Abstract
BackgroundDue to their extensive self-renewal and multilineage differentiation capacity, human embryonic stem cells (hESCs) have great potential for studying developmental biology, disease modeling, and developing cell replacement therapy. The first hESC line was generated in 1998 by culturing inner cell mass (ICM) cells isolated from human blastocysts using an immunosurgery technique. Since then, many techniques including mechanical ICM isolation, laser dissection, and whole embryo culture have been used to derive hESC lines. However, the hESC derivation efficiency remains low, usually less than 50%, and it requires a large number of human embryos to derive a significant number of hESC lines. Due to a shortage of and restricted access to human embryos, a novel approach with better hESC derivation efficiency is badly needed to decrease the number of embryos used.MethodsWe hypothesized that the low hESC derivation efficiency might be due to extensive proliferation of trophoblast (TE) cells which could interfere with ICM proliferation. We therefore developed a methodology to minimize TE cell proliferation by culturing ICM in a feeder-free system for 3 days before transferring them onto feeder cells.ResultsThis minimized trophoblast cell proliferation (MTP) technique could be successfully used to derive hESCs from normal, abnormal, and frozen–thawed embryos with better derivation efficiency of more than 50% (range 50–100%; median 70%).ConclusionsWe successfully developed a better hESC derivation methodology using the “MTP” culture system. This methodology can be effectively used to derive hESCs from both normal and abnormal embryos under feeder-free conditions with higher efficiency when compared with other methodologies. With this methodology, large-scale production of clinical-grade hESCs is feasible.
Highlights
Due to their extensive self-renewal and multilineage differentiation capacity, human embryonic stem cells have great potential for studying developmental biology, disease modeling, and developing cell replacement therapy
This methodology can be effectively used to derive Human embryonic stem cell (hESC) from both normal and abnormal embryos under feeder-free conditions with higher efficiency when compared with other methodologies
Derivation of hESCs by whole embryo culture and mechanical inner cell mass (ICM) dissection To compare the efficiency of our minimized trophoblast cell proliferation (MTP) method with previously available hESC derivation methods, we firstly derived hESCs under feeder-based conditions using two standard hESC derivation methods, WEC and MID (Fig. 1a)
Summary
Due to their extensive self-renewal and multilineage differentiation capacity, human embryonic stem cells (hESCs) have great potential for studying developmental biology, disease modeling, and developing cell replacement therapy. The first hESC line was generated in 1998 by culturing inner cell mass (ICM) cells isolated from human blastocysts using an immunosurgery technique. Many techniques including mechanical ICM isolation, laser dissection, and whole embryo culture have been used to derive hESC lines. To generate hESC lines for various research and clinical applications, a large number of human embryos are usually required (Additional file 1: Table S1) [2, 3]. To derive hESCs, immunosurgery, mechanical ICM dissection (MID), and whole embryo culture (WEC) techniques are developed. Due to a shortage of and restricted access to human embryos, a more efficient procedure for deriving hESCs under xeno-free conditions is critically needed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
