Ah-Young Lee1, Kwi-Hoon Jang1, Chris Hyunchul Jo2,3. 1. Department of Orthopedic Surgery, SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, 20 Boramae-ro 5-gil, Dongjak-gu, Seoul, 07061, Korea. 2. Department of Orthopedic Surgery, SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, 20 Boramae-ro 5-gil, Dongjak-gu, Seoul, 07061, Korea. chrisjo@snu.ac.kr. 3. Department of Translational Medicine, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 03080, Korea. chrisjo@snu.ac.kr.
Abstract
BACKGROUND: Enzymatic digestion and explant method have been widely used for isolating umbilical cord-derived mesenchymal stem cells (UC MSCs), although there is still a strong need for robust protocols for optimal isolation for large-scale stem cell banks. This study aims to establish an explant method for clinical scale production of MSCs from human UC tissue and to characterize UC MSCs isolated and cultured with the explant method. METHODS: UC MSCs were isolated by enzymatic digestion, minimal cube explant (MCE) 1-2, MCE 2-4, and MCE 10 and cultured, respectively. Also, human antibody array and basic fibroblast growth factor (bFGF) secretion in conditioned medium (CM) was analyzed. The cells were evaluated initial cell number, colony forming unit-fibroblast (CFU-F), proliferation capacity, CD marker expression, and multi-lineage differentiation. SA-β-gal assay as well as expression of p16, p21 and p53 was performed by RT-PCR. RESULTS: MCE 2-4 is the most optimized method for isolation of small umbilical cord-derived fast proliferating cells (smumf cells) with the greatest number. MCE 2-4 had the highest secretion of various bioactive factors including bFGF. The MCE 2-4 provided significantly higher CD146 expression than enzymatic digestion, and that expression was maintained until P20. The gene expression of p16, p21, and p53 of smumf cells did not change until P10 and SA-β-gal activity did not increase until P14. CONCLUSION: This study demonstrated that MCE 2-4 provided an optimal environment to isolate MSCs with quantity and quality from human whole UC tissue through secretion of various bioactive factors inherent to UC.
BACKGROUND: Enzymatic digestion and explant method have been widely used for isolating umbilical cord-derived mesenchymal stem cells (UC MSCs), although there is still a strong need for robust protocols for optimal isolation for large-scale stem cell banks. This study aims to establish an explant method for clinical scale production of MSCs from human UC tissue and to characterize UC MSCs isolated and cultured with the explant method. METHODS: UC MSCs were isolated by enzymatic digestion, minimal cube explant (MCE) 1-2, MCE 2-4, and MCE 10 and cultured, respectively. Also, human antibody array and basic fibroblast growth factor (bFGF) secretion in conditioned medium (CM) was analyzed. The cells were evaluated initial cell number, colony forming unit-fibroblast (CFU-F), proliferation capacity, CD marker expression, and multi-lineage differentiation. SA-β-gal assay as well as expression of p16, p21 and p53 was performed by RT-PCR. RESULTS: MCE 2-4 is the most optimized method for isolation of small umbilical cord-derived fast proliferating cells (smumf cells) with the greatest number. MCE 2-4 had the highest secretion of various bioactive factors including bFGF. The MCE 2-4 provided significantly higher CD146 expression than enzymatic digestion, and that expression was maintained until P20. The gene expression of p16, p21, and p53 of smumf cells did not change until P10 and SA-β-gal activity did not increase until P14. CONCLUSION: This study demonstrated that MCE 2-4 provided an optimal environment to isolate MSCs with quantity and quality from human whole UC tissue through secretion of various bioactive factors inherent to UC.
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