Tips and tricks for successfully culturing and adapting human induced pluripotent stem cells

Abstract

Reprogramming somatic cells toward pluripotency became possible over a decade ago. Since then, induced pluripotent stem cells (iPSCs) have served as a versatile and powerful tool not only for basic research but also with the long-term goal of using them in human cell transplantation after differentiation. Nonetheless, downstream applications are frequently blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture and cryopreservation, adaptation to feeder-free conditions, or expansion of the cells. Therefore, in this article we aim to provide other researchers with practical and detailed information to successfully culture and adapt iPSCs. Specifically, we (1) describe the most common problems when in-vitro culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) compare different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that the troubleshooting and recommendations provided in this article can be of use to other researchers working with iPSCs and who may be experiencing similar issues, hopefully enhancing the appeal of this promising cell source to be used for biomedical investigations, such as tissue engineering or regenerative medicine applications. Reprogramming somatic cells toward pluripotency became possible over a decade ago. Since then, induced pluripotent stem cells (iPSCs) have served as a versatile and powerful tool not only for basic research but also with the long-term goal of using them in human cell transplantation after differentiation. Nonetheless, downstream applications are frequently blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture and cryopreservation, adaptation to feeder-free conditions, or expansion of the cells. Therefore, in this article we aim to provide other researchers with practical and detailed information to successfully culture and adapt iPSCs. Specifically, we (1) describe the most common problems when in-vitro culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) compare different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that the troubleshooting and recommendations provided in this article can be of use to other researchers working with iPSCs and who may be experiencing similar issues, hopefully enhancing the appeal of this promising cell source to be used for biomedical investigations, such as tissue engineering or regenerative medicine applications. Induced pluripotent stem cells (iPSCs) have the ability to proliferate indefinitely in culture without reduction of the quality, besides potential of differentiation into any desired cell type.1Rowe R.G. Daley G.Q. Induced pluripotent stem cells in disease modelling and drug discovery.Nat. Rev. Genet. 2019; 20: 377-388Google Scholar Therefore, when first discovered in 2006,2Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Google Scholar these cells were thought to be the “panacea” for biomedical applications. In the case of tissue engineering, iPSCs hold great potential for personalized tissues, which can be used for regenerative medicine and/or in vitro studies to tailor other medical interventions.3Loskill P. Huebsch N. Engineering tissues from induced pluripotent stem cells.Tissue Eng. Part A. 2019; 25: 707-710Google Scholar However, all these advantages and potential applications are blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture, adaptation, and/or expansion of the cells.4Chen K.G. Mallon B.S. McKay R.D.G. Robey P.G. Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.Cell Stem Cell. 2014; 14: 13-26Google Scholar iPSCs' recovery after freezing is still an issue5Nishishita N. Muramatsu M. Kawamata S. An effective freezing/thawing method for human pluripotent stem cells cultured in chemically-defined and feeder-free conditions.Am. J. Stem Cells. 2015; 4: 38-49Google Scholar,6Ye H. Wang Q. Efficient generation of non-integration and feeder-free induced puripotent stem cells from human peripheral blood cells by Sendai virus.Cell. Physiol. Biochem. 2018; 50: 1318-1331Google Scholar and, in addition, it has been reported that iPSC colonies may disappear and break up into single cells during initial colony morphology-based selection.7Pfannkuche K. Fatima A. Gupta M.K. Dieterich R. Hescheler J. Initial colony morphology-based selection for iPS cells derived from adult fibroblasts is substantially improved by temporary UTF1-based selection.PLoS One. 2010; 5: e9580Google Scholar The use of iPSCs in all downstream applications requires the establishment of protocols that will allow large-scale, cost-effective cultivation of cells, without compromising on their quality.8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar Reprogramming methods and iPSC culture strategies initially involved the use of mouse or human feeder layers, thus coinciding with the protocol established by Thomson for in vitro culturing embryonic stem cells (ESCs).9Thomson J.A. Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Google Scholar These feeder cells secrete essential growth factors, extracellular matrix components, and cytokines into the culture media, which support pluripotent cell growth and proliferation.8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar,10Yao S. Chen S. Clark J. Hao E. Beattie G.M. Hayek A. Ding S. Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions.Proc. Natl. Acad. Sci. U S A. 2006; 103: 6907-6912Google Scholar,11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar Although a robust method, feeder-based systems are labor intensive, hard to scale,11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar and can also be a source of animal pathogens and mycoplasma contamination.8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar,11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar,12Mannello F. Tonti G.A. Concise review: no breakthroughs for human mesenchymal and embryonic stem cell culture: conditioned medium, feeder layer, or feeder-free; with human or medium, or that is not Cells. 