60× objective. Scale bars 10 μm.

[0081] FIG. 8 shows iPSCs on h.d. surfaces immunostained with TBX3 monoclonal (Table 2) antibody, using confocal microscopy. TBX3 expression is clearly upregulated on the GDF5 h.d. surface with a particle density in the range of the budding zone, compared to all other surfaces. (A) shows a GDF5 surface with low particle density. (B) is a GDF5 budding zone surface. (C) shows a TGFβ-3 surface with the same particle density as (B). (D) is a TGFβ-3 surface with the same particle density as the picture (B) but with GDF5 (10 ng/ml) added in the differentiation medium. (E) is a surface without biomolecules, coated with Coat-1 (DEF-CS 500 Coat-1, Cellartis, Sweden); and

[0082] FIG. 9 shows histological sections of pellets generated from c-iPSCs (A-C) and, as a control, from patient-derived chondrocytes (D-E). A1, A2 and E1, E2 were stained with Alcian Blue van Gieson that demonstrates the presence of proteoglycans and collagens. A3-A6 & B3-C3 were immunostained for TBX3 which were especially upregulated in the areas with higher amounts of GAG. (A1-A6) show c-iPSCs differentiated on a GDF5 h.d. surface with low particle density. (B1-B3) show c-iPSCs differentiated on GDF5 h.d. surface with budding zone particle density. (C1-C3) are c-iPSCs differentiated on GDF5 gradient surface. (D1-D2) show chondrocyte pellet differentiated on GDF5 gradient surface. (E1-E2) show a chondrocyte pellet with 5% human serum in the medium. In A1-E1, A3-C3, A5-A6, the scale bars indicate 100 μm. In A2-E2, the scale bars indicate 50 μm. In A4, the scale bar indicates 20 μm.

[0083] FIG. 10 shows histological stained sections of pellets generated from c-iPSCs (A) and patient-derived chondrocytes (B-D). It shows the collagen II expression in chondrocytes derived from c-iPSCs first differentiated on a GDF5 gradient (A2) in comparison to the collagen II expression in patient-derived chondrocytes maintained in pellet with 5% serum for two weeks (B2), in patient-derived chondrocytes maintained five days on GDF5 gradient, followed two weeks in pellet in differentiation medium (C2), and in patient-derived chondrocytes maintained five days on TGFβ-3 gradient, followed two weeks in pellet in differentiation medium (D3). A1, B1, C1, and D1 show DAPI stained cell cores. A3, B3, C3, and D3 show the merged channels. Scale bars 20 μm.

EXPERIMENTAL

[0084] The following examples are mere examples and should by no means be interpreted to limit the scope of the invention, as the invention is limited only by the accompanying claims.

Abbreviations

OA Osteoarthritis

[0085] ACI autologous chondrocyte implantation
c-iPSC chondrocyte derived induced pluripotent stem cells
iPSC induced pluripotent stem cells
TGFβ-1 transforming growth factor beta 1
TGFβ-3 transforming growth factor beta 3
GDF5 growth differentiation factor 5
BMP14 bone morphogenetic protein 14
BMPs bone morphogenetic proteins
ECM extracellular matrix
TBX3 T-box transcription factor 3
DAPI 4′,6-diamidino-2-phenylindole
PBS phosphate buffered saline
GAGs glycosaminoglycans
SOX9 Transcription factor SOX9
PDGF platelet-derived growth factor
h.d. surface homogenous density surface

[0086] Cell Culturing

[0087] Method—Generating c-iPSCs from Chondrocytes

[0088] It has previously been shown that chondrocytes from autologous chondrocyte implantation (ACI)-donors can be reprogrammed into iPSCs using a footprint-free method based on mRNA delivery (Borestrom, C., S. Simonsson, et al 2014), a method that was applied to generate c-iPSCs for this study. The iPSCs were derived from chondrocytes from an anonymous female donor.

[0089] In short, the method involves obtaining chondrocytes from ACI-donors, isolating said chondrocytes and expanding them in a chondrocyte medium. Non-integrating mRNA reprogramming technology was used, and mRNA reprogramming was conducted using the Stemgent mRNA Reprogramming Kit according to the manufacturer's instructions, with some minor modifications, performing daily mRNA transfections for 21 days. Clonal iPSC lines were established by picking hESC-like colonies.

[0090] Method—Cell Culturing of c-iPSCs for Control Experiments

[0091] The iPSCs were stored in liquid nitrogen at −196° C. until use. Newly thawed c-iPSCs were seeded for monolayer culturing in cell culture flasks (Corning™ Primaria™ Tissue Culture Flasks, vented, FisherScientific, USA) which were coated specifically for c-iPSCs (DEF-CS 500 Coat-1, Cellartis, Sweden) and placed in a 37° C. humidity chamber at 90% humidity and 5% CO.sub.2. Cell medium (Cellartis® DEF-CS™ 500 Basal Medium with Additives, Cellartis, Sweden), supplemented with additional growth factors, was changed every day and cell passage was performed every third day. All handling of cells was performed in a sterile cell lab area.

