Purpose

This study aims to isolate, characterize, and compare articular cartilage-derived progenitor cells (ACPCs) from healthy and osteoarthritic human cartilage, and to demonstrate the potential to use these ACPCs in addition to primary chondrocytes for one-stage procedures to treat cartilage defects.

Methods and Materials

Cells were isolated from full-thickness healthy (n=6, age 46-49, mean age 48) and osteoarthritic (n=6, age 41-82, mean age 62) human cartilage. Subsequently, ACPCs were isolated from the total cell population by clonal growth after a differential fibronectin adhesion assay. Healthy and osteoarthritic ACPCs were characterized by multilineage differentiation and flow cytometry analysis to assess surface marker expression. Next, ACPCs were cocultured with osteoarthritic chondrocytes in 3D pellet cultures in a range of cell-cell ratios. Pellets were harvested after 28 days and assessed for cartilage-like matrix production using quantitative biochemical analyses for glycosaminoglycans and collagen. (Immuno)histochemistry was performed to visualize proteoglycan and collagen production.

Results

Healthy and osteoarthritic ACPCs were successfully differentiated into the adipogenic and chondrogenic lineage, but failed to produce calcified matrix when exposed to osteogenic induction media. Adipogenic differentiation was more successful in osteoarthritic ACPCs, whereas chondrogenic differentiation was more distinct in healthy ACPCs. Both ACPC populations met the criteria for cell surface marker expression to identify mesenchymal stromal cells (MSCs) as shown by flow cytometry. Cartilage-like matrix production was observed in ACPC pellet cultures, as well as pellets consisting of a coculture of ACPCs and chondrocytes (figure 1).

Conclusion

In conclusion, this study provides further insight into the progenitor cell population in both healthy and osteoarthritic human articular cartilage, which shows similarities to MSCs. Furthermore, ACPCs show potential for use complementary to osteoarthritic chondrocytes in one-stage cartilage repair treatments, as a potential alternative to MSCs.

Purpose

Cell-based therapy for cartilage repair is becoming an established technique for modern healthcare. Unfortunately, there is no consensus on an optimal cell source yet. The study provides a donor-matched quantitative comparison of the connective tissue progenitors (CTPs) derived from cartilage (Outerbridge grade G1-2-3), bone marrow aspirate concentrate (BMC), infrapatellar fat pad (IPFP), synovium (SYN) and periosteum (PERI) with respect to: 1) cell concentration (cells/cc), 2) CTP prevalence (colony forming units per million cells), and 3) biological performance based on in vitro proliferation (cells per colony), migration (colony density) and differentiation (expression of negatively charged extracellular matrix: GAG-ECM) potentials.

Methods and Materials

Ten patients undergoing total knee arthroplasty (mean-age: 59years, females=6) were recruited. Automated ASTM-based quantitative colony forming unit assay was used to compare cell concentration, CTP prevalence, and biological performance across tissue sources. A significance level of 0.05 was used overall. Tukey’s multiple comparison method was used to exam all pairwise comparisons between cell sources.

Results

Cell concentration was highest in G3 (p=0.002) and BMC (p=0.001). CTP prevalence was highest in IPFP (p=0.001), SYN (p=0.003) and G1-2 cartilage (p=0.02). Proliferation potential was highest in SYN (p<0.001) derived CTPs. Migration potential was highest in G1-2-3 (p<0.001). Differentiation potential was highest in G1-2-3 (p<0.001). CTPs derived from any given tissue sources and any given individual were highly heterogeneous in biological performance.

Conclusion

Data presented in this study suggests that cartilage (G1-2-3) is the preferred tissue source for cartilage repair, based on P_CTP and GAG-ECM, followed by SYN, IPFP, BMC and PERI. However, within each tissue source, there exists a subset of CTPs with biological performance similar to G1-2-3 cartilage, particularly in SYN and IPFP. Refinements to current cell expansion strategies, particularly performance-based clonal selection and expansion of preferred CTPs and their progeny can potentially lead to improved cell population with predictive future.