25: Scholar this not allow the of all the to iPSC feeder cells can with and/or H. for human embryonic stem 2006; Scholar is in iPSCs have to be to feeder-free culture when pluripotent cells are from cell culture to to adapt to the to adaptation and of Human Stem Cells in this adaptation is for the cells, in the case of derived cell experiencing differentiation and adaptation and of Human Stem Cells in K. Feeder-free adaptation, culture and of human cells using feeder-free 2010; of human pluripotent stem cells to feeder-free in chemically defined with Mol. Biol. Scholar feeder-free H. Wang Q. Efficient generation of non-integration and feeder-free induced puripotent stem cells from human peripheral blood cells by Sendai virus.Cell. Physiol. Biochem. 2018; 50: 1318-1331Google Wang J. Feeder-free of human fibroblasts with Stem Biol. 2013; K. F. R. Human induced pluripotent stem cells from a and feeder-free Genet. S. of human induced pluripotent stem cells using a feeder-free Stem Biol. 2018; Scholar feeder-based methods are still the most common on which to the H. Wang Q. Efficient generation of non-integration and feeder-free induced puripotent stem cells from human peripheral blood cells by Sendai virus.Cell. Physiol. Biochem. 2018; 50: 1318-1331Google M. R.G. in pluripotent stem and Rev. Scholar In we have the generation of iPSC lines derived from human R. M. F. S. of human induced pluripotent stem cells from 2019; R. M. S. of a human iPS cell from a with no Scholar is that the problems that we to face during the of generation and of the cells were the adaptation to feeder-free conditions. are a of of matrices and culture media to feeder in S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar and S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google H. for human embryonic stem 2006; M. R.G. in pluripotent stem and Rev. Chen K. A. M. Human Stem and pluripotent stem cells as systems for and S. N. K. generation of from human iPS K. A. H. N. H. K. of common embryonic stem cell lines Scholar and S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google H. for human embryonic stem 2006; M. R.G. in pluripotent stem and Rev. Chen K. A. M. Human Stem and pluripotent stem cells as systems for and S. N. K. generation of from human iPS K. A. H. N. H. K. of common embryonic stem cell lines Scholar S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google H. for human embryonic stem 2006; M. R.G. in pluripotent stem and Rev. Chen K. A. M. Human Stem and pluripotent stem cells as systems for and S. N. K. generation of from human iPS K. A. H. N. H. K. of common embryonic stem cell lines Scholar S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google H. for human embryonic stem 2006; M. R.G. in pluripotent stem and Rev. Chen K. A. M. Human Stem and pluripotent stem cells as systems for and S. N. K. generation of from human iPS K. A. H. N. H. K. of common embryonic stem cell lines Scholar S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google H. for human embryonic stem 2006; M. R.G. in pluripotent stem and Rev. Chen K. A. M. Human Stem and pluripotent stem cells as systems for and S. N. K. generation of from human iPS K. A. H. N. H. K. of common embryonic stem cell lines Scholar S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google H. for human embryonic stem 2006; M. R.G. in pluripotent stem and Rev. Chen K. A. M. Human Stem and pluripotent stem cells as systems for and S. N. K. generation of from human iPS K. A. H. N. H. K. of common embryonic stem cell lines when the for troubleshooting, we is that generation and in vitro culture of iPSCs is a and S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google M. K. and of induced pluripotent stem R. Biol. Sci. 2015; Scholar but the the difficulties and problems during the of iPSC lines and working with Therefore, in this article we aim to in this of by the protocols we established for the of iPSC is to (1) which are the most common problems when iPSCs onto feeder cells as well as its possible troubleshooting, and (2) different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that this of problems and possible to have them will to other researchers that have similar when working with iPSCs. fibroblast growth without with and cell cell growth matrix cell matrix with and and no and and freezing and tissue culture tissue culture with and of in into freezing and The use of freezing is freezing can be and in up to of a and from and use not the with and matrix on working on a is in and the by up and a to the to not and and by into in and of to in to a of and into of up to the for or the by of to the of the to an the and for up to we and and use as for that it is no to the human the of and of in the or use the of iPSC a with a to a that for the iPSC colonies working the all of these protocols be in a for are for cell culture and is no of and of the protocols in this has been using iPSC lines in and S. of human induced pluripotent stem cells using a feeder-free Stem Biol. 2018; R. M. S. of a human iPS cell from a with no Scholar a of feeder cells is for cell and of iPSC The protocol we is on the fibroblast cell with but other methods for cell such as can be S. E. F. M. cell and Eng. Rev. 2015; Scholar the culture the cell in with and in a with cell for cell expansion of cells culture are not over the cells will in We not using feeder cells that have been for in to the iPSC colonies to break up into single cells and disappear in the of the from the and the cells with the cell with for