Mesenchymal Stem Cells Derived from Induced Pluripotent Stem Cells are Epigenetically Rejuvenated

Earlier this year, Miltalipov and his research group published a paper in Nature that compared the genetic integrity of embryonic stem cells made from embryos, to induced pluripotent stem cells and embryonic stem cells made from cloned embryos.  All three sets of stem cells seemed to have comparable numbers of mutations, but the induced pluripotent stem cells had “epigenetic changes” that were not found in either stem cell line from cloned or non-cloned embryos.

Genetic characteristics have to do with the sequence of the DNA molecules that make up the genome of an organism.  Epigenetic characteristics have nothing to do with the sequence of DNA, but instead are the result of small chemicals that are attached to the DNA molecule.  These small chemical tags affect gene expression patterns.  Every cell has a specific epigenetic signature.

During development, the cells that will form our eggs and sperm in our bodies, the “primordial germ cells,” begin their lives in the outer layer of the embryo.  During the third week of life, these primordial germ cells or PGCs move like amoebas and wander into the yolk sac wall and collect near the exit of a sac called the “allantois.”  The PGCs are outside the embryo at this time or extraembryonal.  Incidentallyyolk sac is a terrible name for this structure, since it does not produce yolk proteins.  Therefore other textbooks have renamed it the “primary umbilical vesicle,” which is a bit of a mouthful, but it probably better than “yolk sac.”


1 - Primordial germ cells 2 - Allantois 3 - Rectum 4 - Ectoderm 5 - Foregut 6 - Primordial Heart 7 - Secondary yolk sac 8 - Endoderm 9 - Mesoderm 10 - Amniotic cavity
1 – Primordial germ cells
2 – Allantois
3 – Rectum
4 – Ectoderm
5 – Foregut
6 – Primordial Heart
7 – Secondary yolk sac
8 – Endoderm
9 – Mesoderm
10 – Amniotic cavity

The embryo around this time undergoes a bending process as a result of its growth and the head bends toward the tail (known as the cranio-caudal curvature) and then the sides of the embryo fold downwards and eventually fuse (lateral folding).  This bending of the embryo allows the PGCs to wander back into the embryo again between the fourth and sixth week.  The PGCs move along the yolk sac wall to the vitelline and into the wall of the rectum.  After crossing the dorsal mesentery (which holds the developing intestines in place) they colonize the gonadal or genital ridge (which is the developing gonad). During their journey, and while in the gonadal ridge, the PGCs divide many times.

1 - Rectum 2 - Vitelline 3 - Allantois 4 - Nephrogenic cord (pink) 5 - Gonadal ridge (green) 6 - Primordial germ cells (red dots) 7 - Heart prominence
1 – Rectum
2 – Vitelline
3 – Allantois
4 – Nephrogenic cord (pink)
5 – Gonadal ridge (green)
6 – Primordial germ cells (red dots)
7 – Heart prominence

When the PGCs move into the developing gonad, the chemical tags on their DNA are completely removed (rather famous paper – Lee, et al., Development 129, 1807–1817 (2002).  This epigenetic erasure proceeds in order for the PGCs to develop into gametes and then received a gamete-specific set of epigenetic modifications.  These epigenetic modifications also extend to the proteins that package the DNA into chromosomes – proteins called histones.  Specific modifications of histone proteins and DNA lead to gamete-specific expression of genes.  Once fertilization occurs, and the embryological program is initiated, tissue-specific epigenetic modifications are conveyed onto the DNA and histones of particular cell populations.

This is a long-winded explanation, but because many cancer cells have abnormal epigenetic modifications, these epigenetic abnormalities in induced pluripotent stem cells (iPSCs) have been taken with some degree of seriousness.  Although, there is little evidence to date that links the cancer-causing capabilities of iPSCs with specific epigenetic modifications, although it certainly affects the ability of these cells to differentiate into various cell types.

