Chondrogenic Pellet Research Articles

Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: a revision of the prohypertrophic signalling network concept

BackgroundIn vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-β is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-β can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors – compound A (CompA) and LDN-212854 (LDN21) – in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-β receptors during MSC in vitro chondrogenesis.MethodsStandard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-β and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1–34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice.ResultsAll groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups.ConclusionsOverall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-β that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-β-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

Osteoporosis GWAS-implicated DNM3 locus contextually regulates osteoblastic and chondrogenic fate of mesenchymal stem/progenitor cells through oscillating miR-199a-5p levels.

Genome wide association study (GWAS)-implicated bone mineral density (BMD) signals have been shown to localize in cis-regulatory regions of distal effector genes using 3D genomic methods. Detailed characterization of such genes can reveal novel causal genes for BMD determination. Here, we elected to characterize the "DNM3" locus on chr1q24, where the long non-coding RNA DNM3OS and the embedded microRNA MIR199A2 (miR-199a-5p) are implicated as effector genes contacted by the region harboring variation in linkage disequilibrium with BMD-associated sentinel single nucleotide polymorphism, rs12041600. During osteoblast differentiation of human mesenchymal stem/progenitor cells (hMSC), miR-199a-5p expression was temporally decreased and correlated with the induction of osteoblastic transcription factors RUNX2 and Osterix. Functional relevance of miR-199a-5p downregulation in osteoblastogenesis was investigated by introducing miR-199a-5p mimic into hMSC. Cells overexpressing miR-199a-5p depicted a cobblestone-like morphological change and failed to produce BMP2-dependent extracellular matrix mineralization. Mechanistically, a miR-199a-5p mimic modified hMSC propagated normal SMAD1/5/9 signaling and expressed osteoblastic transcription factors RUNX2 and Osterix but depicted pronounced upregulation of SOX9 and enhanced expression of essential chondrogenic genes ACAN, COMP, and COL10A1. Mineralization defects, morphological changes, and enhanced chondrogenic gene expression associated with miR-199a-5p mimic over-expression were restored with miR-199a-5p inhibitor suggesting specificity of miR-199a-5p in chondrogenic fate specification. The expression of both the DNM3OS and miR-199a-5p temporally increased and correlated with hMSC chondrogenic differentiation. Although miR-199a-5p overexpression failed to further enhance chondrogenesis, blocking miR-199a-5p activity significantly reduced chondrogenic pellet size, extracellular matrix deposition, and chondrogenic gene expression. Taken together, our results indicate that oscillating miR-199a-5p levels dictate hMSC osteoblast or chondrocyte terminal fate. Our study highlights a functional role of miR-199a-5p as a BMD effector gene at the DNM3 BMD GWAS locus, where patients with cis-regulatory genetic variation which increases miR-199a-5p expression could lead to reduced osteoblast activity.

Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs

Optogenetics is a rapidly advancing technology combining photochemical, optical, and synthetic biology to control cellular behavior. Together, sensitive light-responsive optogenetic tools and human pluripotent stem cell differentiation models have the potential to fine-tune differentiation and unpick the processes by which cell specification and tissue patterning are controlled by morphogens. We used an optogenetic bone morphogenetic protein (BMP) signaling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We engineered light-sensitive hESCs through CRISPR-Cas9-mediated integration of the optoBMP system into the AAVS1 locus. The activation of optoBMP with blue light, in lieu of BMP growth factors, resulted in the activation of BMP signaling mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells in the dark. Furthermore, cells differentiated with light could form chondrogenic pellets consisting of a hyaline-like cartilaginous matrix. Our findings indicate the applicability of optogenetics for understanding human development and tissue engineering.

Senescence during early differentiation reduced the chondrogenic differentiation capacity of mesenchymal progenitor cells.

Introduction: Mesenchymal stromal/progenitor cells (MSCs) are promising for cartilage cell-based therapies due to their chondrogenic differentiation capacity. However, MSCs can become senescent during in vitro expansion, a state characterized by stable cell cycle arrest, metabolic alterations, and substantial changes in the gene expression and secretory profile of the cell. In this study, we aimed to investigate how senescence and the senescence-associated secretory phenotype (SASP) affect chondrogenic differentiation of MSCs. Methods: To study the effect of senescence, we exposed MSCs to gamma irradiation during expansion or during chondrogenic differentiation (the pellet culture). Western blot analysis was used to evaluate MSCs response to the chondrogenic inductor TGF-β. Results: When senescence was induced during expansion or at day 7 of chondrogenic differentiation, we observed a significant reduction in the cartilage matrix. Interestingly, when senescence was induced at day 14 of differentiation, chondrogenesis was not significantly altered. Moreover, exposing chondrogenic pellets to the medium conditioned by senescent pellets had no significant effect on the expression of anabolic or catabolic cartilage markers, suggesting a neglectable paracrine effect of senescence on cartilage generation in our model. Finally, we show that senescent MSCs showed lower phosphorylated SMAD2 levels after TGFβ1 stimulation than control MSCs. Conclusion: Overall, these results suggest that the occurrence of senescence in MSCs during expansion or early differentiation could be detrimental for cartilage tissue engineering.