Purpose

In view of the importance of the TGFβ pathway in cartilage homeostasis and its deregulation in rheumatic diseases, we aimed at identifying genes modulating by TGFβ3 in the early phases of mesenchymal stromal cells (MSC) differentiation into chondrocytes and deciphering their role in maintaining cartilage phenotype.

Methods and Materials

Transcriptomic analysis was performed on human bone marrow MSC, induced to differentiate into chondrocytes using micropellet culture with/without TGFβ3 and stopped at day 0.5/1/2/3 as compared to d0. Expression of chondrocyte markers and novel genes was quantified by RT-qPCR and western-blotting. Gain- and loss-of-function experiments were performed using recombinant neuromedin B (NMB) and siRNA transfection on MSC induced to chondrogenesis and in mature OA chondrocytes.

Results

Transcriptomic data analysis identified genes significantly modulated in TGFβ3-induced micropellets. We selected NMB as one of the most highly up-regulated gene and as a secreted factor. Expression was increased by a 40-fold factor at d1, stabilized at d2-d3 and returned to basal expression at d21 of chondrogenesis. Enhanced expression of NMB was specific to chondrogenesis. NMB basal expression in MSC positively and significantly correlated with SOX9, COL2B, aggrecan and COL10 expression at d21. Down-regulation of NMB expression in MSCs resulted in partial inhibition of chondrogenesis while addition of rNMB did not impact differentiation. Expression of NMB and NMBR was also detected in chondrocytes. Addition of rNMB did not impact chondrogenic (COL2B, ACAN), hypertrophic (MMP13, AP) and fibrotic (COL1, COL3) markers but NMB inhibition induced these markers. Finally, we showed NMB expression is associated with Ca²⁺ influx required for Sox9-induced chondrogenesis.

Conclusion

NMB induction in the early stages of chondrogenesis is required for MSC differentiation. NMB expression needs to be down-regulated in late stages of chondrogenesis and finely regulated in mature chondrocytes to maintain their phenotype. High expression of NMB in MSC might be a predictive marker of chondrogenic potential.

Purpose

Upregulation of differentiation potential of mesenchymal stem cells contributes to the development of regenerative medicine. Nanosecond pulsed electric fields (nsPEFs) have been shown to influence intracellular organelles and trigger strong biological effects [1, 2]. In current study, we study the effects of nsPEFs on differentiation potential of pig MSCs (Mesenchymal stem cells) in vitro and explore the mechanisms behind the phenomenon.

Methods and Materials

The effects of nsPEFs-preconditioning on the expression levels of tri-lineage differentiation were evaluated by quantitative RT-PCR of marker genes and Histological staining. Mechanisms were explored with gene expression level of OCT4 and NANOG and the methylation level of their promoter. The expression of DNA methylation transferase and global DNA methylation level were performed to further study the mechanisms behind.

Results

nsPEFs-preconditioning with proper parameters (10 ns at 20 kV/cm, 100 ns at 10 kV/cm) significantly potentiated tri-lineage differentiation capacity of MSCs with upregulated genes expression(Fig.1). nsPEFs-preconditioning promotes the formation of glycosaminoglycan for chondrogenic differentiation, calcium nodule for osteogenic differentiation, lipid droplet for adipogenic differentiation through demethylation of OCT4 and NANOG promoter and upregulation of OCT4 and NANOG, followed by downregulation of DNMT1 and global DNA methylation level (Fig.2).

Conclusion

This study demonstrates a unique approach of nsPEFs-treatment to potentiate the tri-lineage differentiation potential of MSCs through demethylation of OCT4 and NANOG which has translational potential for MSCs-based regenerative medicine.

Reference

1. Zhang, K., et al., Nanosecond Pulsed Electric Fields (nsPEFs) Regulate Phenotypes of Chondrocytes through Wnt/β-catenin Signaling Pathway. Scientific Reports, 2014. 4: p. 5836.

