The role of single cell derived vascular resident endothelial progenitor cells in the enhancement of vascularization in scaffold-based skin regeneration
“…[35][36][37][38] Their presence in circulation is also used as a surrogate marker for a variety of pathologies, including heart disease and cancer. [39][40][41] However, their widespread use for diverse applications and experimental investigations is often limited by the low abundance of this population.…”
Endothelial progenitor cells (EPCs) play a significant role in multiple biological processes such as vascular homeostasis, regeneration, and tumor angiogenesis. This makes them a promising cell of choice for studying a variety of biological processes, toxicity assays, biomaterial-cell interaction studies, as well as in tissueengineering applications. In this study, we report the generation of two clones of SV40-immortalized EPCs from umbilical cord blood. These cells retained most of the functional features of mature endothelial cells and showed no indication of senescence after repeated culture for more than 240 days. Extensive functional characterization of the immortalized cells by western blot, flow cytometry, and immunofluorescence studies substantiated that these cells retained their ability to synthesize nitric oxide, von Willebrand factor, P-Selectin etc. These cells achieved unlimited proliferation potential subsequent to inactivation of the cyclin-dependent kinase inhibitor p21, but failed to form colonies on soft agar. We also show their enhanced growth and survival on vascular biomaterials compared to parental cultures in late population doubling. These immortalized EPCs can be used as a cellular model system for studying the biology of these cells, gene manipulation experiments, cell-biomaterial interactions, as well as a variety of tissue-engineering applications.
“…[35][36][37][38] Their presence in circulation is also used as a surrogate marker for a variety of pathologies, including heart disease and cancer. [39][40][41] However, their widespread use for diverse applications and experimental investigations is often limited by the low abundance of this population.…”
Endothelial progenitor cells (EPCs) play a significant role in multiple biological processes such as vascular homeostasis, regeneration, and tumor angiogenesis. This makes them a promising cell of choice for studying a variety of biological processes, toxicity assays, biomaterial-cell interaction studies, as well as in tissueengineering applications. In this study, we report the generation of two clones of SV40-immortalized EPCs from umbilical cord blood. These cells retained most of the functional features of mature endothelial cells and showed no indication of senescence after repeated culture for more than 240 days. Extensive functional characterization of the immortalized cells by western blot, flow cytometry, and immunofluorescence studies substantiated that these cells retained their ability to synthesize nitric oxide, von Willebrand factor, P-Selectin etc. These cells achieved unlimited proliferation potential subsequent to inactivation of the cyclin-dependent kinase inhibitor p21, but failed to form colonies on soft agar. We also show their enhanced growth and survival on vascular biomaterials compared to parental cultures in late population doubling. These immortalized EPCs can be used as a cellular model system for studying the biology of these cells, gene manipulation experiments, cell-biomaterial interactions, as well as a variety of tissue-engineering applications.
“…This collateral artery was used for all biochemical and histological analyses except for those experiments where isolated vascular resident progenitor cells in culture were utilized as described in the Materials and Methods and Results sections. Based upon the generation of monoclonal antibodies against membrane preparations of this collateral vessel and immunohistochemical screening for collateral selectivity we generated a monoclonal antibody (CTA 157-2) that bound to the cell membrane of cells in the endothelial lining of growing collateral vessels as well as of isolated vascular resident endothelial progenitor cells (VR-EPCs) that were recently characterized by our group [18]. Interestingly CTA 157-2 was found to bind and activate extracellular proteasome.…”
Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa β3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo.
“…To date, the body of literature concerning the use of vascular cells in combination with tissue-specific cells for regenerative medicine applications remains limited. However, recently, endothelial cells of various origins were incorporated into tissue-engineered constructs to promote myocardial regeneration [Tulloch et al, 2011], dermal tissue regeneration [Zhang et al, 2011], and skeletal muscle growth [Levenberg et al, 2005], which resulted in improved integration into the host. Tulloch et al [2011] demonstrated that the addition of endothelial cells to the human cardiac cells resulted in an increase in lumen containing structures in vitro and a contribution to functional vasculature in vivo.…”
Section: Applications In Regenerative Medicinementioning
Although a ‘vascular stem cell’ population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources including human embryonic stem cells and induced pluripotent stem cells. We review the vascular potential of these human pluripotent stem cells, the mechanisms by which they are induced to differentiate toward a vascular endothelial cell fate, and their applications in regenerative medicine.
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