The Upscale Manufacture of Chondrocytes for Allogeneic Cartilage Therapies.

Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of the current two-stage autologous procedures. Upscale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. In this study, we assess a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum®) for adult chondrocyte manufacture. Chondrocytes were isolated from knee arthroplasty-derived cartilage (n = 5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL-supplemented cultures were then expanded in the Quantum bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterized in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays), and single telomere length analysis. Quantum expansion of chondrocytes resulted in 86.4 ± 38.5 × 106 cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 106 cells. This related to 3.0 ± 1.0 population doublings in the Quantum bioreactor, compared with 2.1 ± 0.6 and 1.3 ± 1.0 on TCP in hPL- and FBS-supplemented media, respectively. Quantum- and TCP-expanded cultures retained equivalent chondropotency and mesenchymal stromal cell marker immunoprofiles, with only the integrin marker, CD49a, decreasing following Quantum expansion. Quantum-expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture, however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c, and CD51/61 compared with FBS cultures. Quantum expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum hollow-fiber bioreactor. This rapid, upscale expansion does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavorable in terms of retaining chondrogenic capacity.

Human fetal cartilage-derived chondrocytes and chondroprogenitors display a greater commitment to chondrogenesis than adult cartilage resident cells.

Obtaining regeneration-competent cells and generating high-quality neocartilage are still challenges in articular cartilage tissue engineering. Although chondroprogenitor cells are a resident subpopulation of native cartilage and possess a high capacity for proliferation and cartilage formation, their potential for regenerative medicine has not been adequately explored. Fetal cartilage, another potential source with greater cellularity and a higher cell-matrix ratio than adult tissue, has been evaluated for sourcing cells to treat articular disorders. This study aimed to compare cartilage resident cells, namely chondrocytes, fibronectin adhesion assay-derived chondroprogenitors (FAA-CPCs) and migratory chondroprogenitors (MCPs) isolated from fetal and adult cartilage, to evaluate differences in their biological properties and their potential for cartilage repair. Following informed consent, three human fetal and three adult osteoarthritic knee joints were used to harvest the cartilage samples, from which the three cell types a) chondrocytes, b) FAA-CPCs, and MCPs were isolated. Assessment parameters consisted of flow cytometry analysis for percentage expression of cell surface markers, population doubling time and cell cycle analyses, qRT-PCR for markers of chondrogenesis and hypertrophy, trilineage differentiation potential and biochemical analysis of differentiated chondrogenic pellets for total GAG/DNA content. Compared to their adult counterparts, fetal cartilage-derived cells displayed significantly lower CD106 and higher levels of CD146 expression, indicative of their superior chondrogenic capacity. Moreover, all fetal groups demonstrated significantly higher levels of GAG/DNA ratio with enhanced uptake of collagen type 2 and GAG stains on histology. It was also noted that fetal FAA CPCs had a greater proliferative ability with significantly higher levels of the primary transcription factor SOX-9. Fetal chondrocytes and chondroprogenitors displayed a superior propensity for chondrogenesis when compared to their adult counterparts. To understand their therapeutic potential and provide an important solution to long-standing challenges in cartilage tissue engineering, focused research into its regenerative properties using in-vivo models is warranted.

In Vitro Mineralisation of Tissue-Engineered Cartilage Reduces Endothelial Cell Migration, Proliferation and Tube Formation.