that cells are not still to the culture the an cells are from the culture the with a of cell in a and for to a cell the cell in of the In this protocol we used a this protocol for the first and to possible in the we that by as the cell is by and using cells to and for the cells with the and cells in a of and the in the for and for the in cell to a of the cells in a using the cells be also as the cells using and a the cell for the of freezing to cell and for cells, and it on cells in freezing we the of freezing to cell to this it is to as as possible the are in the of the cells to is for the of Mol. Biol. of the cell in a freezing and for the to feeder cells by the in a the be with of the only a of not the in the or of the may this the with and the the cell with a and in a to a the freezing medium, with the cell for the and cells in of to the in culture of iPSCs onto feeder we that with cells be used to to of a culture of of the of for on to to that the the of the and for a the the feeder cells of a of feeder of feeder and the feeder cells as but not to the of the the in a to of the feeder the with feeder cells in of for and the iPSCs colony to allow the feeder cells to in culture and over the iPSCs. the iPSC colonies with the colonies with and of cells with A. M.J. E. A. F. R. J. and generation of induced pluripotent stem cells from human S. M. F. A. M. and for human induced pluripotent stem One. Scholar and that and on the the and its with of in the feeder that have been in The of be on the of to colonies a iPSC colonies well of a when the of the to which colony will be of in the will to the growth and cytokines by the feeder cells. not the in the the iPSC colonies to be and the the feeder to the break up the iPSC colonies into and them by using a and its as in and to iPSCs it is of not to the of the of colonies into a well with feeder cells in a iPSC colonies well of a this iPSC colonies to in culture and with to colonies and, as differentiation. that of the may be a is it to the and it in a to the the the for we not all the medium, but to of of iPSCs lines are established by human colonies on the these in with the growth the first after of the somatic cells. In this iPSC colonies be first after in culture, on the with to colony for and the iPSCs. the to colony for and the iPSCs. in the are up in and working the as in this we of freezing media, Stem and in for and the colonies from are and colonies are for when the iPSC lines are established the can be and with colonies that not been can be used for as in the iPSC the to the break up all the colonies in well as in the the colony of well with a and the to the for and the of well to be the to the cell the with iPSCs, the with and the of the with the the in a to the cell from the iPSCs in of and up and to with a cell are the cell to with a this the cells are in with and be and of the cells to is for the of Mol. Biol. the the cell in a freezing and for the to a for long-term we this or after have been with to colony for iPSCs. the to colony for in the the protocol similar to the for iPSCs. However, of and of the all the colonies in well were up and of of in the desired of iPSCs from the and on the the iPSCs by the in the a of is in the this the with and the the and of in a the cell to the in the iPSCs that cell are not into single the for the and cell in of of the iPSC is to well of a with feeder cells and of the culture the iPSCs in the and and for the of we not all the but of of the culture the to iPSC in this we describe and different culture media or with different matrix and to the method to adapt the iPSCs to feeder-free of the different matrices and culture media for the adaptation of the iPSCs to feeder-free culture with and without of of colonies after the first but not after the with and without of conditioned from human embryonic stem of colonies after the first but with and without of conditioned from human embryonic stem colony cell possible to without with and without of conditioned from human embryonic stem of colonies not with and without of conditioned from human embryonic stem of colonies to the culture but and cells were with and without of conditioned from human embryonic stem of colonies not with and without of conditioned from human embryonic stem of colonies not of the colony with and without of conditioned from human embryonic stem a of the colony with and without of with and without of conditioned from human embryonic stem in a media is from conditioned media, which is by from a of as the protocol in of a of feeder also of the cells and to the cells in culture with to have a of and culture them in without to of iPSCs onto feeder also of feeder cells on in a to the of the the conditioned in a and it without can be for up to its the conditioned a to cell and of for the different cell in a culture media, different media were in this the and defined culture by it in without and in a as by the of in of without and by up and of to well of a that all the is for in other culture to for the the and is the iPSCs in of cell culture the as in the as in of iPSCs onto feeder also of feeder cells on in other culture to of culture the iPSCs. the of be we use a working of as by the and the are to a in other culture to a of the and the of to a of with and and by up and of the to well of a all the is for the with a and is the iPSCs in of cell culture in with of in of with by up and of may be of to well of a that all the is for in other culture to by the and the iPSCs in of cell culture is is the from the by it in a in with and and and by up and of to well of a that all the is in other culture to the the and the iPSCs in of cell culture is culture medium, growth and/or the the culture in as in the and culture media the iPSC colonies that are