WO2011144901A1 - Expansion and directed differentiation of epidermal neural crest stem cells - Google Patents

Expansion and directed differentiation of epidermal neural crest stem cells Download PDF

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WO2011144901A1
WO2011144901A1 PCT/GB2011/000763 GB2011000763W WO2011144901A1 WO 2011144901 A1 WO2011144901 A1 WO 2011144901A1 GB 2011000763 W GB2011000763 W GB 2011000763W WO 2011144901 A1 WO2011144901 A1 WO 2011144901A1
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cells
neural crest
stem cells
crest stem
differentiation
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WO2011144901A4 (en
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Maya Sieber-Blum
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The University Of Newcastle Upon Tyne
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Definitions

  • the present invention relates to directed differentiation of epidermal neural crest stem cells, and more specifically human epidermal neural crest stem cells (hEPI- NCSC), for example to their differentiation into dopaminergic neurons, dopaminergic neuronal progenitors, sensory neurons, sympathetic neurons, cholinergic neurons, melanocytes, Schwann cells, smooth muscle, osteogenic differentiation into osteocytes, bone and cartilage and adrenergic differentiation.
  • Embryonic neural crest stem cells (NCSC's) physiologically give rise to a wide array of different cell types and tissues.
  • Neural crest cells give rise to the autonomic and enteric nervous systems, most primary sensory neurons and their glia, craniofacial bone/cartilage, smooth musculature of the cardiac outflow tract and great vessels, endocrine cells such as the adrenal medulla and C-cells of the thyroid , and melanocytes if the skin and internal organs. This is especially significant for the purposes of cell-based therapies and biomedical engineering of tissues. Neural crest-derived multipotent stem cells persist in the periphery during embryogenesis and in the adult organism.
  • EPI-NCSC epidermal neural crest stem cells
  • EPI-NCSC skin-derived precursors
  • Yu et a/ (2010) reported that other human hair follicle-derived neural crest-like cells formed spheres in culture following enzymatic digestion of intact hair follicles, and they expressed neuronal markers when injected into the mouse brain.
  • these cells differ from hEPI-NCSC, as they are isolated from entire hair follicles, do not show any migratory behaviour, and express non-crest markers. It is therefore questionable as to whether Yu et al describe a substantially pure population of neural crest derived stem cells.
  • hEPI- NCSC have the distinct advantage that due to their migratory ability they can be isolated with ease as a highly pure population of multipotent stem cells of neural crest origin.
  • the neural crest is a transient embryonic structure that arises during neurulation at the boundary of the neural plate and the somatic ectoderm. Neural crest cells subsequently delaminate from the forming neural tube and migrate to various locations in the embryo to generate a wide array of progeny. Neural crest derivatives include craniofacial bone/cartilage, meninges, tooth papillae, the autonomic and enteric nervous systems, most primary sensory ganglia, endocrine cells such as the adrenal medulla, smooth musculature of the cardiac outflow tract and great vessels and pigment cells (melanoyctes) of the skin and internal organs.
  • hEPI-NCSC Human epidermal neural crest stem cells
  • hEPI-NCSCs are adult stem cells but are of embryonic origin, the neural crest. The cells reside in a postnatal location, the bulge of the hair follicle. They are multipotent and able to generate all major neural crest derivatives, including neurons, nerve supporting cells, bone/cartilage cells, smooth muscle cells, and melanocytes. They are readily accessible in the hairy skin, for example from pubic skin, scalp skin or other suitable areas, by minimally invasive procedures.
  • hEPI-NCSCs can be isolated as a highly pure population of stem cells, and they can be expanded ex vivo into millions of cells, without losing stem cell markers. They express the neural crest stem cell molecular signature as well as other neural crest and global stem cell genes. The inventors have previously been involved in providing methods for isolating cultures of hEPI-NCSCs, and these are discussed in detail in US Application Number 1 1/376498.
  • the inventors have determined in the Wnt1-cre::R26R compound transgenic mouse, in which all neural crest cells and their derivatives are specifically and indelibly labelled by expression of beta-galactosidase, that the bulge of hair follicles contains neural crest stem cells, and they have developed a technique for the explantation and culture of the bulge of human hair.
  • Wataya et al (2008) discusses the efficacy of cell replacement therapy in Parkinson's disease, concluding that several studies on foetal tissue (mouse and human) have already established the quantity and purity of transplanted cells necessary for meaningful therapeutic application.
  • the same paper also highlights the various groups that have already succeeded in differentiating various types of neurons including telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons, from human embryonic stem cells.
  • telencephalic cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons
  • this paper acknowledges a number of problems inherent within the existing methods for differentiation on top of the known general concerns in relation to the use of embryonic stem cells.
  • Jin et al (2008) has similarly shown that brain-derived neurotrophic factor (BDNF) can up-regulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells, and that co-culturing rat ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow up-regulates TH and dopamine (DA) expression.
  • BDNF brain-derived neurotrophic factor
  • VMCs rat ventral mesencephalic cells
  • DA dopamine
  • Park et al (2008) have also described the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases.
  • Deierborg et al (2008) provide an excellent review of several exciting regenerative approaches to PD developed over the last 20 years, and Levy et al (2008) specifically assesses the developmental potential of h SC to replace the midbrain dopamine neurons selectively lost in PD. To date, such approaches have typically only involved embryonic and/or foetal derived neural stem cells, thereby severely restricting successful human application.