[0092] Method—Cell Culturing of Chondrocytes for Control Experiments

[0093] The chondrocytes used for the pellet-experiments were provided from three combined anonymous male donors, with written consent. Chondrocytes were expanded in a monolayer in chondrocyte medium consisting of Dulbecco's modified Eagle's medium/F12 (DMEM/F12, ThermoFisher Scientific, USA) supplemented with 0.5 mL 8 mM L-ascorbic acid (Sigma Aldrich, USA), 1% penicillin-streptomycin (Sigma Aldrich, USA) and 10% human serum, at 37° C. in 5% CO.sub.2 and 90% relative humidity. Medium was changed three times per week. All handling of cells was performed in a sterile cell lab area.

[0094] Preparation of Gradient Surfaces of GDF5, TGFβ-1 and TGFβ-3

[0095] Method

[0096] Cline Scientific's Nano Gradients and Nano Surfaces (Cline Scientific AB, Sweden) were used as a tool in the differentiation process. The gradient surface may be a nano gradient surface as disclosed in EP 2 608 896 B1. Such nano gradient surface has a continuous gradient of deposited and electrically charged nanoparticles. The nanoparticles preferably have an average diameter between 10 and 60 nm, and the average centre-to-centre distance of the nanoparticles is typically about 10 to 60 nm in one end of the gradient and about 100 to 150 nm in the other end of the gradient.

[0097] A particle density of nanoparticles on the gradients were in the range of from 3 to 3000 particles/μm.sup.2, determined with Scanning Electron Microscopy (SEM) using a Zeiss Ultra 55 operating at an accelerating voltage of 3.00 kV. Images were acquired in the secondary/backscattered electron mode using the In Lens detector. The nanoparticles used were gold nanoparticles and the surface was a silica surface. The substrate 100 is shown schematically in FIGS. 2a-2e.

[0098] According to a functionalization protocol, thiolated streptavidin 60 (SH-streptavidin, ProteinMods, USA) was applied to the gold nanoparticles 20, as shown in FIG. 2c. After sufficient incubation in room temperature, superfluous streptavidin 60 was rinsed off with Phosphate Buffered Saline (PBS) (Phosphate Buffered Saline, Amresco, USA).

[0099] Thereafter, laminin 521 (Biolaminin 521 LN, BioLamina, Sweden) was applied to coat parts 120 of the glass surfaces 110 between the nanoparticles. After incubation (as above), superfluous laminin 40 was rinsed off with PBS. The substrate 100 and its prepared surface 110 having a gradient of gold nanoparticles 20, having thiolated streptavidin 60 attached to said nanoparticles 20, and where the surfaces 110 in between the nanoparticles 20 are coated with laminin 40 is shown in FIGS. 2c and 2d.

[0100] Previous experiments with glass surfaces showed that the c-iPSCs have an impaired capacity to attach to glass. Therefore, laminin was attached to the gradient surface between the biomolecule functionalized particles, successfully ameliorating cell adhesion (Aumailley, M. 2013). In addition, laminin will overall enhance the binding of iPSCs to the surface and is thought to stimulate cell differentiation.

[0101] GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA) and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin. The biotinylation was performed with a superfluous amount of biotin (EZ-Link™ Sulfo-NHS-LC-Biotin, Thermo Fischer Scientific, USA) to make sure that biotin was attached to all available sites of the proteins GDF5, TGFβ-1 and TGFβ-3.

[0102] The excess amount of biotin that was not attached was discarded using repeated wash and centrifugation according to Thermo Fisher's biotinylation protocol. The success of the biotinylation was controlled using a HABA test (HABA/Avidin Reagent, Sigma Aldrich, USA). The HABA test ensured that the number of biotins attached to the proteins were within specified limits according to the producers.

[0103] The biotinylated proteins 80 with a known concentration, 40 nM in suspension, were attached to the streptavidin 60 coated gold nanoparticles 20 and incubated at 4° C. overnight. Similarly, to the previous steps, superfluous proteins (GDF5, TGFβ-1 and TGFβ-3) were rinsed off with PBS. After functionalisation the surfaces were stored in 4° C. until c-iPSCs were seeded and differentiated on the gradient surfaces. A functionalised gradient surface 100 is shown schematically in FIG. 2e.

[0104] During SEM analysis of the gradient surfaces, the particle density where the c-iPSCs had a response that was of interest were identified (500 to 1500 particles/μm.sup.2). Densities within the range of interest were reproduced on new homogenous density (h.d.) surfaces (without a gradient pattern) (Nano Surfaces, Cline Scientific AB, Sweden).