Tissue engineering bone via endochondral ossification requires the generation of a cartilage template which undergoes vascularisation and remodelling. While this is a promising route for bone repair, achieving effective cartilage vascularisation remains a challenge. Here, we investigated how mineralisation of tissue-engineered cartilage affects its pro-angiogenic potential. To generate in vitro mineralised cartilage, human mesenchymal stromal cell (hMSC)-derived chondrogenic pellets were treated with β-glycerophosphate (BGP). After optimising this approach, we characterised the changes in matrix components and pro-angiogenic factors by gene expression analysis, histology and ELISA. Human umbilical vein endothelial cells (HUVECs) were exposed to pellet-derived conditioned media, and migration, proliferation and tube formation were assessed. We established a reliable strategy to induce in vitro cartilage mineralisation, whereby hMSC pellets are chondrogenically primed with TGF-β for 2 weeks and BGP is added from week 2 of culture. Cartilage mineralisation determines loss of glycosaminoglycans, reduced expression but not protein abundance of collagen II and X, and decreased VEGFA production. Finally, the conditioned medium from mineralised pellets showed a reduced ability to stimulate endothelial cell migration, proliferation and tube formation. The pro-angiogenic potential of transient cartilage is thus stage-dependent, and this aspect must be carefully considered in the design of bone tissue engineering strategies.

MegaPro, a clinically translatable nanoparticle for in vivo tracking of stem cell implants in pig cartilage defects.

Rationale: Efficient labeling methods for mesenchymal stem cells (MSCs) are crucial for tracking and understanding their behavior in regenerative medicine applications, particularly in cartilage defects. MegaPro nanoparticles have emerged as a potential alternative to ferumoxytol nanoparticles for this purpose. Methods: In this study, we employed mechanoporation to develop an efficient labeling method for MSCs using MegaPro nanoparticles and compared their effectiveness with ferumoxytol nanoparticles in tracking MSCs and chondrogenic pellets. Pig MSCs were labeled with both nanoparticles using a custom-made microfluidic device, and their characteristics were analyzed using various imaging and spectroscopy techniques. The viability and differentiation capacity of labeled MSCs were also assessed. Labeled MSCs and chondrogenic pellets were implanted into pig knee joints and monitored using MRI and histological analysis. Results: MegaPro-labeled MSCs demonstrated shorter T2 relaxation times, higher iron content, and greater nanoparticle uptake compared to ferumoxytol-labeled MSCs, without significantly affecting their viability and differentiation capacity. Post-implantation, MegaPro-labeled MSCs and chondrogenic pellets displayed a strong hypointense signal on MRI with considerably shorter T2* relaxation times compared to adjacent cartilage. The hypointense signal of both MegaPro- and ferumoxytol-labeled chondrogenic pellets decreased over time. Histological evaluations showed regenerated defect areas and proteoglycan formation with no significant differences between the labeled groups. Conclusion: Our study demonstrates that mechanoporation with MegaPro nanoparticles enables efficient MSC labeling without affecting viability or differentiation. MegaPro-labeled cells show enhanced MRI tracking compared to ferumoxytol-labeled cells, emphasizing their potential in clinical stem cell therapies for cartilage defects.

Nsun4 and Mettl3 mediated translational reprogramming of Sox9 promotes BMSC chondrogenic differentiation

The chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) has been used in the treatment and repair of cartilage defects; however, the in-depth regulatory mechanisms by which RNA modifications are involved in this process are still poorly understood. Here, we found that Sox9, a critical transcription factor that mediates chondrogenic differentiation, exhibited enhanced translation by ribosome sequencing in chondrogenic pellets, which was accompanied by increased 5-methylcytosine (m5C) and N6-methyladenosine (m6A) levels. Nsun4-mediated m5C and Mettl3-mediated m6A modifications were required for Sox9-regulated chondrogenic differentiation. Interestingly, we showed that in the 3’UTR of Sox9 mRNA, Nsun4 catalyzed the m5C modification and Mettl3 catalyzed the m6A modification. Furthermore, we found that Nsun4 and Mettl3 co-regulated the translational reprogramming of Sox9 via the formation of a complex. Surface plasmon resonance (SPR) assays showed that this complex was assembled along with the recruitment of Ythdf2 and eEF1α-1. Moreover, BMSCs overexpressing Mettl3 and Nsun4 can promote the repair of cartilage defects in vivo. Taken together, our study demonstrates that m5C and m6A co-regulate the translation of Sox9 during the chondrogenic differentiation of BMSCs, which provides a therapeutic target for clinical implications.

