Regeneration of a Functional Pulmonary Vascular Bed

Abstract

A method for vascular regeneration comprises delivering endothelial cells to a lung scaffold, delivering perivascular cells to the lung scaffold, and providing a multiphase culture program to the scaffold. The multiphase culture program comprises a first phase including delivering an angiogenic medium, e.g., having 40-100 ng/ml of pro-angiogenic factors, and a second phase including delivering a stabilization medium, e.g., having 0.5-2% of serum and 1-20 ng/ml of angiogenic factors.

What is claimed is: 
     
         1 . A method for vascular regeneration, comprising:
 (a) delivering endothelial cells to a lung scaffold;   (b) delivering perivascular cells to the lung scaffold;   (c) providing a multiphase culture program to the scaffold, the multiphase culture program comprising:
 (1) a first phase including delivering an angiogenic medium having 40-100 ng/ml of pro-angiogenic factors, and 
 (2) a second phase including delivering a stabilization medium having 0.5-2% of serum and 1-20 ng/ml of angiogenic factors. 
   
     
     
         2 . The method of  claim 1 , wherein the pro-angiogenic factors include at least one of recombinant human VEGF, bFGF, ANG1, EGF and PDGF. 
     
     
         3 . The method of  claim 1 , wherein the stabilization medium includes at least one of forskolin or hydrocortisone. 
     
     
         4 . The method of  claim 1 , further comprising maintaining the lung scaffold in a bioreactor surrounding the lung scaffold, the bioreactor comprising a tracheal line, an arterial line, and a venous line. 
     
     
         5 . The method of  claim 4 , wherein the lung scaffold comprises an airway and vasculature, the method further comprising:
 (a) connecting the airway to the tracheal line;   (b) connecting the lung scaffold to the arterial line and to the venous line; and   (c) seeding the lung scaffold with cells over the arterial line and the venous line.   
     
     
         6 . A method for vascular regeneration comprising:
 (a) delivering HUVECs and perivascular supporting hMSCs to a lung scaffold;   (b) delivering an angiogenic medium to the lung scaffold during a first phase; and   (c) delivering a stabilization medium to the lung scaffold during a second phase.   
     
     
         7 . The method of  claim 6 , further comprising maintaining the lung scaffold in a bioreactor surrounding the lung scaffold, the bioreactor comprising a tracheal line, an arterial line, and a venous line, wherein the HUVECs and perivascular supporting hMSCs are delivered through the arterial line and the venous line. 
     
     
         8 . A method for differentiating endothelial and perivascular cells from human induced pluripotent stem cells (hiPSCs) comprising:
 (a) culturing the hiPSCs in the presence of at least one GSK3 inhibitor;   (b) culturing the hiPSCs in the presence of a complete differentiating medium;   (c) culturing the hiPSCs with the differentiating medium supplemented with a TGF-β1 inhibitor; and   (d) separating hiPSC-derived perivascular progenitor cells (hiPSC-PPCs) and hiPSC-derived endothelial cells (hiPSC-ECs).   
     
     
         9 . The method of  claim 8 , wherein the at least one GSK3 inhibitor is CHIR99021. 
     
     
         10 . The method of  claim 8 , wherein the at least one TGF-β1 inhibitor is SB431542. 
     
     
         11 . The method of  claim 8 , further comprising maintaining hypoxic culture conditions of 4% or less of 02. 
     
     
         12 . The method of  claim 8 , further comprising measuring a plateau in an increase of endothelial coverage defined by CD31 and VE-cadherin expression to indicate sufficient vascular and the end of a first phase of culture. 
     
     
         13 . An airway organ bioreactor apparatus for vascular regeneration, comprising:
 a lung chamber;   at least one ingress line communicably coupled to the lung chamber and configured for delivering endothelial cells to a lung scaffold and delivering perivascular cells to the lung scaffold; and   a control module coupled to a pump, the control module configured to cause administration of a multiphase culture program to the lung scaffold, the multiphase culture program comprising:
 in a first phase, delivering an angiogenic medium having 40-100 ng/ml of pro-angiogenic factors, and 
 in a second phase delivering a stabilization medium having 0.5-2% of serum and 1-20 ng/ml of angiogenic factors.