1. The study aimed to reprogram differentiated dermal papilla (DP) cells from mouse hair follicles into pluripotent stem cells by injecting them into mouse blastocysts to generate chimeric embryos. 2. DP cells from GFP mice were injected into blastocysts. Injected and intact embryos were transferred to surrogate mothers. Resulting fetuses were examined for contribution of DP cells. 3. Some fetuses showed contribution of DP cells to embryonic tissues including ectoderm. DP cells were also found mosaicly distributed in liver, stomach, bone marrow, and cartilage tissues of fetuses, as well as in hair follicles. However, only a minority of injected DP cells

1. Reprogramming hair follicles cells to stem cells like
phenotype by injection into blastocysts
1. Introduction
A.Madich, G.Richardson, C.Jahoda, School of Biological and Biomedical Science, Durham University, South Road, Durham DH1 3LE
The “gold-standard test” for pluripotency is the
ability of a cell to contribute extensively to all adult
cell types, including the germ line.
• Differentiated adult cells can be transformed into
pluripotent cells when aggregated with ES cells,
suggesting ES factors may be essential for
conferring pluripotency
• Although important regulatory transcription
factors have been discovered, information can still
be gained through studying embryonic stem cells
using traditional means.
• Here we describe work aimed at changing
differentiated dermal papilla (DP) cells from mouse
hair follicles into pluripotent stem cells, by
generating chimeric embryos, and to understand
control mechanisms of cell fate during early
embryo development.
2. Material and Methods
• Embryo collection and culture: fully expanded
3.5 pcd blastocysts were collected from CD-1
females induced to superovulation or after natural
mating.
• Cells for blastocyst injection: A cell line of DP
cells were derived from hair follicles of
fluorescently labelled GFP CD-1 mice. Typically
cells had a diameter of 5 m to 15 m in culture.
• Generation of chimeric mice: 8-10 DP cells
were injected into the blastocavity of each
blastocyst, using an Eppendorf Systems
micromanipulator.
• Embryo transfer: after brief cultivation in
KSOM at 37C, 5% CO2 injected blastocysts and
control (intact) embryos were transferred to foster
CD-1 mothers under anesthesia.
•Biopsy: foetal samples were fixed, embedded in
OCT and sectioned vertically with respect to the
skin surface. Serial 8m sections were taken at
24-32 m intervals.
• Immunostaining / immunofluorescence: to
visualize daughter DP-GFP cells the sections
were stained using a polyclonal GFP-antibody.
• In vitro cultivation: injected blastocysts were
cultivated on a monolayer of feeder mouse
embryonic fibroblasts in ESC medium with 20%
FCS during 9-13 days.
1. Reynolds, AJ & Jahoda, CAB, Inductive properties of hair follicle cells. Ann NY Acad Sci, 1991 642, 226–242.
2. Jahoda, CAB, Horne, KA, Oliver, RF, Induction of hair growth by implantation of cultured dermal papilla cells. Nature, 1984 311, 560–562.
3. Elliot, K, Stephenson, TJ, Messenger, AG, Differences in hair follicle dermal papilla Volume are due to extracellular matrix volume and cell number: implication for the control of hair follicle
size and androgen responses. Embryonic differentiation. Journal of Investigative Dermatology, 1999 113, 873–877.
We are grateful to LSSU of Durham University for technical assistance and support
a) cppendorf Systems d) Common
scheme for blastocysts injection
3. Results
References and Acknowledgments
2. Sometimes the extra elastic features of trophoblast wouldn’t allow
penetration of the pipette and injected cells were deposited between
the trophoblast and zona pellucida (2a, 6). This could result in a mosaic
trophoblast or loss of injected cells.
3. The argument exists that the embryo’s own blastomeres are more
viable and can have a suppression effect on injected cells reducing the
contribution of these cells to the postimplantation epiblast.
• Nevertheless, 28 transfers gave a rise to 24 full-grown foetuses (5.3%
of transferred embryos). Transfers of intact embryo led to more than
50% implantations.
• Some mouse embryos bearing fluorescent cells had been visualized
at traditional resolution (3a-c).