2. Ning, T., et al., Nanosecond pulsed electric fields enhanced chondrogenic potential of mesenchymal stem cells via JNK/CREB-STAT3 signaling pathway. Stem cell research & therapy, 2019. 10(1): p. 45.

Purpose

Combination of chondrocytes with autologous or allogeneic mesenchymal stem cells (MSCs) has enabled the development of single-stage cell-based cartilage regeneration procedures, with proven clinical success. With repeat long-term sampling, the MSCs population dies and disappears. Characterization of the mechanism and role of MSC death in the therapeutic effect of MSCs and chondrocytes is currently unknown and hence the aim of the current study.

Methods and Materials

One human chondrocyte donor was cultured with 3 different human adipose derived MSCs at distinct ratios (100% chondrocytes, 80% aMSCs + 20% chondrocytes, 100% aMSCs) for 7 days in pellets (figure 1a). The cells were cultured under hypoxia (2% oxygen) in chondrocyte proliferation medium. RNA sequencing and bioinformatic analysis was performed followed by western blot and immunofluorescence.

Results

Up and down regulation of specific genes was detected using MORPEUS heat map analysis (figure 1b). Among the 362 significantly up regulated genes, 92 were present in all the 3 co-cultures (figure 1c). These genes were analysed using ClueGo software to determine their GO-pathway. To further asses the interaction between the up regulated genes STRING software was used. Of the 92 genes, 51 had interconnections. Of this subgroup, a literature research showed that 73% of these genes play a role in cell death pathways like autophagy and/or apoptosis (figure 2).

To determine which of the two pathways was involved in cell death, the protein levels of cleaved caspase 3 as marker for apoptosis and P62, LC3I & LC3II as markers for autophagy were analysed. The results showed no support for activation of caspase-dependent cell death at the protein level. Instead markers for autophagy were clearly and substantially upregulated.

Conclusion

In single-stage cell-based cartilage regeneration procedures, MSCs disappear by a caspase independent autophagy pathway. We postulate that the self-sacrifice of the MSCs by autophagy generates a chondrocyte stimulatory environment boosting cartilage formation.

Purpose

Purpose：Mesenchymal stem cells (MSCs) gradually lose multilineage differentiation potentials when cultured in vitro, which hinders their subsequent applications. Though much efforts have been enforced to take this challenge, progresses are limited. In our previous research, nanosecond pulsed electrical fields (nsPEFs）enhanced multilineage differentiation potential of MSCs. We tried to explore if concomitant applications of nsPEFs could enhanced differentiation potentials of MSCs during prolonged in vitro culture.

Methods and Materials

Materials & Methods: Pig mesenchymal stem cells cultured on conductive films were treated with nanosecond pulsed electrical fields (nsPEFs). After optimizing parameters (100ns 10kv/cm, 10ns 20kv/cm, judged by gene expression of Oct4A, Nanog and Sox2) of nsPEFs on conductive film, concomitant applications of multiple nsPEFs treatment (four times) through conductive film were performed.

Results

Results: In this study, pre-conditioning of mesenchymal stem cells with nanosecond pulsed electrical fields (nsPEFs) through gas cuvettes significantly enhanced the expression of Oct4A (3.8 fold of 100ns 10kv/cm, 3.4 fold of 10ns 20kv/cm), Nanog (3.2 fold of 100ns 10kv/cm, 2.8 fold of 10ns 20kv/cm) and Sox2 (1.8 fold of 100ns 10kv/cm, 2.0 fold of 10ns 20kv/cm). Furthermore, after optimizing parameters of nsPEFs on conductive film (100ns 10kv/cm) and time interval (3d between each nsPEFs treatment), concomitant applications of multiple nsPEFs treatment (four times) enhanced gene expression of Oct4A (3.5~4.5fold), Nanog (3.5~4fold) and Sox2 (1.3~2fold) significantly while maintained this high level gene expression for nearly 30 days of MSCs, much longer than single nsPEFs treatment (normal 7 days). Differentiation potential was significantly increased in both single and multiple nsPEFs treatment, judged by chondrogenic (3-fold of Sox9), osteogenic (6-fold Runx2) and adipogenic (4-fold of PPAR-γ) marker gene.