[0105] H.d. surfaces with a particle density lower (400 particles/μm.sup.2) than the particle density of interest (500 to 1500 particles/μm.sup.2) were used as controls.

[0106] Gradient surfaces with only streptavidin and laminin were used as reference surfaces.

[0107] Several h.d. surfaces within the range of 500 to 1500 particles/μm.sup.2 were reproduced and tested, among others 575 particles/μm.sup.2, 652 particles/μm.sup.2, 729 particles/μm.sup.2, 768 particles/μm.sup.2, 792 particles/μm.sup.2, 859 particles/μm.sup.2, 1017 particles/μm.sup.2, 1027 particles/μm.sup.2, 1083 particles/μm.sup.2, 1145 particles/μm.sup.2, and 1367 particles/μm.sup.2.

[0108] The h.d. surfaces and control surfaces were prepared using the same method as described above.

[0109] Results

[0110] Throughout the experiments, the gradient surfaces were necessary material to study how the cells are affected by morphogen protein at different concentrations.

[0111] A montage of SEM pictures taken at different positions with 1 mm intervals along the substrate 100 having a gradient surface 110 is provided in FIG. 1. A schematic view of such substrate 100 with the gradient surface 110 is provided in FIG. 2a.

[0112] FIG. 2e shows a schematic illustration of how the morphogens 80 are attached to the gold nanoparticles 20 in a gradient pattern via the linker streptavidin 60 and with laminin 40 between particles to ameliorate cell adhesion.

[0113] As described above, after functionalization of the gradients, seeding and differentiation of c-iPSCs, the gradient surfaces were scanned to visualize the cells. New h.d. surfaces having particle densities in the range where c-iPSCs had shown favourable response were produced, based on the cell response found on the gradient surfaces.

[0114] The resulting cell behaviour from gradient surfaces and h.d. surfaces functionalized with morphogen proteins and laminin are presented below.

[0115] Differentiation on Gradient Surfaces and h.d. Surfaces, and c-iPSC Analysis on Gradient Surfaces and h.d. Surfaces

[0116] Method—Differentiation on Surfaces

[0117] Each gradient surface and h.d. surface were seeded with 50 000 cells/cm.sup.2 and incubated in 6 well plates at 37° C. in a humidity chamber for two hours. Thereafter, 2 ml maintenance medium was applied to each well. The maintenance medium was a commercially available medium (Cellartis® DEF-CS™ 500 Basal Medium with Additives, Cellartis, Sweden).

[0118] The next day, 2 ml chondrocyte differentiation medium, a previously published modified medium (Nguyen, Hagg et al. 2017) (Table 1), was added with different compositions depending on which morphogen the gradient surface was functionalized with.

[0119] To gradient surfaces functionalized with TGFβ-1, TGFβ-3 was added in the differentiation medium.

[0120] To gradient surfaces functionalized with TGFβ-3, TGFβ-1 was added to the differentiation medium.

[0121] GDF5 gradient surfaces had both TGFβ-1 and TGFβ-3 added in the differentiation medium, as they are known to be essential for differentiation of c-iPSCs into chondrocytes.

[0122] The differentiation medium was changed every second day during the five-day differentiation period.

[0123] Method c-iPSC Analysis

[0124] In order to analyse a middle step of the differentiation process, the results from the differentiation on the surfaces (both controls, gradient surfaces and h.d. surfaces) were analysed. Hence, this method step is not necessary to differentiate the c-iPSCs to chondrocytes but serves to provide insight into the differentiation process in the first place.

[0125] Previous preliminary studies performed by the inventors using c-iPSCs and patient-derived chondrocytes for differentiation on gradient surfaces showed that especially chondrocytes were difficult to maintain attached to the gradient surface after five days. Therefore, after five days of differentiation on the gradient surfaces in differentiation medium, the cells were fixed with Histofix™ (Histolab Products AB, Sweden), nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (ProLong™ Gold Antifade Mountant with DAPI, Thermo Fisher Scientific, USA), and the surfaces were mounted on glass slides. Cell analysis was performed using the fluorescence imaging microscope IN Cell Analyzer 6000 (IN Cell Analyzer 6000, GE Healthcare, United Kingdom), Eclipse 90i (Eclipse 90i, Nikon Instruments, Japan) and Confocal microscope (Nikon A1, Nikon Instruments, Japan).

[0126] The results from the cell analysis for the different surfaces produced are presented below.

