Periodic Reporting for period 1 - CAREQiPSC (Cartilage derived from equine induced pluripotent stem cells: an in vitro and ex vivo One Medicine approach for osteoarthritis)
Periodo di rendicontazione: 2022-02-01 al 2024-01-31
Osteoarthritis is a degenerative joint pathology that constitutes the first cause of disability in older adults and accounts for more than 80% of chronic lameness in horses. Effective treatments are not available and developing them is of utmost importance. For this purpose, stem cell therapy in under investigation. Induced pluripotent stem cells (iPSCs) are obtained by ‘reprogramming’ adult cells into pluripotent stem cells that resemble those in the embryo. These iPSCs can provide unlimited cells for therapy with ability to differentiate into almost any type of cell, while no carrying ethical concerns.
However, pre-clinical knowledge is limited to small animals (rodents and rabbits) with low translational potential, as their anatomy and biomechanics differ from those in human. Larger species such as the horse better resembles human joint’s features and can also benefit from these therapies, since they are also veterinary patients (One Medicine). To develop this approach, the first mandatory step is obtaining suitable cartilage from equine iPSCs, but we still lack critical knowledge on the biology of these cells and their differentiation process into cartilage (chondrogenesis).
Thus, the overall goal of CAREQiPSC is to examine approaches to obtaining cartilage from equine iPSCs and to explore its therapeutic potential, as a necessary step to advance osteoarthritis treatments for both equines and humans. There are four specific goals that underpin this objective:
1) Establishing equine iPSCs from new types of equine cells that may retain higher chondrogeneic ‘memory’. Cord blood cells and articular chondrocytes were initially selected, based on findings in human iPSCs.
2) Deriving mesenchymal-like cells as an intermediate population between equine iPSCs and chondrocytes.
3) Comparing the chondrogeneic ability of these mesenchymal cells under different in vitro three-dimensional culture systems that are designed to promote their differentiation.
4) Exploring the therapeutic potential of the cells in an ex vivo cartilage explant system.
Two main conclusions have been obtained, providing evidence for the first time about:
1) Equine iPSCs can be generated more efficiently from cells of early developmental stages, which show higher proliferation and higher expression of pluripotent markers.
2) Chondrogenesis of equine iPSCs can be induced following different pathways, yet protocols require further fine-tuning to be adapted to the equine species.
However, pre-clinical knowledge is limited to small animals (rodents and rabbits) with low translational potential, as their anatomy and biomechanics differ from those in human. Larger species such as the horse better resembles human joint’s features and can also benefit from these therapies, since they are also veterinary patients (One Medicine). To develop this approach, the first mandatory step is obtaining suitable cartilage from equine iPSCs, but we still lack critical knowledge on the biology of these cells and their differentiation process into cartilage (chondrogenesis).
Thus, the overall goal of CAREQiPSC is to examine approaches to obtaining cartilage from equine iPSCs and to explore its therapeutic potential, as a necessary step to advance osteoarthritis treatments for both equines and humans. There are four specific goals that underpin this objective:
1) Establishing equine iPSCs from new types of equine cells that may retain higher chondrogeneic ‘memory’. Cord blood cells and articular chondrocytes were initially selected, based on findings in human iPSCs.
2) Deriving mesenchymal-like cells as an intermediate population between equine iPSCs and chondrocytes.
3) Comparing the chondrogeneic ability of these mesenchymal cells under different in vitro three-dimensional culture systems that are designed to promote their differentiation.
4) Exploring the therapeutic potential of the cells in an ex vivo cartilage explant system.
Two main conclusions have been obtained, providing evidence for the first time about:
1) Equine iPSCs can be generated more efficiently from cells of early developmental stages, which show higher proliferation and higher expression of pluripotent markers.
2) Chondrogenesis of equine iPSCs can be induced following different pathways, yet protocols require further fine-tuning to be adapted to the equine species.
Main results achieved include:
- Generation and characterisation of equine iPSCs:
- Cord blood cells and articular chondrocytes could only be partially reprogrammed, but a novel source of equine mesenchymal stem cells derived from early embryos (eMSCs) were successfully reprogrammed
- Differences between the equine cells tested were studied: eMSCs showed higher proliferation and higher expression of pluripotent markers, which is potentially linked to their higher reprogramming efficiency
- Equine iPSCs were characterised by gene and protein expression of pluripotent markers and ability to differentiate into cells from the three germ layers
- Differentiation of equine iPSCs into chondrocytes:
- Two protocols were tested: obtaining an intermediate mesenchymal population, and directed chondroprogenitor induction
- Equine iPSCs acquired chondrocyte-like morphology and increased their expression of chondrogeneic markers, but were not able to fully differentiate. This is likely due to the continuous transgene expression and/or to the use of human iPSC protocols
Results of the project have been disseminated at scientific meetings:
- International Society of Cell and Gene Therapy 2023 Meeting (Paris, France).
- RegenBell Symposium on Stem Cells and Regenerative Medicine 2023 (Barcelona, Spain).
An abstract is under consideration for presentation at:
- European College of Veterinary Surgeons Annual Meeting 2024 (Valencia, Spain).
Two original articles are under preparation, each one covering one of the main conclusions of the project.
One review article was published to give a comprehensive update on the state of the art of veterinary iPSCs:
- Barrachina et al., 2023. Induced pluripotent stem cells in companion animals: how can we move the field forward? doi.org/10.3389/fvets.2023.1176772
An overview of the project was given in three invited talks:
- 15th Annual Conference of the Graduate Centre of Life Sciences (Justus Liebig University). 2022. Giessen, Germany.