Conclusion

Conclusion: Our study suggested that multiple nsPEFs treatment through conductive film could enhance the multilineage differentiation potential of MSC for long time scale.

Purpose

Cartilage regeneration is the future of regenerative medicine, as this can provide a massive relief to degenerative diseases such as osteoarthritis and rheumatoid arthritis. My research project involves working on reprogrammed human induced pluripotent stem cells (hiPSCs) acquired from patients suffering from a rare genetic disease known as pseudo chondrodysplasia. This will also give an insight into production of defective cartilage through various phases of growth. Using Crispr Cas9 approach, gene editing of the mutant type hiPSCs will be carried out in future.

This poster show different techniques been utilized in our laboratory to convert induced pluripotent stem cells to mesenchymal stem cells which closely resembles chondrocytes and ultimately forming a growth plate like cartilage structure. Pluripotency was checked through immunofluorescence, flow cytometry and Q-PCR as this will determine the fate of the hiPSCs.

Methods and Materials

Materials:

Reprogrammed iPSCs, Tesr 8 media, MeSen Pro RS media, chondro basal media, growth factors

Method: Stem cells were grown in TESR-8 media and were differnetiated by adding a mesodemal media Mesen Pro RS and cutured untill they form MSC like cells these were then condensed into 3 dimensional growth plate like cartilage pellets by using basal media and growth factors like TGF-b and BMP and maintained for 21 days.

Results

The healthy normal pellets stained positive with alcian blue and Safrinin O and the mutant pellets when growth factors were added on addition there has been a slight rescue of the abnormal phenotype. Cartilage ,markers like collagen 2, aggrecan and COMP were detetced in the healthy pellets and was reduced in untreated mutant pellets but on addition of growth factors to the mutant pellets there was an increase in the cartilage markers.

Conclusion

growth factors increases the growth plate like cartilage formation.

hiPSCs from healthy person formed normal cartilage and hiPSCs from disease patient formed abnormal cartilage.

Purpose

Osteoarthritis (OA) is a degenerative and inflammatory joint disease, due to the high levels of pro-inflammatory cytokines and catabolic enzymes. The scarce regenerative capacity of cartilage and the limited outcome of the therapies are increasing the interest in mesenchymal stem cells (MSCs). Furthermore, in literature it is yet observed that platelet-rich plasma (PRP), thanks to its regenerative and anti-inflammatory abilities, is a recommended treatment for OA, as well as being a low-cost biological and not invasive remedy. This study investigates the chondrogenic ability of MSCs from adipose tissue (ADSCs) and bone marrow (BMSCs) in an OA-like microenvironment; evaluates the anti-inflammatory and regenerative roles of blood autologous protein solution (APS), enriched of anti-inflammatory cytokines and growth factors.

Methods and Materials

BMSC and ADSC micromasses, stimulated with interleukin (IL)1β and tumor necrosis factor (TNF)α or synovial fluid from OA patients, were cultured for 4 weeks in presence or absence of APS conditioned medium (CM). Their differentiation toward chondrogenic lineage was evaluated with histology, immunohistochemistry, histomorphometry and molecular biology approaches.

Results

ADSCs and BMSCs were well organized in compact micromasses, with a more abundant matrix in ADSCs and cellularity in BMSCs (Figure 1). Gene expression of SOX9 and ACAN decreased in presence of inflammatory factors and did not recover with CM (in ADSC and BMSC). Conversely, the addition of CM reduced Metalloproteinases (MMP13), collagen (COL1a1), COLL X and COLL I in ADSCs and BMSCs. COL2a1 and MMP13 were respectively more and less expressed in ADSCs than in BMSCs in all culture conditions. Moreover, CM appeared to have more effect in cells treated with IL1β and TNFα than those treated with synovial fluid.