Selection of Highly Proliferative and Multipotent Meniscus Progenitors through Differential Adhesion to Fibronectin: A Novel Approach in Meniscus Tissue Engineering

Meniscus injuries can be highly debilitating and lead to knee osteoarthritis. Progenitor cells from the meniscus could be a superior cell type for meniscus repair and tissue-engineering. The purpose of this study is to characterize meniscus progenitor cells isolated by differential adhesion to fibronectin (FN-prog). Human osteoarthritic menisci were digested, and FN-prog were selected by differential adhesion to fibronectin. Multilineage differentiation, population doubling time, colony formation, and MSC surface markers were assessed in the FN-prog and the total meniscus population (Men). Colony formation was compared between outer and inner zone meniscus digest. Chondrogenic pellet cultures were performed for redifferentiation. FN-prog demonstrated multipotency. The outer zone FN-prog formed more colonies than the inner zone FN-prog. FN-prog displayed more colony formation and a higher proliferation rate than Men. FN-prog redifferentiated in pellet culture and mostly adhered to the MSC surface marker profile, except for HLA-DR receptor expression. This is the first study that demonstrates differential adhesion to fibronectin for the isolation of a progenitor-like population from the meniscus. The high proliferation rates and ability to form meniscus extracellular matrix upon redifferentiation, together with the broad availability of osteoarthritis meniscus tissue, make FN-prog a promising cell type for clinical translation in meniscus tissue-engineering.

In vitro detection of structural changes in chondrogenic mesenchymal stem cell pellets at the nano-scale using nano-sensitive optical coherance tomography

In vitro detection of structural changes in chondrogenic mesenchymal stem cell pellets at the nano-scale using nano-sensitive optical coherance tomography

Cell Source-Dependent In Vitro Chondrogenic Differentiation Potential of Mesenchymal Stem Cell Established from Bone Marrow and Synovial Fluid of Camelus dromedarius.

Simple SummaryThis is the first study to demonstrate the establishment and subsequent analysis of attributes, including the chondrogenic capacity of mesenchymal stem cells (MSCs) from bone marrow (BM) and synovial fluid (SF) from the same donor Camelus dromedarius. MSCs of SF origin were notably more efficient in their chondrogenic capacity and represent a potential source for camel regenerative medicine addressing chondrocyte-related problems.Mesenchymal stem cells (MSCs) are promising multipotent cells with applications for cartilage tissue regeneration in stem cell-based therapies. In cartilage regeneration, both bone marrow (BM-MSCs) and synovial fluid (SF-MSCs) are valuable sources. However, the cellular characteristics and chondrocyte differentiation potential were not reported in either of the camel stem cells. The in vitro chondrocyte differentiation competence of MSCs, from (BM and SF) sources of the same Camelus dromedaries (camel) donor, was determined. Both MSCs were evaluated on pluripotent markers and proliferation capacity. After passage three, both MSCs showed fibroblast-like morphology. The proliferation capacity was significantly increased in SF-MSCs compared to BM-MSCs. Furthermore, SF-MSCs showed an enhanced expression of transcription factors than BM-MSCs. SF-MSCs exhibited lower differentiation potential toward adipocytes than BM-MSCs. However, the osteoblast differentiation potential was similar in MSCs from both sources. Chondrogenic pellets obtained from SF-MSCs revealed higher levels of chondrocyte-specific markers than those from BM-MSCs. Additionally, glycosaminoglycan (GAG) content was elevated in SF-MSCs related to BM-MSCs. This is, to our knowledge, the first study to establish BM-MSCs and SF-MSCs from the same donor and to demonstrate in vitro differentiation potential into chondrocytes in camels.

Adipose Derived Mesenchymal Stem Cells from a Hypoxic Culture Reduce Cartilage Damage.

The method to benifit tissue engineering of adipose-derived stem cells (ADSCs) to cartilage has been an objective of intense research in treating increasing cartilage-related disease. In this study, whether hypoxic expansion would enhance the proliferation and in vitro chondrogenic differentiation of ADSCs was studied, and then hypoxic expansion was applied to reduce cartilage damage in a rat model in vivo. Hypoxic expansion increased the proliferation and decreased the expression of aging-related genes, including p16, p21, and p53, of human ADSCs in comparison with normoxic expansion. In addition, the γH2AX expression was reduced in the hypoxic ADSCs. The chondrogenic markers were enhanced in the hypoxic ADSC differentiated chondrogenic pellets, including SOX9 on day 7 and gene expressions of COL 2 and COL 10 on day 21. To determine the in vitro chondrogenic differentiation potential of ADSCs, ADSC differentiated 21-day chondrogenic pellets were stained by Alcian blue staining and the immunostaining of COL 2 and COL 10, the results of which confirmed the enhancement of differentiation potential after the hypoxic expansion. Moreover, cartilage injury in a rat model was reduced by hypoxic ADSC treatment that was determined by histological and immunohistochemical staining detections. The effects of hypoxic expansion of ADSCs and bone marrow-derived stem cells (BMSCs) on chondrogenic differentiation potential were also compared. Smaller sizes were presented in the in vitro hypoxic BMSC differentiated chondrogenic pellets, whereas the chondrogenic marker expressions were significantly higher than those of the hypoxic ADSCs. However, there was no significant difference between the treatments of the hypoxic ADSCs and BMSCs in the cartilage injury in vivo. In conclusion, hypoxic expansion increases the chondrogenic differentiation potential of ADSCs and BMSCs in vitro and enhances them to reduce cartilage damage in vivo. Although the hypoxic BMSCs showed compact chondrogenic pellet formation and higher potential of chondrogenesis, the easy access and large resources of ADSCs still uplifted the application.