TABLE-US-00001 TABLE 1 Differentiation medium.sup.1 Component Concentration Company DMEM, high glucose, L- 4500 mg/L Gibco Glutamine and HEPES. PEST added Insulin-Transferrin- 1/0.55/0.00067 g/L Thermo Fisher Scientific Selenium Ascorbic acid 14 μg/ml Sigma Aldrich Dexamethasone 0.1 μM Merck Linoleic acid  5 μg/ml Sigma Aldrich Sodium Pyruvate 1 mM Sigma Aldrich TGFβ-1 10 ng/ml R&D Systems TGFβ-3 10 ng/ml R&D Systems .sup.1Components and concentrations of the used for differentiation of c-iPSCs and for pellet culture. The first six components were always included in the medium. TGFβ-1 and TGFβ-3 were added to the medium dependent on which molecule was functionalized on the surface. TGFβ-1 was not included in the medium for a TGFβ-1 gradient/surface and TGFβ-3 was not included in the medium for a TGFβ-3 gradient/surface. Both TGFβ-1 and TGFβ-3 were added in the medium for GDF5 gradients/surfaces.

[0127] Results

[0128] Laminin Control Gradient Surfaces

[0129] Laminin gradient surfaces (without morphogens) were used as controls to compare against the cellular responses from the morphogen gradient surfaces and h.d. surfaces. Cells on all replicated laminin gradient surfaces resulted in formation of condensed semi-circular structures consisting of cell free cavities, as shown in areas A1 and A2 in FIG. 3.

[0130] GDF5 Gradients and h.d. Surfaces

[0131] Compared to results on laminin control gradient surfaces, c-iPSCs differentiated on GFD5 gradient surfaces resulted in another type of cell behaviour, condensed cell clusters/aggregates (termed budding zones), at a particle density of 500 to 1500 particles/μm.sup.2 (FIG. 4), determined using SEM (FIGS. 1a and 1b). Larger cell masses were observed over the entire surface, but in the budding zone, small cell budding clusters were formed.

[0132] The differentiation process of iPSCs into chondrocytes is initiated by mesenchymal condensation, meaning reduction of intercellular spaces and is followed by continued differentiation. Cellular condensation is an important part of the differentiation process during chondrogenesis and skeletogenesis. To achieve a condensation process, the composition of the cellular microenvironment is crucial (Tacchetti, Tavella et al. 1992).

[0133] New homogenous density surfaces (h.d. surface) with a particle density in the range of the budding zone (500 to 1500 particles/μm.sup.2) was used to create surfaces with a homogenous GDF5 density (FIG. 5). FIG. 5A shows a GDF5 h.d. surface with a particle density at 400 particles/μm.sup.2, i.e., lower than the particle density at the observed budding zone. As seen in FIG. 5B, the same cell budding phenomena as observed on the gradient surfaces was also observed on an h.d. surface having a particle density of 900 particles/μm.sup.2. For comparison, on the h.d. surfaces with the lower particle density (400 particles/μm.sup.2) (FIG. 5A), cells formed larger gatherings as well but no indications of budding formations.

[0134] Conclusion

[0135] The result found for the GDF5 gradient surfaces was especially noteworthy, where cell budding zones were observed in a specific density range (500 to 1500 particles/μm.sup.2), implicating the role of GDF5 at a certain concentration in a condensing budding formation. As described above, such condensation and budding formation is an indicative factor, though not confirmative, of that chondrogenesis has been induced.

[0136] The results were affirmed and repeated on specific h.d. surfaces where the cells behaved in the same way as observed on gradient surfaces, generating budding areas and a condensed structure. The appearance of cell clusters on both GDF5 gradient surfaces and h.d. surfaces provide important information for inducing the condensation and budding formation in chondrocyte differentiation, indicating an early stage in the formation of condensed chondrogenic structures in c-iPSCs. These results suggest that the cell budding responses are dependent on specific GDF5 concentrations and that the particular density interval was essential to induce cellular clusters.

[0137] TGFβ-1 Gradients and h.d. Surfaces

[0138] c-iPSCs on all replicated TGFβ-1 gradient surfaces formed semi-circular structures in areas where the particle density was low, a behaviour comparable to control gradient surfaces with only laminin. At higher TGFβ-1 densities, this cell response was not observed; the cell development stimulated by TGFβ-1 resulted in evenly distributed cells. The cell responses were studied further on h.d. surfaces without a gradient, where separate surfaces with low and high particle density were used to compare the cellular responses when exposed to different single morphogen densities. At low TGFβ-1 h.d. surfaces (600 particles/μm.sup.2), cells tended to form the mentioned semi-circular structures consisting of cell free cavities covering the h.d. surfaces (FIG. 5C). At high TGFβ-1 h.d. surfaces (2000 particles/μm.sup.2), the cells were evenly distributed over the h.d. surfaces (FIG. 5D).

[0139] Conclusion

[0140] It has been suggested that TGFβ, as well as GDF5, induce condensation (Wang, Rigueur et al. 2014). Surprisingly, c-iPSCs grown on TGFβ-1 gradient surfaces did not show condensational tendencies and did not respond similar to cells stimulated with GDF5 as no budding zone was observed on the TGFβ-1 gradient surfaces. Since TGFβ-1 is suggested as relevant to condensation specifically, the lack of budding was somewhat surprising.

