Biology subjects

Prithiviraj, S.

Publications and source records attributed to Prithiviraj, S..

4 recordsLinked to original sources

bioRxiv2025

Pre-clinical validation of in vitro engineered but cell-free human tissue grafts for skeletal regeneration

Tissue engineering strategies predominantly consist of the autologous generation of living substitutes capable of restoring damaged body parts. Persisting challenges with patient-specific approaches include inconsistent performance, high costs and delayed graft availability. Towards developing a one-for-all solution, a more attractive paradigm lies in the exploitation of dedicated cell lines for the fabrication of human tissue grafts. Following decellularization, this new class of biomaterials relies on the sole extracellular matrix and embedded growth factors instructing endogenous repair. This conceptual approach was previously validated using a custom mesenchymal line for the manufacturing of human cartilage, exhibiting remarkable osteoinductive capacity following lyophilization. Key missing criteria to envision clinical translation include proper decellularization as well as stringent assessment of both immunogenicity and regenerative performance. Here, we report the engineering and subsequent decellularization of human cartilage tissue with minimal matrix impairment. Ectopic evaluation in immunocompetent and immunocompromised animals reveal preservation of osteoinductivity predicted by macrophage kinetic of polarization. By establishing in vitro human allogeneic co-culture models, we evidenced the immuno-evasive properties of cell-free human cartilages, controlling macrophages and dendritic cells maturation as well as T cell activation. Lastly, regenerative performance was stringently assessed in an immunocompetent rat orthotopic model whereby decellularized human cartilage grafts achieved morphological and mechanical restoration of all critical-sized femoral defects. Taken together, our study compiles robust safety and efficacy pre-requisites prompting a first-in-human trial for engineered and decellularized human tissue grafts.

bioengineering↗

bioRxiv2025

Human mitochondrial transfer modeling reveals biased delivery from mesenchymal-to-hematopoietic stem cells

Within the bone marrow (BM), the intercellular communication between hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem/stromal cells (MSCs) is critical for the life-long maintenance of functional hematopoiesis. In recent years, the transfer of mitochondria between MSCs and HSPCs has emerged as a key aspect of this communication, occurring both in stress and homeostatic conditions. In human, the mesenchymal-to-hematopoietic transfer process and functional impact remain cryptic, primarily due to a lack of robust models. To this end, we here describe the development and exploitation of iMSOD-mito, an immortalized human MSCs line bearing an inducible mCherry mitochondrial tag. Co-culture with primary healthy HSPCs or a leukemic cell line revealed a high mitochondrial transfer rate (>15%), exclusively relying on cell-to-cell contact. While all CD34+ blood cells received mitochondria, a preferential transfer towards phenotypic hematopoietic stem cells was identified. Similarly, using primary MSCs with genetically labelled mitochondria we confirmed a transfer to all CD34+ populations, albeit occurring at a lower frequency than with the iMSOD-mito (3.38%). By engineering 3D bone marrow niches in perfusion bioreactor, this transfer rate could be significantly increased, while the biased towards HSC as receiver was maintained. Functionally, mitochondria-receiving cells exhibited an increased mitochondria membrane potential and reactive oxygen species (ROS) production, which in HSPCs was associated with retained quiescence in single cell divisional assay. In summary, we propose the iMSOD-mito as a standardized tool to model human mesenchymal-to-hematopoietic mitochondria transfer in 2D or 3D culture systems. Our work prompts the study of mitochondria transfer in both healthy or disease conditions, towards the design of regenerative therapies or identification of new targets in a malignant context.

cell biology↗

bioRxiv2023

Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines

Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECM), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred standardization, the availability of such cell lines opened avenues for the design of tailored eECMs by applying dedicated genetic tools. In this study, we demonstrated the exploitation of CRISPR/Cas9 as a high precision system for editing the composition and function of eECMs. Human mesenchymal stromal/stem cell (hMSC) lines were modified to knockout VEGF and RUNX2 and assessed for their capacity to generate osteoinductive cartilage matrices. We report the successful editing of hMSCs, subsequently leading to targeted VEGF and RUNX2-knockout cartilage eECMs. Despite the absence of VEGF, eECMs retained full capacity to instruct ectopic endochondral ossification. Conversely, RUNX2-edited eECMs exhibited impaired hypertrophy, reduced ectopic ossification and superior cartilage repair in a rat osteochondral defect. In summary, our approach can be harnessed to identify the necessary eECM factors driving endogenous repair. Our work paves the road towards the compositional eECMs editing and their exploitation in broad regenerative contexts.

bioengineering↗

bioRxiv2021

Engineering of human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia and solid tumor metastasis

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Pre-clinical research relies on xenograft mouse models, precluding the human-specific functional interactions of stem cells with their bone marrow microenvironment. Human mesenchymal cells can be exploited for the in vivo engineering of humanized ossicles (hOss). Those mini-bones provide a human niche conferring engraftment of human healthy and malignant blood samples, yet suffering from major reproducibility issue. Here, we report the standardized generation of hOss by developmental priming of a custom-designed human mesenchymal cell line. We demonstrate superior engraftment of cord blood hematopoietic cells and primary acute myeloid leukemia samples, but also validate our hOss as metastatic site for breast cancer cells. Finally, we report the first engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically reported osteolytic lesions. Collectively, our hOss constitute a powerful standardized and malleable platform to model normal hematopoiesis, leukemia and solid tumor metastasis.

bioengineering↗