Our hair, and that of any mammal, is surprisingly complex. No substitute (e.g., faux fur or faux wool) has been able to replicate fully the desirable material properties of hair, wool or fur. Largely this is because the advantages of wool (and other mammalian hairs) over synthetic and plant-based fibres are locked up in specific organised combinations of structure and proteins. Wool fibres are more complex than a uniform mixture of the 150 or so different proteins from which they are composed. It is the way in which combinations of proteins are laid down into nanometre-scale and micrometre-scale building blocks that is the key to fibre properties.

In short, we cannot make a synthetic fibre that closely mimics wool fibres even if we wanted to; only a wool follicle can make a wool fibre. However, it should be possible to grow a wool follicle in the laboratory, which can then grow wool fibres. Our approach is to use cutting-edge techniques in which multiple single stem cells are brought together in the right conditions so that they spontaneously grow into a three-dimensional miniature tissue (called an organoid), in this case a wool follicle.

FIG. 1: (A) Sheep iPSCs form embryoid bodies in suspension culture following withdrawal of hFGF, hLIF and Dox from culture media. Immunofluorescence staining shows differentiation of sheep iPSCs give rise to cells expressing markers of the three germ layers: (B) β III-Tubulin, (C) Desmin, and (D) Cytokeratin. Hematoxylin and eosin staining of teratomas derived from sheep iPSCs reveals the presence of tissues from all three germ layers: (E) glandular epithelium (endoderm), (F) muscle (mesoderm), and (G) neural epithelium (ectoderm). (H) Sheep iPSCs at passage 15 showed a normal karyotype of 54XX. Scale bars:  = 50 µm *

Various studies showed that sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. The generation of sheep iPSCs places sheep on the front lines of large animal tissue replications such as wool. *


FIG. 2: Immunofluorescence staining demonstrates that sheep iPSC colonies are positive for expression of Oct4, Sox2, and Nanog, as well as the surface marker SSEA-4. Colonies were not observed to express SSEA-1, SSEA-3, Tra-1-60, or Tra-1-81. Scale bars: = 50 mm.*

*Sheep Fibroblasts into Pluripotency under a Drug-Inducible Expression of Mouse-Derived Defined Factors Yang Li et al 2011

Whilst our WOOLOID™️ R&D we will conduct the following morphological assessments:
1. External structure and surfaces of Wooloids described following fixation, staining of lipid-rich bodies, critical point drying and scanning electron microscopy (SEM). Output: Observation and description of surface features and possible, or lack of, fibre exit points or growth directions.

 2. Internal structure of Wooloids determined using transmission electron microscopy (TEM). 
 Analysis with focus on interfaces between follicles and support material. Output: Observation and description of Wooloid internal structural features including sub-structure interfaces and accessory structures (e.g., sebaceous glands, follicle entrance, dermal sheath). 

3. TEM of follicle structure following full or partial extraction of follicle from Wooloid. Analysis of follicle biology, cell lines, developmental zones and surrounding structure. Output: Observation and description of Wooloid developmental features. 

4. Extraction and structural analysis of single fibres using SEM and TEM. Surface features of Wooloid fibres and sheep wool from cell donor animals compared using SEM with respect to cuticle scale pattern, cuticle cell interfaces and damage and adhered material (e.g., inner-root sheath breakdown products). Cross sections analysed using TEM to examine the cuticle structure, cortex (including patterns of keratin intermediate filaments) and medulla. Overall organisation of Wooloid fibre compared to known wool phenotype (e.g., lustrous, crimped etc.). Outputs: Observation and description of Wooloid fibre structural features with comparison to natural sheep wool.



SEM IMAGE OF WOOL SURFACE CUTICLE (LEICHESTER)

FIG. 3:  Scanning electron micrographs (SEM) wool surface cuticle of 18 micrometre diameter English Leicester wool fibres.