- CALIN Masterclass series on veterinary regenerative medicine. 2022. Galway, Ireland (online).
- Webinar series of the Equine Practitioners Spanish Society. 2023. Spain (Online).
- Generation and characterisation of equine iPSCs:
- Cord blood cells and articular chondrocytes could only be partially reprogrammed, but a novel source of equine mesenchymal stem cells derived from early embryos (eMSCs) were successfully reprogrammed
- Differences between the equine cells tested were studied: eMSCs showed higher proliferation and higher expression of pluripotent markers, which is potentially linked to their higher reprogramming efficiency
- Equine iPSCs were characterised by gene and protein expression of pluripotent markers and ability to differentiate into cells from the three germ layers
- Differentiation of equine iPSCs into chondrocytes:
- Two protocols were tested: obtaining an intermediate mesenchymal population, and directed chondroprogenitor induction
- Equine iPSCs acquired chondrocyte-like morphology and increased their expression of chondrogeneic markers, but were not able to fully differentiate. This is likely due to the continuous transgene expression and/or to the use of human iPSC protocols
Results of the project have been disseminated at scientific meetings:
- International Society of Cell and Gene Therapy 2023 Meeting (Paris, France).
- RegenBell Symposium on Stem Cells and Regenerative Medicine 2023 (Barcelona, Spain).
An abstract is under consideration for presentation at:
- European College of Veterinary Surgeons Annual Meeting 2024 (Valencia, Spain).
Two original articles are under preparation, each one covering one of the main conclusions of the project.
One review article was published to give a comprehensive update on the state of the art of veterinary iPSCs:
- Barrachina et al., 2023. Induced pluripotent stem cells in companion animals: how can we move the field forward? doi.org/10.3389/fvets.2023.1176772
An overview of the project was given in three invited talks:
- 15th Annual Conference of the Graduate Centre of Life Sciences (Justus Liebig University). 2022. Giessen, Germany.
- CALIN Masterclass series on veterinary regenerative medicine. 2022. Galway, Ireland (online).
- Webinar series of the Equine Practitioners Spanish Society. 2023. Spain (Online).
CAREQiPSC aims at providing critical knowledge to facilitate in vivo application of iPSCs in the horse to treat osteoarthritis, which will both advance the veterinary field and will generate preclinical information transferable to human therapy. Osteoarthritis affects more than 303 million people worldwide and constitutes the single most common cause of disability in older adults, representing a huge socio-economic burden associated with reduced life quality and direct and indirect estimated costs greater than €11,000 annually per patient. In horses, osteoarthritis is responsible for up to 83.5% of chronic lameness. Due to the main use of these animals in sports, such pathology constitutes an important burden for the equine industry, which has an annual economic impact in Europe of over €100 billion, with an additional turnover of >€25 billion in horseracing betting. Actually, the equine sports and leisure market in Europe is the largest in the world and promotes EU regional sustainable growth.
Therefore, the lack of effective treatments for osteoarthritis is a timely issue that has led to interest in stem cell therapies, of which iPSCs hold great promise as they can provide an ethical and unlimited source of chondrocytes for cartilage repair. However, this is a new field with many challenges yet to face. While in vitro experiments and in vivo studies in rodents have provided valuable knowledge, further pre-clinical research in large animals is critical for clinical translation. The horse is both a highly valuable model and a potential beneficiary as patient, but very little progress has been made in obtaining cartilage from equine iPSCs.
This project provides fundamental knowledge to advance in this direction and facilitate human translation by using a Comparative Medicine approach. Specifically, different types of equine cells have been barely compared to obtain iPSCs, and there are no reports comparing pre-natal, peri-natal and adult equine cells. In fact, the equine parental cells covered by this project have not been explored before. Furthermore, there is not a clearly superior protocol for reprogramming equine iPSCs and this project helps developing more consistent and repeatable methodologies. Cartilage derivation from equine iPSCs has not been consistently reported, so assessing different methodologies and characterising the cartilage obtained facilitates establishing suitable protocols to promote the chondrogenesis of these cells, which is fundamental for both pre-clinical and clinical application. In this way, equine and human medicine are both advanced by helping each other under an innovative One Medicine approach.
Therefore, the lack of effective treatments for osteoarthritis is a timely issue that has led to interest in stem cell therapies, of which iPSCs hold great promise as they can provide an ethical and unlimited source of chondrocytes for cartilage repair. However, this is a new field with many challenges yet to face. While in vitro experiments and in vivo studies in rodents have provided valuable knowledge, further pre-clinical research in large animals is critical for clinical translation. The horse is both a highly valuable model and a potential beneficiary as patient, but very little progress has been made in obtaining cartilage from equine iPSCs.
This project provides fundamental knowledge to advance in this direction and facilitate human translation by using a Comparative Medicine approach. Specifically, different types of equine cells have been barely compared to obtain iPSCs, and there are no reports comparing pre-natal, peri-natal and adult equine cells. In fact, the equine parental cells covered by this project have not been explored before. Furthermore, there is not a clearly superior protocol for reprogramming equine iPSCs and this project helps developing more consistent and repeatable methodologies. Cartilage derivation from equine iPSCs has not been consistently reported, so assessing different methodologies and characterising the cartilage obtained facilitates establishing suitable protocols to promote the chondrogenesis of these cells, which is fundamental for both pre-clinical and clinical application. In this way, equine and human medicine are both advanced by helping each other under an innovative One Medicine approach.