[0141] At lower TGFβ-1 densities along the gradient surface, cells tended to form another structure in the shape of smaller clusters in a semicircular shape. However, this is more likely due to the effect of laminin. The low h.d. surfaces (600 particles/μm.sup.2) resulted, as expected from results from gradient surfaces, in that cells formed cell-free cavities, while cells on high h.d. surfaces (2000 particles/μm.sup.2) were evenly distributed, and no cavities were formed.

[0142] As cells on all replicated laminin gradient surfaces induced the same pattern seen at lower TGFβ-1 densities, it is most likely that low TGFβ-1 densities are not enough to override the effect of laminin. At higher densities, the effect of TGFβ-1 takes over and the cell uniformity is evident.

TGFβ-3 Gradients and h.d. Surfaces

[0143] c-iPSCs on TGFβ-3 gradient surfaces did not generate any visual condensing cell response, i.e., the induced cell development resulted in evenly distributed cells. To compare and confirm the results, h.d. surfaces were used at high and low morphogen densities. Cells were evenly distributed over the gradient surfaces as well as the h.d. surfaces throughout the differentiation both at low (600 particles/μm.sup.2) and high (1900 particles/μm.sup.2) density (FIGS. 5E and 5F). No budding zone was observed and no tendency to otherwise condense; the cells were evenly distributed at microscopic and ocular inspection.

[0144] Conclusion

[0145] Results from gradient surfaces with TGFβ-3 did not show any indications of budding formation or condensed structures. Thus, the conclusion is that TGFβ-3 is not as relevant as GDF5 for condensation and to generate budding zones, though being important for the cell development and differentiation.

[0146] As shown in FIGS. 5E and 5F, the h.d. surfaces with a high (1900 particles/μm.sup.2) and a low (600 particles/μm.sup.2) particle density showed no specific cell responses such as observed primarily for GDF5, but also for TGFβ-1. In comparison to TGFβ-1, low densities of TGFβ-3 were not overridden by laminin and no cavities were formed, thus confirming TGFβ-3 to be a stronger influence than TGFβ-1 in this setting.

[0147] Overall, it may be concluded that no budding formation was observed for either TGFβ-1 or TGFβ-3, and when comparing the three different proteins, (GDF5, TGFβ-1, and TGFβ-3) it was clear that GDF5 induced condensation of cells to a higher extent than TGFβ-1 and TGFβ-3.

[0148] GDF5 and TGFβ-1 are both molecules that are involved in cellular condensation (Francis-West, Abdelfattah et al. 1999, Coleman, Vaughan et al. 2013; Kim et al. 2014). However, only on the GDF5 surfaces cells formed condensed budding structures. TGFβ-3 have shown to inhibit cellular condensation, which may correlate to the results in our in vitro setting (Jin et al. 2010). Thus, the similarities between TGFβ-1 and TGFβ-3 were somewhat surprising as well as the lack of similarities between the results for TGFβ-1 and GDF5.

[0149] Immunostaining on c-iPSC Seeded Gradient Surfaces and Histological Pellet Sections

[0150] Method

[0151] c-iPSCs on GDF5 gradient surfaces were stained for the proteins SOX9 and TBX3. Histological pellet sections where stained for TBX3. Also, TGFβ-3 h.d. surfaces were used as controls, as TGFβ-3 is known to provide increased aggrecan expression. A differentiation medium comprising GDF5 was added to the TGFβ-3 h.d. control surfaces to verify the importance of the surface densities of GDF5 and not only the presence of GDF5 per se. Paraffin embedded histological sections were deparaffinised and dehydrated prior immunostaining. Cells on surfaces and in pellets sections were permeabilised prior to immunostaining using 0.1% tritonX-100 (TritonX-100, Sigma Aldrich, USA) in PBS and incubated in room temperature for 10 minutes.

[0152] After permeabilisation, cells were incubated in blocking medium (2% BSA (Bovine Serum Albumin, Sigma Aldrich, USA), 0.1% tritonX-100, 100 mM glycine (Glycin, Riedel-de Haen A G, Germany) dissolved in PBS) for 15 minutes, followed by incubation in 500 μl of diluted specific primary antibody (Table 2) at 4° C. overnight.

[0153] The next day, cells were rinsed and washed with PBS three times for 3 minutes. Thereafter, cells were incubated with blocking medium for 15 minutes followed by incubation with a secondary antibody for 2 hours in a humidity chamber (Table 2). After three rinsing/washing steps in PBS, each for 3 minutes, the cells and pellet sections were mounted with ProLong™ Gold Antifade Mountant media (ProLong™ Gold Antifade Mountant with DAPI, Thermo Fisher Scientific, USA) and were imaged using fluorescence microscopy (Eclipse 90i, Nikon Instruments, Japan) and confocal microscopy (Nikon A1, Nikon Instruments, Japan).

