Sandeep Kumar Vishwakarma1, Chandrakala Lakkireddy1, Avinash Bardia1, Nagarapu Raju1, Syed Ameer Basha Paspala1, Md Aejaz Habeeb1, Aleem Ahmed Khan2. 1. Central Laboratory for Stem Cell Research & Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad 500058, Telangana, India; Dr. Habeebullah Life Sciences, Attapur, Hyderabad 500030, Telangana, India. 2. Central Laboratory for Stem Cell Research & Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad 500058, Telangana, India; Dr. Habeebullah Life Sciences, Attapur, Hyderabad 500030, Telangana, India. Electronic address: aleem_a_khan@rediffmail.com.
Abstract
BACKGROUND: The present study has been aimed to identify molecular dynamics of pancreatic transcription factors (pTFs) during events of directed trans-differentiation of human hepatic progenitor cells (hHPCs) into insulin producing cells (InPCs) within bioengineered humanized neoorgan. The study demonstrates applicability of acellularized whole splenic scaffold (ASOS) to generate insulin producing humanized transplantable neoorgan through activation of pancreatic transcription factors. METHODS: An efficient acellularization process was developed for xenogeneic rat spleen using change in different gradients of reagents perfusion through splenic artery for varying time points. The acellularized xenogeneic spleen scaffold was characterized thoroughly for preservation of extra-cellular matrix and retention of organ specific vasculature and mechanical properties. Further scaffolds were sterilized and repopulated with hHPCs which were triggered using a stage wise induction with growth factors and hyperglycemic challenge for trans-differentiation into InPCs. Dynamics of pTFs alone or simultaneously during induction process was identified using gene expression analysis and immunological staining. RESULTS: The cells within the engineered neoorgan respond to growth factors and extrinsic hyperglycemic challenge and generate large number of InPCs under controlled dynamic regulation of pTFs. Highly controlled regulation of pTFs generates higher percentage of Nkx-6.1+/C-peptide+ cells within the engineered splenic scaffolds. Generation of high percentage of insulin and C-peptide positive cells in three-dimensional organ architecture responded better to hyperglycemic stimuli and produced higher quantity of insulin than 2D-culture system. CONCLUSION: The present study provides a novel platform for designing effective regenerative strategies using whole organ scaffolds to control hyperglycemia under tight regulation of pTFs using humanized neoorgan system.
BACKGROUND: The present study has been aimed to identify molecular dynamics of pancreatic transcription factors (pTFs) during events of directed trans-differentiation of human hepatic progenitor cells (hHPCs) into insulin producing cells (InPCs) within bioengineered humanized neoorgan. The study demonstrates applicability of acellularized whole splenic scaffold (ASOS) to generate insulin producing humanized transplantable neoorgan through activation of pancreatic transcription factors. METHODS: An efficient acellularization process was developed for xenogeneic rat spleen using change in different gradients of reagents perfusion through splenic artery for varying time points. The acellularized xenogeneic spleen scaffold was characterized thoroughly for preservation of extra-cellular matrix and retention of organ specific vasculature and mechanical properties. Further scaffolds were sterilized and repopulated with hHPCs which were triggered using a stage wise induction with growth factors and hyperglycemic challenge for trans-differentiation into InPCs. Dynamics of pTFs alone or simultaneously during induction process was identified using gene expression analysis and immunological staining. RESULTS: The cells within the engineered neoorgan respond to growth factors and extrinsic hyperglycemic challenge and generate large number of InPCs under controlled dynamic regulation of pTFs. Highly controlled regulation of pTFs generates higher percentage of Nkx-6.1+/C-peptide+ cells within the engineered splenic scaffolds. Generation of high percentage of insulin and C-peptide positive cells in three-dimensional organ architecture responded better to hyperglycemic stimuli and produced higher quantity of insulin than 2D-culture system. CONCLUSION: The present study provides a novel platform for designing effective regenerative strategies using whole organ scaffolds to control hyperglycemia under tight regulation of pTFs using humanized neoorgan system.
Authors: Gabriel Alexander Salg; Nathalia A Giese; Miriam Schenk; Felix J Hüttner; Klaus Felix; Pascal Probst; Markus K Diener; Thilo Hackert; Hannes Götz Kenngott Journal: J Tissue Eng Date: 2019-10-30 Impact factor: 7.813
Authors: Cataldo Pignatelli; Francesco Campo; Alessia Neroni; Lorenzo Piemonti; Antonio Citro Journal: Transpl Int Date: 2022-08-25 Impact factor: 3.842