Screening for Growth-Factor Combinations Enabling Synergistic Differentiation of Human MSC to Nucleus Pulposus Cell-Like Cells

Background: Multiple studies have examined the potential of growth factors (GF) to enable mesenchymal stromal cells (MSC) to nucleus pulposus (NP) cell-like cell differentiation. Here we screened a wide range of GF and GF combinations for supporting NP cell-like cell differentiation. Methods: Human MSC were stimulated using 86 different GF combinations of TGF-β1, -2, -3, GDF5, -6, Wnt3a, -5a, -11, and Shh. Differentiation potency was assessed by alcian blue assay and NP cell marker expression (e.g., COL2A1, CD24, etc.). The top four combinations and GDF5/TGF-β1 were further analyzed in 3D pellet cultures, on their ability to similarly induce NP cell differentiation. Results: Almost all 86 GF combinations showed their ability to enhance proteoglycan production in alcian blue assay. Subsequent qPCR analysis revealed TGF-β2/Wnt3a, TGF-β1/Wnt3a, TGF-β1/Wnt3a/GDF6, and Wnt3a/GDF6 as the most potent combinations. Although in pellet cultures, these combinations supported NP marker expression, none showed the ability to significantly induce chondrogenic NP matrix production. Only GDF5/TGF-β1 resulted in chondrogenic pellets with significantly enhanced glycosaminoglycan content. Conclusion: GDF5/TGF-β1 was suggested as an optimal GF combination for MSC to NP cell induction, although further assessment using a 3D and in vivo environment is required. Wnt3a proved promising for monolayer-based NP cell differentiation, although further validation is required.

Unravelling the role of osteoprotegerin in osteoarthritis by applying a human isogenic induced pluripotent stem cell model

Purpose: Founded by the hypothesis that the OA disease pathway of the previously identified high impact gain-of-function mutation in TNFRSF11B could be extrapolated to common, age-related, OA disease phenotypes, we set out to functionally study its effect. To this end, we established in vitro hiPS derived spherical chondrogenic and osteogenic pellets and compared TNFRSF11B mutated cells generated from affected family members to CRISPR/Cas9 repaired isogenic control cells. Given that the mutant OPG decoy receptor more efficiently antagonizes osteoclastogenesis, we expect that the mutation affects matrix mineralization hence inflicting the observed chondrocalcinosis phenotype. Methods: Using skin fibroblasts, hiPSCs of an affected family member were created (FOA) and differentiated to induced mesenchymal stromal stem cells (iMSCs). These were differentiated for 6 weeks to deposit neo-cartilaginous and neo-osseous pellets or neo-bone in parallel with a CRISPR-Cas9 repaired hiPSC line (FOA-R) serving as an isogenic control. Presence of glycosaminoglycans was determined by Alcian blue while mineralization was determined by Alizarin red staining. Gene expression analyses were performed to study anabolic markers and hypertrophy (ie, COL2A1, COL1A1, COL10A1) and matrix mineralization (ie, ALPL, MGP). Results: As demonstrated by the intensity of Alcian blue and Alizarin red staining of spherical neo-cartilaginous and neo-osseous pellets, respectively (Figure 1) the bi-directional differentiation via iPSC derived iMSCs appeared successful. Additionally, while only subtle differences in intensity were observed for the Alcian blue staining, the Alizarin red staining intensity in FOA mutated neo-osseous pellets was clearly higher as that of the FOA-repaired pellets, indicating increased matrix mineralization (Figure 1). Gene expression analyses of neo-cartilaginous pellets, showed, however, that in the presence of the FOA TNFRSF11B mutation, MGP levels were considerably decreased (FD=0.14 P=1.6x10-13), indicating cell signaling towards matrix mineralization also in the mutated neo-cartilaginous tissues. In line with this, neo-cartilaginous tissues from FOA mutated cells compared to FOA repaired cells expressed lower levels of COL2A1 (FD=-6.8, P-value=3.9x10-3) concomitant with higher levels of COL1A1 (FD=1.7, P-value=7.4x10-3). Additionally, in the FOA mutated neo-osseous pellets gene expression analysis showed a higher expression of RUNX2 (FD=2.6, P-value= 8.29x10-3) and a trend towards a higher expression of COL1A1 (FD=1.54, P-value= 0.079) and ALPL (FD=1.57, P-value=0.055) relative to FOA repaired neo-osseous pellets.supplements. Conclusions: Employing hiPS-cells from early-onset OA family members (FOA) and CRISPR/Cas9 repaired isogenic controls (FOA-R) in an established spherical 3D in vitro chondrogenic and osteogenic pellet model confirmed that the previously identified gain of function mutation in TNFRSF11B encoding OPG acts via increased matrix mineralization of cartilage and bone. In cartilage, likely, this is modulated via MGP expression as suggested by the significant lower levels in the mutant cells. This may raise the possibility to slow down OA progression in early-onset family members with the use of vitamin K.

