Derivation and Culture of Induced Pluripotent Stem Cells on an Artificial Substrate


When mouse embryonic stem cells were first derived in 1981 independently by Gail Martin at UCSF and Evans and Kaufman at Cambridge University, the inner cell mass cells from the blastocyst-stage mouse embryos were cultured on a layer of mouse skin cells that had been treated with a drug that prevented them from dividing or with radiation that did the same. These single layers of mouse skin fibroblasts secreted growth factors that prevented the embryonic stem cells from differentiating and drive them to divide. These layers of cells were known as “feeder” cells, because the secretions of the cells fed the growing embryonic stem cells.

When James Thomson at the University of Wisconsin derived the first human embryonic stem cell lines in 1998, he also used mouse feeder cells to keep the cells growing and undifferentiated. Once the embryonic stem cells were taken from this culture system, they began to differentiate.

Human and mouse embryonic stem cells. (A) Colony of Human Embryonic Stem Cells (Cat# GSC-1103) growing on mitotically arrested feeder layers (Cat Nr GSC-6001M); colony morphology is characteristic of undifferentiated human ES cells. (B) Mouse Embryonic Stem Cells (Cat# GSC-5002) in culture.
Human and mouse embryonic stem cells. (A) Colony of Human Embryonic Stem Cells (Cat# GSC-1103) growing on mitotically arrested feeder layers (Cat Nr GSC-6001M); colony morphology is characteristic of undifferentiated human ES cells. (B) Mouse Embryonic Stem Cells (Cat# GSC-5002) in culture.

However, it became equally clear that using mouse feeder cells represented a problem if human embryonic stem cells were going to be used for clinical purposes because animal cells can harbor occult viruses and other infectious agents that can infect human cells. Also, animal cells possess unusual sugars that are transferred to human cells when they are together in culture. Such foreign sugars can elicit robust immune responses against the cells if they are used for clinical purposes See Martin et al., Nature Medicine 2005; 11:228-232; and Stacey et al., Journal of Biotechnology 2006;125:583-588). Therefore, it became clear that finding ways to grow embryonic stem cells in the absence of feeder lines was an important goal if these cells were going to be used for clinical purposes.

Several laboratories successfully derived so-called “Xeno-free” embryonic stem cells by using protein substrata to grow the cells. These protein substrata included matrigel (animal), human laminin, E-cadherin, and vitronectin (see Xu C,, et al (2001) Nat Biotechnol 19:971–974; Miyazaki T,, et al. (2008) Biochem Biophys Res Commun 375:27–32; Nagaoka M,, et al. (2010) BMC Dev Biol 10:60; Chen G,, et al. (2011) Nat Methods 8:424–429). When Yamanaka and his colleagues discovered procedures for making human induced pluripotent stem cells, once again, feeder lines were initially used, but feeder-free protocols were also developed for deriving xeno-free induced pluripotent stem cells (iPSCs; see Chen G,, et al. (2011) Nat Methods 8:424–429; Nakagawa M,, et al. (2014) Sci Rep 4:3594).

A new report from Luis Gerardo Villa-Diaz, Jin Koo Kim, Joerg Lahann, and Paul H. Krebsbach from the University of Michigan, Ann Arbor, Michigan, has described a way to derive and grow human iPSCs on a completely synthetic substratum. This substratum, poly2-(methacryloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide, or PMEDSAH, forms a hydrogel that is completely synthetic. Therefore, the cells do not touch anything made from genetically manipulated cells or animal products.

Krebsbach and his group used fibroblasts from human gum tissue as their cell source. These cells were reprogrammed into iPSCs by means of infection with recombinant Sendai viruses. These viruses cause expression of the four genes required for reprogram cells (Oct4, Klf4, Sox2, and c-Myc), but they do not insert their viral genomes into the chromosomes of the host cell. Therefore, these viruses only express the reprogramming factors transiently, and afterwards, no trace of them can be found in the iPSC line, provided you properly screen for the absence of the virus.  The reprogrammed cells were grown on the PMEDSAH and the cells not only were reprogrammed on this substratum, but also grew on it rather well.

The gum-based fibroblasts were nicely reprogrammed and made iPSCs that expressed all the right genes and produced tumors called teratomas when implanted into nude mice. The teratoma-production assay is an important test for pluripotency, because teratomas are tumors that consist of the mishmash of different tissue types. The fact that implanted cells produce these tumors with a mixed cell population of such wildly different cell types is an important indication of their pluripotency.

