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 37C, 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 8m 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 3c 3a

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