Conclusion

From these preliminary results it seems that both ADSCs and BMSCs might contribute to cartilage regeneration in a joint affected by OA and that APS might be useful in this regard.

Purpose

Articular cartilage injuries can lead to significant joint morbidity. Although marrow stimulation procedures such as microfracture are a popular treatment option, results are often suboptimal, with the resultant neocartilage exhibiting a more fibrous than hyaline phenotype. Micronized matrix allograft cartilage has emerged as a promising new adjunct to augment microfracture procedures by promoting a greater degree of hyaline cartilage regeneration and improved long-term outcomes. However, little is known about the effects of micronized matrix allograft cartilage on a cellular level. The purpose of this study was to investigate the effects of micronized matrix allograft cartilage (BioCartilage) on multipotent stem cell differentiation.

Methods and Materials

Human mesenchymal stem cells (MSCs) were treated with BioCartilage (micronized matrix allograft cartilage; Arthrex Inc., Naples, FL) and/or human platelet-rich plasma (PRP). Chondrogenic differentiation was assessed using polymerase chain reaction (PCR) to measure expression of type II collagen (COL2), aggrecan (AGG), and SOX9 marker genes. Human adipose-derived stem cells (ADSCs) were also treated with BioCartilage; osteocalcin (OCN), alkaline phosphatase (ALP), and RUNX2 marker genes were used to measure osteogenic differentiation, and CD31 and VEGF were used to measure angiogenic differentiation.

Results

In the MSCs, BioCartilage resulted in significant increases in COL2 and SOX9 expression at various timepoints.(Figures 1, 2).

BioCartilage combined with PRP also demonstrated a synergistic effect on increasing AGG expression. In the ADSCs, BioCartilage did not affect OCN or RUNX2 expression, but did result in a decrease in ALP expression. BioCartilage also resulted in a decrease in CD31 expression, but an increase in VEGF expression.

Conclusion

This study supports the theory that micronized matrix allograft cartilage (BioCartilage) promotes hyaline cartilage differentiation of stem cells. The data also suggests that BioCartilage inhibits bone and blood vessel formation, which is important for maintaining cartilage regeneration in microfracture procedures.

Purpose

The absence of a long term treatment for OA has led to the development of new approaches for early intervention. Mechanical regulators, such as TRPV4, play a key role in OA and are potential targets for early treatments. We demonstrate a new technology to remotely mechano-activate ion channels such as TRPV4 to control MSc and chondrogenic differentiation and maturation.

Methods and Materials

MScs from bone marrow and other sources cultured as pellets (4x10⁵ cells) and micromass cultures (5x10⁴ cells). In some cases, cells were previously transfected with GFP- collagen type 2 reporters. Cells were labelled with Nanomag (250 nm) with carboxylic coating and linked to the specific antibody for each group: TRPV4 and RGD. Cells were cultured for 21 days in chondrogenic, chondrogenic without TGF-B3 and basal media.The tagged cells were submitted to a 1h daily cyclical magnetic field controlled by a MICA bioreactor (MICA Biosystems Ltd) or static. Chondrogenic differentiation was monitored with temporal key markers.

Results

In response to the mechano-activation of TRPV4, we observed an increase in Collagen 2 and enhanced early expression onset in transfected MSCs at 14 days in culture. Histological and molecular analysis demonstrated a similar pattern for Collagen 2 and aggrecan up-regulation in MSCs. Enlarged pellet size was observed in the TRPV4 tagged chondroprogenitors compared to controls with an increase in uniform expression patterns (Figure 1). Elevated proliferation levels were found in the activated ion channel group. Further work underway includes ex vivo models to demonstrate remote control.

Conclusion

The development of allogeneic injectable solutions for early OA therapy could provide solutions which would improve long lasting motility with ageing. MICA approaches using MNPs tagged to receptor targets has the potential to generate these new therapies. The use of ion channels such as TRPV4 as targets for enhancing chondrogenesis is being explored further in ex vivo and animal models.