to and as in of iPSCs onto feeder also iPSC the iPSC from to colonies to well of a of culture with of as a and the of colonies well is cell is after the first to feeder-free culture conditions. when using the no to the culture a is it to the and it in a to the the colonies with it is possible to the after the first of the iPSCs the colonies are and with a of the first after feeder-free adaptation of the iPSCs, the colonies using the as in of iPSCs onto feeder also iPSC the different can be used to the cells, in or single cells on the of cells the with for or with cell for to or cell after the or cell the cells are not from the culture the iPSC colonies by culture when culture medium, we it with a to that the cell are not into single the cell with a to and it to a and it up and to a to that the of the is colony with cell be to the the cell by the of the and without the the iPSC in the of of a and in a for cell in other culture to we with a of or the iPSC colonies are in well to or of a and this as the feeder-free cultures of iPSCs are a the iPSCs with in for the by of culture the cell in a cells can be using and a the for to a cell and in the desired of for downstream of single iPSCs has been when in that the of the fibroblast cell of cells, in with cells Therefore, this with the protocol were established as effective to the of feeder cells. The of feeder cells that were in the culture for culturing and the iPSC colonies to a of feeder cells a of feeder cells were in of a of the iPSC culture onto feeder cells, it using the protocol in the of iPSCs onto feeder of iPSC colonies after were to colonies onto feeder cells. These colonies the desired iPSC colonies with and of cells with A. M.J. E. A. F. R. J. and generation of induced pluripotent stem cells from human S. M. F. A. M. and for human induced pluripotent stem One. Scholar and were to on the colonies are not after or colonies or of colonies are common the and only colonies to a will the of the is that the of colonies to be to be after the of is to the iPSCs. Therefore, we not to use feeder cells that have been in for of of colonies when the of iPSCs. is that the of iPSC colonies have the of as and not that the recovery after improved when the in the for the culture In addition, when of well is not all the is but of is of the freezing protocol freezing and the freezing of and recovery after when iPSCs were using the freezing and the recovery and colonies in culture, as as after the when using the colonies to after the freezing protocol established using the and the in the but protocols are for iPSCs, as F. S. of pluripotent stem cell cultures for Scholar to of the iPSCs after that only colonies are and not of the well the culture is of of human embryonic and induced pluripotent stem the of colonies may be In iPSC colonies to of the cells after well of colonies is to recovery after from be colonies when the still we different of freezing/thawing well of a of a or of a that the of the iPSC colonies have the of to single cells the freezing and but have it to be to use freezing that a such as the of were as in with were used for the first adaptation the first with a the iPSC to the the as of cells that the colony Nonetheless, when the using or colony not to the and as of of to the culture improved and of the iPSC colonies when as R. S. of single human embryonic stem Scholar when using However, from the the colony in the culture and not to the Therefore, it not possible to or iPSCs with this The culture used in with the first culture with of as by the In this iPSC colony were iPSCs in culture and colonies in in the of the which adaptation were using medium, and with of in with different and In these colony not to the after the first The were when the matrix with the culture used in with the cells to the culture but and cells were colonies were not and with In the culture iPSCs cells as colonies after the first and it possible to and However, colonies of the S. M. F. A. M. and for human induced pluripotent stem One. Scholar by the defined and to the iPSC were to the culture of a matrix and the culture cell and colony in the culture were after the first the the of cells that to the to proliferate as colonies with defined and to after the first a The using the and iPSCs with the colony In to these the first in feeder-free were with the the cells been for to cells were in to that iPSC colonies culture with of only when cells were the we initially this protocol to be for culturing and the iPSCs in feeder-free conditions. However, colonies of a and we a similar protocol but with a different of culture media and matrix (1) the first in feeder-free were with the (2) from the iPSCs were by using cell culture with of only when cells were this method, colonies with defined and were and cell and differentiation are Therefore, it the established method for culturing and the iPSCs in feeder-free conditions. not to the after of the iPSC colonies on feeder-free to of the iPSC colony is the are the cells will not be to the and will to the or single lines with in feeder-free Stem Scholar are to have that the first to in a cell and adaptation In addition, of the culture with or may also and of the iPSC colonies when R. S. of single human embryonic stem Scholar a of cells in the culture is during the first These cells disappear with the of the may be as by the iPSC lines may be by the of the culture when to a feeder-free a the culture used in the feeder-based with the culture of the feeder-free to this We the from the from the of and the for their We also the from of for their We also of and of the of and and and the of and are by a from and and of a from and and of the and and of the article for all of the all The no

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