A secretome analysis of chondrogenic three-dimensional pellet for its potential therapeutic effects on osteoarthritis

Purpose: Osteoarthritis (OA) is a common degenerative joint disorder characterized by the gradual degeneration of articular cartilage and low-grade inflammation in the joint. Recent strategies for OA treatment include the use of different therapeutics based on cell, gene, and tissue engineering. Previously, we established a 3D culture method for an autologous chondrocyte implantation (ACI) therapy which demonstrated significant chondrogenic capacity capable of filling cartilage lesions in the articular joint.

Optimization of lyophilized hyperacute serum as a regenerative therapeutic in osteoarthritis

Purpose: Blood-derived products are known to be involved in musculoskeletal regeneration by promoting cell proliferation and modulating inflammation. By altering the cell-hostile microenvironment we are pursuing to stop or even reverse the degenerative process in the joints of osteoarthritic patients. Hyperacute serum is a blood-derived product that retains the regenerative potential of platelet rich plasma (PRP) overcoming its variability disadvantages. Hyperacute serum obtained by squeezing a fibrin clot and its composition has been compared to PRP, showing that it better resembles cellular and molecular concentrations of in vitro and in vivo samples. To expand the potential therapeutic use, a new formulation method as lyophilized powder was established. This freeze-dried format will enable allogenic therapy with a highly regenerative, standardized and stable blood product. In contrast to freshly isolated hyperacute serum, which allows for autologous therapy, where the donors and the patients are the same individuals, most of the time elderly OA patients; the allogenic serum is a nutrient-enriched version as it is obtained from young healthy donors. Moreover, it is manufactured by pooling multiple donor´s serum, which solves the problem of donor variability. Hyperacute serum in lyophilized format can be stored for longer periods of time, therefore, administration to patients is facilitated. Two different manufacturing processes lead to the development of two different versions of lyophilized hyperacute serum. Our aim is to test the regenerative capacities of the two new lyophilized hyperacute sera in the context of osteoarthritis (OA) alone and in combination with hyaluronic acid (HA). Methods: Two-dimensional (2D) cultures of primary chondrocytes were kept in culture under different supplementation conditions, including three versions of hyperacute serum (one freshly isolated and two lyophilized), PRP, FCS and combinations of hyperacute serum+HA. These 2D cultures were analyzed with the XTT viability assay. 3D chondrogenic spheroids from primary-isolated chondrocytes were developed and kept in culture for 1 week within the listed culture conditions. In addition to XTT viability analysis , collagen type II and aggrecan production and secretion within the extracellular matrix, confocal microscopy together with immunofluorescence were applied. Results: XTT assays (sample size=5) of the 2D cultures were performed at the following time points: day 3, day 5 and day 7. When comparing the different formats of hyperacute serum at day 3, we have observed that 10% lyophilized extra-filtered hyperacute serum promotes cell viability in a comparable level to 10% FCS (1,87 and 1,57 a.u. respectively, ns). Moreover, increasing the concentration of this hyperacute serum to 20% leads to 2,27 a.u.; however, this increase did not reach the level of statistical significance. In previous publications, it was shown that freshly isolated hyperacute serum had a high performance in promoting cell viability as well as in affecting gene expression of genes that play a role in osteoarthritis, by holding back inflammation (TNFα, Il-1β, Il-1ra), promoting tissue regeneration (COL1A, osteonectin) and preventing bone degradation (RANKL). However, lyophilized extra-filtered hyperacute serum did not present the same strong effect. We hypothesize that due to the further filtration process, nutrients including growth factors were removed, which had a clear impact in promoting cell viability. Nevertheless, filtered lyophilized hyperacute serum still showed a comparable effect as FCS, known to be a gold standand in cellular supplementation. Due to the loss of nutrient supply, a non-filtered lyophilized hyperacute serum with a different manufacturing process was produced. Same experiments were repeated demonstrating that non-filtered lyophilized hyperacute serum shows a significant promotion of cell viability (2,93 a.u., P=0,0313), similarly to freshly isolated hyperacute serum or PRP (3,26 a.u. and 3,15 a.u., respectively; ns.). When testing the effect of HA alone (2,19 a.u., ns) or in combination with extra-filtered or non-filtered lyophilized hyperacute sera, it showed no significant differences in cell viability due to a possible added effect on such hypercute sera (1,83 a.u. and 3,39 a.u., respectively; ns.). Further gene expression analysis need to be performed in order to better understand the mechanisms involved in each of the conditions. XTT data from days 5 and 7 of culture supported the findings concerning HA and the significant difference between the two lyophilized versions of hyperacute sera; however, it revealed higher cell viability when cultures were supplemented with 10% PRP and 10% freshly isolated hyperacute serum. Confocal microscopy of 3D chondrogenic pellets has shown that production and secretion of collagen type II and aggrecan, two main structural proteins of the cartilage, increased in 3D chondrogenic spheroids supplemented with the different formulations of hyperacute sera as well as FCS. Conclusions: Non-filtered lyophilized hyperacute serum maintains the supplementation capacity in 2D and 3D chondrogenic cultures, as shown in the past by the freshly isolated format. The implementation of hyaluronic acid to such blood-derived products does not show, for now, any added effect. Non-filtered lyophilized hyperacute serum is a stable and standardized blood product with regenerative potential in the context of OA.