[0154] Results

[0155] The c-iPSC behaviour resulting in a budding zone, found solely on GDF5 coated surfaces, were further studied using immunostaining for specific protein expressions, as described in the method section above.

[0156] Transcription factor SOX9 (SOX9) is well known to be an important regulating protein in chondrocyte differentiation, mesenchymal condensations and for formation and secretion of cartilage structural proteins. SOX9 is expressed in chondroprogenitors and differentiated chondrocytes but not in hypertrophic chondrocytes, indicating its importance in early cartilage development (Hino, Saito et al. 2014, Lefebvre and Dvir-Ginzberg 2017). Mutations in the SOX9 gene that leads to altered levels of SOX9 proteins is suggested to be linked to skeletal- and degeneration diseases (Lefebvre and Dvir-Ginzberg 2017). SOX9 is a chondrogenic marker and is therefore expected to be present during differentiation (Hino, Saito et al. 2014, Lefebvre and Dvir-Ginzberg 2017).

[0157] T-box transcription factor 3 (TBX3) is considered important for, and significantly expressed during, limb formation in early development (Sheeba and Logan 2017). Mutations, which have resulted in decreased protein expressions, are found to cause developmental defects such as ulnar-mammary syndrome (Bamshad, Le et al. 1999).

[0158] Hence, the presence of SOX9 and TBX3 are of high relevance when determining if differentiation of stem cells into chondrocytes have taken place.

[0159] Immunostaining using selected antibodies (Table 2) visualized expression of SOX9 and TBX3 respectively, in c-iPSCs differentiated on GDF5 gradient surfaces. All cell aggregates contained high SOX9 expressions (FIG. 6).

[0160] TBX3 expression was indeed observed to be specifically expressed in the buds found in the budding zone (500 to 1500 particles/μm.sup.2), primarily in the nuclei but also in forms of highly stained vesicles around nuclei (FIG. 7).

[0161] To validate these elevated levels of TBX3 observed in buds on GDF5 gradient surfaces, h.d. surfaces with a particle density corresponding to the budding zone were used.

[0162] For comparison, h.d. surfaces with GDF5 at low particle density (i.e., lower than the particle density within the GDF5 budding zone—400 particles/μm.sup.2), TGFβ-3 (at the same particle density as within the GDF5 budding zone—500 particles/μm.sup.2), TGFβ-3 surfaces (at the same particle density as within the GDF5 budding zone—500 particles/μm.sup.2) with GDF5 added to the medium, and finally, glass slides coated with Coat-1 without added biomolecules were used as controls.

[0163] It was found that the TBX3 expression in cells differentiated on TGFβ-3 surfaces was more upregulated compared to all other controls, but still less than results from GDF5 surfaces. This finding confirms that TBX3 is more expressed at the GDF5 levels of the budding zone, as shown in FIG. 8.

[0164] Cell condensation is seen to some extent (FIG. 8), but not in the way that budding zones are observed on the GDF5 gradient or h.d. surfaces within the budding zone range (500 to 1500 particles/μm.sup.2).

[0165] c-iPSC histological pellets were also stained for TBX3 using immuno-histochemistry (FIG. 9 A3-6, B3, C3). TBX3 were specifically induced in the area of the pellets with upregulated amounts of GAG, i.e., the induction of TBX3 could be specifically correlated to expression of GAG and was not correlated with the generally elevated expression of collagens.

TABLE-US-00002 TABLE 2 Antibodies and isotype control antibodies.sup.2 Target Primary antibody Isotype control Secondary antibody TBX3 Anti-TBX3 Mouse IgG1 kappa Alexa Fluor 546, Antibody, LS- Isotype Control, Invitrogen A11030, C133955, Nordic 14-4714-82, ThermoFisher Biosite eBioscience, USA Scientific, USA TBX3 TBX3 polyclonal Alexa Fluor 594, antibody, Invitrogen Invitrogen 42-4800, A-21207, ThermoFisher ThermoFisher Scientific, USA Scientific, USA SOX9 Anti-SOX9, normal mouse Alexa Fluor 546, ab76997, Abcam, IgG, sc-2025, Invitrogen A21133, United Kingdom Santa Cruz, USA ThermoFisher Scientific, USA .sup.2Antibodies and isotype control antibodies used for immunostaining of cells on GDF5 gradients.

[0166] Pellet Cultures from GDF5 Gradient Surfaces, GDF5 h.d. Surface and Chondrocyte Control Experiments

[0167] Method—GDF5 Surface Pellets and Chondrocyte Pellet Control Experiments

[0168] The method described below was conducted using: [0169] GDF5 gradient surfaces seeded with c-iPSCs; [0170] GDF5 h.d. surfaces seeded with c-iPSCs—as control, having nanoparticles in a particle density of 440 particles/μm.sup.2; [0171] GDF5 h.d. surfaces seeded with c-iPSCs—having nanoparticles in a particle density within budding zone range. Such surface had particle densities as disclosed in the section “Preparation of gradient surfaces of GDF5, TGFβ-1 and TGFβ-3”.