A paper has just come from the laboratory of Wolfgang Wagner from the Aachen University Medical School, in Aachen, Germany that derived iPSCs from mesenchymal stem cells from human bone marrow, and then in a cool one-step procedure, differentiated these cells into mesenchymal stem cells (MSCs).  These  iPS-MSCs looked the same, and acted the same in cell culture as the parent MSCs, and had the same gene expression profiles as primary MSCs.  However, all age-related and tissue-specific epigenetic patterns had been erased by the reprogramming process.  This means that all the tissue-specific, senescence-associated, and age-related epigenetic patterns were erased during reprogramming.  Another feature of these iPS-MSCs is that they lacked but the ability to down-regulate the immune response, which is a major feature of MSCs.

Thus, this paper by the Wagner lab shows that MSCs derived from iPSCs are rejuvenated by the reprogramming process.  Also, the donor-specific epigenetic features are maintained, which was also discovered by Shao and others last year.  This suggests that epigenetic abnormalities are not an inherent property of the derivation of iPSCs, and that this feature is not an intractable characteristic of iPSCs derivation and may not prevent these cells from being successfully and safely used in the clinic.  However, this might be a cell type-specific phenomenon.  Also, the loss of the immune system regulatory capabilities of these iPS-MSCs is troubling and this requires further work.


Identifying the Actors Who Play the Part During Reprogramming

A remarkable paper in the journal Nature by Claudia Doege and others (Nature 488, 652-655 (2012)) has revealed the mechanism by which cells are reprogrammed to induced pluripotent stem cells (iPSCs).

Fully differentiated cells have those genes that induce pluripotency (that is, the ability to form any cell type in the adult body) completely shut off (see Takahashi and Yamanaka, Cell 126, 663-676 (2006)). However, if four different genes are introduced into these differentiated cells, namely Oct4, Sox2, c-Myc and Klf4, then the differentiated cell de-differentiates into an iPSC. However, how these genes do this has been rather elusive. However the Doege et al. paper has elucidated our understanding of this process.

To begin, we must understand that gene expression is jointly controlled by two classes of proteins and these include transcription factors, which bind to targets in DNA and activate DNA, and epigenetic regulators that alter the proteins that package DNA (histones). Doege and others have identified two epigenetic regulators called Parp1 and Tet2 that stimulate the expression of the dormant pluripotency genes in differentiated cells that convert them into iPSCs.

What do these proteins do? Parp1 and Tet2 induce the removal of a chemical tag (H3K27me3, for those who are interested) from those histones associated with pluripotency genes and induce the addition of a different chemical tag (H3K4me2, again for the interested). The first chemical tag on the histones shut down gene expression, but the second type of chemical tag induce gene expression.

Doege and his colleagues showed that these epigenetic changes occur before increased expression is detected in two pluripotency genes (Nanog and Esrrb). These epigenetic changes are highly correlated with the binding of the transcription factor Oct4 (also known as POU5F1). Oct4, you see, activates the expression of Parp1, and after the histones are properly modified, Oct4 can bind to the promoter of these genes and activate their expression.

This report shows, for the first time, that epigenetic regulators are equally as important as transcription factors in the status switch from differentiated state to iPSC. According to the accompanying summary of Doege’s article by Kyle Loh and Bing Lim, “reprogramming transcription factors liaise with endogenous epigenetic regulators to execute reprogramming.”

Source – Loh and Lim, Epigenetics: Actors in the cell reprogramming drama. Nature 488,599–600 (30 August 2012); doi:10.1038/488599a

Loh and Lim point out that this work also raises new questions. For example, how do Parp1 and Tet2 specifically activate these pluripotency genes rather than affecting the genome globally? Are there cell-type specific epigenetic regulators? Does this mechanism work for other cell types as well? Does this explain why some cell types become iPSCs so much more efficiently than others? Doege et al. have written an incredible paper that blasts open the door of on our understanding of iPSC formation. This should provide new insights into reprogramming in general.