High impact mutation FN1 affects cartilage integrity via aberrant binding to collagen type II

Purpose: By applying exome sequencing to an extended early-onset osteoarthritis (OA) family followed by linkage analysis, a high impact, likely causal mutation (C518F) was identified in the fibronectin (FN1) gene. Founded by the hypothesis that the underlying pathway in which this high impact mutation is causing OA may likely be extrapolated to common, age related, OA disease pathways we here studied the effects of the identified high impact mutation on neo-cartilage formation. Hereto the identified FN1 mutation was introduced in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome engineering technology while employing these cells to an established 3D in vitro chondrogenesis model. Methods: hiPSCs were electroporated with gRNA/Cas9 complex targeting FN1 and single-strand oligodeoxynucleotide template containing the specific FN1 mutation, after which the hiPSCs were plated at low seeding density to obtain clonal colonies. After 14 days, the multicellular colonies were picked and transferred into a 96-wells plate for clonal screening and expansion. Positive clones were identified by PCR followed by HincIII digestion and confirmed by Sanger sequencing. Mesodermal lineage differentiation towards induced chondroprogenitor cells (iCPCs) was performed by stepwise changes of the culture medium. After 14 days, iCPC aggregates were manually picked and chondrogenesis was initiated in 3D using chondrogenic differentiation medium. Pellets were refreshed twice per week and medium and pellets were collected following 5 weeks of chondrogenesis. Simultaneously, conditioned medium of the wildtype and mutant FN1 pellets was collected. After concentrating the medium, a collagen type II solid-phase binding assay was performed, to compare binding affinity of wildtype and mutant FN1 to collagen type II. Results: Sequencing confirmed that out of 274 hiPSC clones 14 (5.1%) were heterozygous and 3 (1.1%) were homozygous for the C518F FN1 mutation. To investigate the effect of the mutation on chondrogenesis, in vitro derived neo-cartilage constructs of wildtype, heterozygous, and homozygous FN1 mutant hiPSCs were generated. As can be seen in Figure 1, we observed a different morphology in the 3D neo-cartilage pellets formed by homo- and heterozygous FN1 mutated cells as compared to those of wildtype, being that the mutant chondrogenic pellets are more protrusive and frail. Alcian blue and collagen type II staining revealed that the frail matrix was not cartilage. On the other hand, neo-cartilage produced by the iCPCs showed comparable staining intensities between the mutant and wildtype groups. To study whether the C518F mutation, located in the gelatin-binding domain of fibronectin, affected binding to collagen type II, we next determined binding affinity between collagen type II and wildtype, heterozygous and homozygous mutated fibronectin. As shown in Figure 2, we observed a significant (P = 0.002) dose response reduction in binding as function of C518F mutated fibronectin. To study the effect of the mutation on integrity of neo-cartilage formed, we next explored chondrocyte signaling as result of the mutation by performing RT-qPCR. To adjust for differences in efficiency between neo-cartilage formation we used COL2A1 and COL1A1 as housekeeping genes of cartilage tissue. Notably, ADAMTS-5 and RUNX2 showed a dose response significant upregulation, while ACAN, and FN1 expression were downregulated with the C518F mutated as compared to wildtype neo-cartilage chondrocytes. Conclusions: By introducing a high impact OA mutation in human iPSCs while utilizing them in an established 3D in vitro chondrogenesis model we showed that the binding capacity between fibronectin and collagen type II is essential for proper cartilage formation. Moreover, as reflected by the aberrant chondrocyte signaling towards a hypertrophic state, such improper binding makes the cartilage prone to an OA state. Together our work merits further exploration of fibronectin as potential target for therapeutic interventions. We advocate that restoring or maintaining proper binding between fibronectin and collagen type 2 should be the focus of such a quest.