[0172] After five days of differentiation on GDF5 gradient surfaces or h.d. surfaces, c-iPSCs were removed from each respective surface using trypsin-EDTA (0.05%) (ThermoFisher Scientific, USA).

[0173] The trypsin-EDTA reaction was quenched with human serum and the cells were transferred into 15 ml conical tubes and centrifuged for 5 minutes at 1200 g in 2 ml differentiation medium with addition of both TGFβ-1 and TGFβ-3 (Table 1), to form a three-dimensional (3D) structure (also referred to as a “pellet” herein), which were to be further differentiated in a differentiation medium.

[0174] Said pellets were then stored in 96 well plates in 200 μl differentiation medium (Table 1) in a 37° C. incubator with 5% CO.sub.2 and were differentiated for five weeks. The medium was changed every third day.

[0175] To compare results from the pellets with differentiated c-iPSC derived from GDF5 gradient surfaces and GDF5 h.d. surfaces (using the method presented above), two control setups were employed. The aim of the control setups was to compare the results from the three pellets with differentiated c-iPSC with pellets derived from healthy actual chondrocytes to verify that chondrocytes derived from c-iPSC behaved in the same way as ordinary, patient-derived chondrocytes, as the observed expression of SOX9 and TBX3 is not sufficient to conclude this, although being strongly indicative. Chondrocytes cultured as described in the paragraph “Cell culturing of chondrocytes for control experiments” were used in the two control setups. In the first control experiment, chondrocytes were seeded to a GDF5 gradient surface and later formed into a pellet with the method described above. In the second control, chondrocytes were formed through a conventional pellet culture method not using a surface or gradient surface but with 5% human serum added to the differentiation medium. Pellets from the two controls were formed using the method described above.

[0176] Chondrocyte pellets were differentiated for two weeks since it has been shown that after two weeks, chondrocytes from OA donors have started to express high levels of both collagen type II and proteoglycans (Tallheden, Bengtsson et al. 2005). After the differentiation period, all pellets were fixated with Histofix™ (Histolab Products AB, Sweden) overnight and then stored in 70% ethanol.

[0177] Thereafter, all pellets were sent to HistoCenter (Möndal, Sweden) for sectioning and staining with Alcian Blue van Gieson to visualize the extracellular matrix (ECM) composition of the pellets. The pellet compositions were analysed using a light microscope (Eclipse 90i, Nikon Instruments, Japan). Histological sections were stained with Alcian Blue van Gieson and displayed areas with a high amount of both blue colored glycosaminoglycans (GAGs) and red/purple coloured collagens.

[0178] Results

[0179] As described in the method section above, cells were differentiated on GDF5 gradient surfaces and GDF5 h.d. surfaces (one particle density from the range of the budding zone, 500 to 1500 particles/μm.sup.2, and one density with a lower particle density as a control, 400 particles/μm.sup.2) before being cultured as 3D micromass pellets suspended in differentiation medium (Table 1). A control having a particle density higher than 1500 particles/μm.sup.2 was not tested partly due to that at higher concentrations than 1500, cells have a tendency to come off the substrate. As can be seen in FIG. 4, cell growth is present also at higher particle densities, however, no budding formation is observed. Hence, on an h.d. surface, cells have difficulty to attach to the surface as a result of the higher particle densities causing such reference/control specimen to be unfit for control experiments.

[0180] The blue colour was mainly concentrated to one area of the pellets while the red/purple was expressed around the blue area and mostly in the distinct edges.

[0181] Two different chondrocyte pellets were used as controls. One using a GDF5 gradient surface and the same differentiation medium as the c-iPSC pellets (Table 1) (FIGS. 9 D1 & D2) and the other without GDF5 gradient or h.d. surface but the addition of 5% human serum to the medium (FIGS. 9 E1 & E2). Both chondrocyte control pellets seem to, compared to the c-iPSC pellets, have a more compact pellet structure. However, according to the colouring, the structural components in all pellets are similar.

[0182] The coloring of the c-iPSC pellets obtained from GDF5 gradient surfaces and h.d. surfaces were compared to chondrocyte control pellets and all indicated the presence of both GAGs (blue) and collagens (red/purple). This implies that the differentiation of the c-iPSC pellets using the protocol including GDF5 gradients and surfaces drives the cells meritoriously towards the chondrocyte linage.