Evaluation of pluripotency of human induced pluripotent stem cells (iPSCs) derived and cultured on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The pluripotency of the three human iPSCs derived and cultured on PMEDSAH was tested by embryoid body (EB) formation, directed in vitro cell lineage differentiation, and teratoma induction. (A): Representative micrograph of EBs from human foreskin fibroblast induced pluripotent stem cells 9 months after derivation and continuous in vitro culture. (B): Graph showing relative RNA transcription levels of genes expressed in cells after directed in vitro differentiation of human iPSCs on PMEDSAH-GPs. (C–E): Representative micrographs of directed in vitro cell lineage differentiation on PMEDSAH-GPs of human iPSCs 9 months after derivation and continuous in vitro culture. Neural differentiation (ectoderm) was achieved after treatment with Noggin (B, C). Definitive endoderm/pancreatic differentiation was induced by activin A treatment (B, D). Mesoderm lineage was obtained after treatment with activin A and BMP4 to induce cardiac muscle differentiation (B, E). Teratoma formation was performed 6 months after derivation and continuous in vitro culture of human iPSCs. (F–H): Representative micrographs of neurons (F), gut glandular epithelium (G), and cartilage (H) identified in teratomas. Scale bars = 200 μm (A), 100 μm (C–E), and 50 μm (F–H). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.
Evaluation of pluripotency of human induced pluripotent stem cells (iPSCs) derived and cultured on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The pluripotency of the three human iPSCs derived and cultured on PMEDSAH was tested by embryoid body (EB) formation, directed in vitro cell lineage differentiation, and teratoma induction. (A): Representative micrograph of EBs from human foreskin fibroblast induced pluripotent stem cells 9 months after derivation and continuous in vitro culture. (B): Graph showing relative RNA transcription levels of genes expressed in cells after directed in vitro differentiation of human iPSCs on PMEDSAH-GPs. (C–E): Representative micrographs of directed in vitro cell lineage differentiation on PMEDSAH-GPs of human iPSCs 9 months after derivation and continuous in vitro culture. Neural differentiation (ectoderm) was achieved after treatment with Noggin (B, C). Definitive endoderm/pancreatic differentiation was induced by activin A treatment (B, D). Mesoderm lineage was obtained after treatment with activin A and BMP4 to induce cardiac muscle differentiation (B, E). Teratoma formation was performed 6 months after derivation and continuous in vitro culture of human iPSCs. (F–H): Representative micrographs of neurons (F), gut glandular epithelium (G), and cartilage (H) identified in teratomas. Scale bars = 200 μm (A), 100 μm (C–E), and 50 μm (F–H). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Even more importantly perhaps is the genetic integrity of these cells. Karyotypes of these iPSC lines (karyotypes lay our the chromosomes of the cell to see if there are a normal number of chromosomes and if the chromosomes appear normal) revealed that they were beautifully normal. However, hCGH (array-based comparative genomic hybridization) analysis, which uses specific chromosome-specific probes to finding missing or duplicated bits of chromosomes that are too small to see in a karyotype revealed a few missing and added bits to the genomes of all three derived iPSC lines. None of these were in known cancer loci.  As shown in the figure below, these lines had only a few mutations.  The karyotypes used cells from nine months after their derivation.  Thus these cells proved to be rather stable.

Genetic stability of the human iPSCs derived on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The genomic stability of the three human iPSC lines derived on PMEDSAH-GPs was tested 9 months after derivation and continuous in vitro culture. (A): Representative standard G-banding metaphase karyotyping of one of the three human iPSCs derived on PMEDSAH-GPs showing normal male karyotype. (B): Ideogram summarizing chromosome losses and gains (left and right, respectively) of the three human iPSCs as detected by high-resolution array-based comparative genomic hybridization. No mutations are localized in chromosome loci where genes related to stem cells, cancer, or culture adaptation are localized. Abbreviations: chr, chromosome; CNG, copy number gain; CNL, copy number loss; hFF, human foreskin fibroblast; hGF, human gingival fibroblast; iPSC, induced pluripotent stem cell.
Genetic stability of the human iPSCs derived on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The genomic stability of the three human iPSC lines derived on PMEDSAH-GPs was tested 9 months after derivation and continuous in vitro culture. (A): Representative standard G-banding metaphase karyotyping of one of the three human iPSCs derived on PMEDSAH-GPs showing normal male karyotype. (B): Ideogram summarizing chromosome losses and gains (left and right, respectively) of the three human iPSCs as detected by high-resolution array-based comparative genomic hybridization. No mutations are localized in chromosome loci where genes related to stem cells, cancer, or culture adaptation are localized. Abbreviations: chr, chromosome; CNG, copy number gain; CNL, copy number loss; hFF, human foreskin fibroblast; hGF, human gingival fibroblast; iPSC, induced pluripotent stem cell.

This paper demonstrates that it is possible to generate transgene-free, stable iPSCs on a synthetic substrate.  This type of platform has the potential to meet the good manufacturing practices that must be used to make products for clinical use.

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Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).