• Two 14 pcd chimeras had significant contribution of DP-GFP cells to
their embryonic ectoderm (3d-g), but other foetuses indicated a
predominant migration of daughter cells to the epiblast that occurs at
gastrulation as observed in other studies.
Head E17
1. 648 mouse embryos were
harvested from donors, 598
were injected with DP-GFP
cells: 43 at morulae stage
(Mo) and 555 at blastocyst
stage (Bl). 434 injected
embryos were transferred to
recipient mice and 164 were
allowed to develop in vitro.
50 embryos were transferred
intact as a control.
1a. 3.5 pcd Bl prepared for injection
1b. Injection causes a brief collapse
of the embryos which does not
influence further development
1c
C
2a 2b
2c 2d
2e 2f
a b
a) Diagram of
Dermal Papilla
b) Dermal papilla
in culture
1d
•Reprogrammed individual DP cells of hair follicle derivation aggregated with other “carrier”
blastomeres appear, in some cases, to be able to contribute to the resulting foetuses and to
form as inner cell mass and trophoectoderm lineages. However, only a minority of cells have
this capability.
•DP cell injections into mouse blastocysts have little detrimental effect on the overall
development of embryos.
• Our findings suggest DP-GFP cells are likely to be present in epiblast-
derived lineages and these, obviously, may influence the development of
the epiblast-derived components.
• GFP signal was detected in both the embryonic and extraembryonic
tissues and was mosaically distributed in the liver, stomach, bone
marrow, head and body cartilage tissues (see next column).
• GFP antibody labelling with haematoxylin-eosin staining allowed us to
observe GFP signals in skin, namely in hair follicles.
3e
10
3d
A
9
The embryos in more than
40% of foster mothers
showed varied signs of
arrested development like
encapsulation, absorbed
embryos or a diminution of
embryonic tissues appearing
like small haematomas. 5
dead foetuses with
development arrested at 8-9
pcd and 5 extremely small
conceptuses in advanced
stage of being absorbed
were found.
3b 3c3a
3f 3g
1. Introduction
2. Material and methods
1c. Embryo
transfer to
uterine horn
of pseudo-
pregnant
mother
1d. Living
offspring
obtained after
transfer of
injected
embryos
1a
1b
Signs
of embryo
development
arrested
after transfer
of injected
embryos:
1e. Haematomas
(8mm)
1f. Absorbed
placenta
tissue (5mm)
1g. Encapsulated
embryo
1e
1f
1g
c
d
2g
2h
2i
2j
2a,2c, 2e) hatched mouse blastocysts
2b, 2d, 2f) DP-GFP cells (green) in blastocavity under fluorescence
2g-j) mouse blastocysts with DP-GFP (green) cells inside
3a-c) Fluorescent cells contributing
to embryonic ectoderm can be
visualized at traditional resolution
x20
3d-g) Use of confocal imaging (M1
AXIO) allowed us to recognise DP-
GFP cells in the ectroderm of
14dpc foetuses.
4 Developmental pluripotency of reprogrammed derma papilla (GFP antibody label, brown) cells
17 dpc, brain and cartilage of head 17 dpc, brain 17 dpc, cartilage tissues of head 15 dpc, tissue of visceral organ
17 dpc, bone marrow, central cord
15 dpc, bone marrow, chest 15 dpc, hair folicle in body area 15 dpc, hair follicles in head area
15 dpc, bone marrow, leg
14 dpc, labirinthical layer of placenta
17 dpc, liver
Conclusion
5 Confirming identity of
the embryonic tissue
derived from chimeric
foetuses carrying
reprogrammed DP
cells:
5a. Skin and muscle
tissue, 17 pcd
5b. Bone tissue and
braine, 17 pcd
5c. Connective
tissues, 15pcd
5d. Bone marrow,
cartilage and
connective tissue,
17pcd
5e. Skin, 15 pcd
5f. Bone marrow and
cartilage tissue.
5g. Muscle, 15pcd
GREEN = GFP Cells
RED =
Autofluorescence
GREEN/RED overlap
= Autofluorescence
5a
5b
5c
5d
5e 5f 5g