[0183] Further, antibody staining (primary antibody collagen II; MP Biomedicals) also showed that differentiation of the c-iPSC using the protocol including GDF5 gradients (cf. FIG. 10A) results in expression of type II collagen, similar to chondrocytes maintained in serum (cf. FIG. 10B). Interestingly, chondrocytes maintained in the absence of serum, but first differentiated on a GDF5 gradient, essentially maintained the ability to express type II collagen (FIG. 10C). This ability was however lost in chondrocytes maintained also in the absence of serum, but first differentiated on a TGFβ-3 gradient (FIG. 10D), further indicating the need of GDF5 to drive the cells towards cartilage and not e.g., bone tissue.

REFERENCES

[0184] Aumailley, M. (2013). “The laminin family.” Cell Adh Migr 7(1): 48-55. [0185] Bamshad, M., T. Le, W. S. Watkins, M. E. Dixon, B. E. Kramer, A. D. Roeder, J. C. Carey, S. Root, A. Schinzel, L. Van Maldergem, R. J. Gardner, R. C. Lin, C. E. Seidman, J. G. Seidman, R. Wallerstein, E. Moran, R. Sutphen, C. E. Campbell and L. B. Jorde (1999). “The spectrum of mutations in TBX3: Genotype/Phenotype relationship in ulnar-mammary syndrome.” Am J Hum Genet 64(6): 1550-1562. [0186] Borestrom, C., S. Simonsson, L. Enochson, N. Bigdeli, C. Brantsing, C. Ellerstrom, J. Hyllner and A. Lindahl (2014). “Footprint-free human induced pluripotent stem cells from articular cartilage with redifferentiation capacity: a first step toward a clinical-grade cell source.” Stem Cells Transl Med 3(4): 433-447. [0187] Coleman, C. M., E. E. Vaughan, D. C. Browe, E. Mooney, L. Howard and F. Barry (2013). “Growth differentiation factor-5 enhances in vitro mesenchymal stromal cell chondrogenesis and hypertrophy.” Stem Cells Dev 22(13): 1968-1976. [0188] Francis-West, P. H., A. Abdelfattah, P. Chen, C. Allen, J. Parish, R. Ladher, S. Allen, S. MacPherson, F. P. Luyten and C. W. Archer (1999). “Mechanisms of GDF-5 action during skeletal development.” Development 126(6): 1305-1315. [0189] Goldring M B. Chondrogenesis, chondrocyte differentiation and articular cartilage metabolism in health and osteoarthrosis. Therapeutic Advances in Musculoskeletal Disease. 2012 August; 4(4): 269-285. [0190] Hino, K., A. Saito, M. Kido, S. Kanemoto, R. Asada, T. Takai, M. Cui, X. Cui and K. Imaizumi (2014). “Master regulator for chondrogenesis, Sox9, regulates transcriptional activation of the endoplasmic reticulum stress transducer BBF2H7/CREB3L2 in chondrocytes.” J Biol Chem 289(20): 13810-13820. [0191] Kim Y I. Ryu J., Yeo J E., Choi Y J., Kim Y S., Ko K. and Koh Y. Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells. Biochemical and Biophysical Research Communications. Volume 450, Issue 4, 8 Aug. 2014, Pages 1593-1599. [0192] Lefebvre, V. and M. Dvir-Ginzberg (2017). “SOX9 and the many facets of its regulation in the chondrocyte lineage.” Connect Tissue Res 58(1): 2-14. [0193] Nguyen, D., D. A. Hagg, A. Forsman, J. Ekholm, P. Nimkingratana, C. Brantsing, T. Kalogeropoulos, S. Zaunz, S. Concaro, M. Brittberg, A. Lindahl, P. Gatenholm, A. Enejder and S. Simonsson (2017). “Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink.” Sci Rep 7(1): 658. [0194] Jin E J., Park K S., Kim D., Lee Y S., Sonn J., Chang J., Bang O., and Kang, S S., TGF-beta 3 inhibits chondrogenesis by suppressing precartilage condensation through stimulation of N-cadherin shedding and reduction of cRREB-1 expression. Molecules and cells. 29. 2010. 425-32. [0195] Sheeba, C. J. and M. P. Logan (2017). “The Roles of T-Box Genes in Vertebrate Limb Development.” Curr Top Dev Biol 122: 355-381. [0196] Tacchetti, C., S. Tavella, B. Dozin, R. Quarto, G. Robino and R. Cancedda (1992). “Cell condensation in chondrogenic differentiation.” Exp Cell Res 200(1): 26-33. [0197] Tallheden T., Bengtsson C., Brantsing C., Sjögren-Jansson E., Carlsson L., Peterson L., Brittberg M., and Lindahl A. Proliferation and differentiation potential of chondrocytes from osteoarthritic patients. Arthritis Research & Therapy. 2005; 7(3): R560-R568. [0198] Wang, W., D. Rigueur and K. M. Lyons (2014). “TGFbeta signaling in cartilage development and maintenance.” Birth Defects Res C Embryo Today 102(1): 37-51.