Characterization of Early-Onset Finger Osteoarthritis-Like Condition Using Patient-Derived Induced Pluripotent Stem Cells.

Early osteoarthritis (OA)-like symptoms are difficult to study owing to the lack of disease samples and animal models. In this study, we generated induced pluripotent stem cell (iPSC) lines from a patient with a radiographic early-onset finger osteoarthritis (efOA)-like condition in the distal interphalangeal joint and her healthy sibling. We differentiated those cells with similar genetic backgrounds into chondrogenic pellets (CPs) to confirm efOA. CPs generated from efOA-hiPSCs (efOA-CPs) showed lower levels of COL2A1, which is a key marker of hyaline cartilage after complete differentiation, for 21 days. Increase in pellet size and vacuole-like morphologies within the pellets were observed in the efOA-CPs. To analyze the changes occurred during the development of vacuole-like morphology and the increase in pellet size in efOA-CPs, we analyzed the expression of OA-related markers on day 7 of differentiation and showed an increase in the levels of COL1A1, RUNX2, VEGFA, and AQP1 in efOA-CPs. IL-6, MMP1, and MMP10 levels were also increased in the efOA-CPs. Taken together, we present proof-of-concept regarding disease modeling of a unique patient who showed OA-like symptoms.

Initial WNT/β-Catenin Activation Enhanced Mesoderm Commitment, Extracellular Matrix Expression, Cell Aggregation and Cartilage Tissue Yield From Induced Pluripotent Stem Cells.

Mesodermal differentiation of induced pluripotent stem cells (iPSCs) in vitro and subsequent specification into mesodermal derivatives like chondrocytes is currently afflicted with a substantial cell loss that severely limits tissue yield. More knowledge on the key players regulating mesodermal differentiation of iPSCs is currently needed to drive all cells into the desired lineage and to overcome the current need for intermediate cell selection steps to remove misdifferentiated cells. Using two independent human iPSC lines, we here report that a short initial WNT/β-catenin pulse induced by the small molecule CHIR99021 (24 h) enhanced expression of mesodermal markers (PDGFRα, HAND1, KDR, and GATA4), supported the exit from pluripotency (decreased OCT4, SOX2, and LIN28A) and inhibited ectodermal misdifferentiation (reduced PAX6, TUBB3, and NES). Importantly, the initial CHIR pulse increased cell proliferation until day 14 (five-fold), adjusted expression of adhesion-related genes (CDH3 up, CDH6 down) and increased extracellular matrix (ECM)-related gene expression (COL6, COL1, COL3, COL5, DCN, NPNT, LUM, MGP, MATN2, and VTN), thus yielding more matrix-interacting progenitors with a high aggregation capability. Enhanced contribution to chondrogenic pellet formation increased the cell yield after eight weeks 200-fold compared to controls. The collagen type II and proteoglycan-positive area was enlarged in the CHIR group, indicating an increased number of cartilage-forming cells. Conclusively, short initial WNT activation improved mesoderm commitment and our data demonstrated for the first time to our knowledge that, acting via stimulation of cell proliferation, ECM expression and cell aggregation, WNT pulsing is a key step to make cell selection steps before chondrogenesis obsolete. This advanced understanding of the WNT/β-catenin function is a major step toward robust and efficient generation of high-quality mesodermal progenitors from human iPSCs and toward rescuing low tissue yield during subsequent in vitro chondrogenesis, which is highly desired for clinical cartilage regeneration, disease modeling and drug screening.