  • Skeletal abnormalities, bone trauma, osteoarthritis and deteriorating joints are examples of conditions, which severely compromise an individual's quality of life and ability to perform daily tasks of living. Derivation of replacement bone and cartilage is thus of particular interest. Osteogenic differentiation by various methods has been reported for mesenchymal stem cells, adipose cells and bone marrow stem cells with others also reporting osteogenic differentiation of NCSC derived from human embryonic stem cells (hESC), SKPs, mouse ESC and cord blood.
  • hEPI-NCSC The advantage of hEPI-NCSC is that the neural crest has the physiological ability to generate bone, as a subset of cranial bones are of neural crest origin. Likewise, melanoyctes are established neural crest derivatives.
  • hEPI-NCSCs are identified as being cells which express, to variable degrees and in a donor dependent manner, the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28 , OCT-4/POU5F1 and NANOG.
  • a method for inducing or stimulating differentiation of a population of stem cells which includes the step of; culturing a population of epidermal neural crest stem cells in the presence of added reagents, said reagents selected to induce or stimulate differentiation of the cells into one or more pre-determined, identifiable cell type or cell types.
  • the predetermined, identifiable cell type is a neural crest derivative.
  • hEPI-NCSC can be harvested as a highly pure population of stem cells without an overt need for further purification, as non-stem cells are not proliferative and within a short time are outgrown by the stem cells and left behind upon subculture. Furthermore, optimization of culture conditions provided bulge explants that produce emigrating hEPI-NCSC with high yield. Ease of isolation, high yield, and rapid ex vivo expansion, along with high purity of the cell population are highly desirable features of hEPI-NCSC and important considerations for cost-effective stem cell production.
  • Neural crest derivatives include craniofacial bone/cartilage, meninges, tooth papillae, the autonomic and enteric nervous systems, most primary sensory and their nerve-supporting glia, endocrine cells such as the adrenal medulla, smooth musculature of the cardiac outflow tract and great vessels and pigment cells (melanoyctes) of the skin and internal organs.
  • differentiated populations of cells would be useful. For example, they could be used in various therapies to replace or supplement a patient's own cells (such as the use of replacement supplementary dopaminergic neurons in the treatment of Parkinson's disease).
  • the differentiated cell populations/differentiated hEPI-NCSCs could also be used in high throughput drug screening. Predetermined cell populations, which can be substantially pure, could be produced and used for specific testing or screening purposes/ drug discovery programmes.
  • the method is for inducing or stimulating differentiation of epidermal neural crest stem cells into one or more of dopaminergic neurons, dopaminergic neuronal progenitors, melanocytes, Schwann cells, smooth muscle, and osteocytes, bone and/or cartilage, sensory neurons, sympathetic neurons or cholinergic neurons.
  • the epidermal neural crest stem cells are a substantially pure population of epidermal neural crest stem cells.
  • the epidermal neural crest stem cells are a substantially pure, expanded population of epidermal neural crest stem cells.
  • the method is for inducing or stimulating differentiation of human epidermal neural crest stem cells.
  • the method comprises the steps of; - isolating anagen phase hair follicles from a subject
  • anagen phase hair follicles are in early or late anagen phase.
  • the added reagents are selected from the groups;
  • dibutyryl cyclic AMP (a) dibutyryl cyclic AMP (db c-AMP) whereby they become immune- fluorescent for neuron-specific beta-Ill tubulin and tyrosine hydroxylase (TH); or
  • TPA (12-0- tetra-decanoylphorbol-133-acteate (PMA), (phorbol 12- myristate 13-acetate), cholera toxin and endothelin-3;
  • EPI-NCSCs differentiating into different cell types, for example, BMP2 (10 ng/ml) for bone differentiation; NGF (20 ng/ml), TGF- 2 (1 ng/ml) and forskolin (10 ⁇ ) for neural differentiation; and Neuregulin-1 (10nM) plus CNTF (10ng/ml) for Schwann cell differentiation.
  • BMP2 (10 ng/ml) for bone differentiation
  • NGF (20 ng/ml)
  • TGF- 2 (1 ng/ml) and forskolin (10 ⁇ ) for neural differentiation
  • Neuregulin-1 (10nM) plus CNTF (10ng/ml) for Schwann cell differentiation.
  • the method includes the step of expanding the population of isolated cells prior to the sub-culturing step.
  • the expansion step is necessary in cases where the resulting cells are to be used for therapeutic applications. It has been acknowledged in the art that there are significant difficulties associated with expanding stem cell populations of any type whilst retaining their essential "sternness" and multipotency.
  • Expansion is typically obtained by culturing the isolated epidermal neural crest stem cells.
  • the culture media for expansion may comprise proliferation media such as NeuroCult XF ®, StemPRO® SC SFM, FGF2, EGF, FBS (foetal bovine serum), ITS+3 (contains insulin, transferrin, sodium selenite as well as oleic acid, linoleic acid, and BSA), GlutaMAX ®, Amphotericin, Penicillin/Streptomycin.
  • proliferation media such as NeuroCult XF ®, StemPRO® SC SFM, FGF2, EGF, FBS (foetal bovine serum), ITS+3 (contains insulin, transferrin, sodium selenite as well as oleic acid, linoleic acid, and BSA), GlutaMAX ®, Amphotericin, Penicillin/Streptomycin.
  • the isolated cells undergo a 2-step sub-culturing process, the first sub-culturing step being culturing the cells in a Neural Progenitor (NP) medium and the second sub-culturing step being culturing the cells in a patterning factor (PF) medium.
  • NP Neural Progenitor
  • PF patterning factor
  • the isolated cells are sub-cultured in a PF medium comprising SHH, FGF-8, GDNF and TGF-beta2.
  • the NP medium comprises;
  • the NP medium comprises;
  • NT-3 (10 ng/ml)
  • Sub-culturing the cells in a neural progenitor medium effectively converts the multipotent stem cells to a neural stem cell like state. Further culturing in a patterning factor medium then directs differentiation of said cells. This is particularly useful when the aim is to produce a population of dopaminergic neurons or dopaminergic neuronal progenitors.
  • the patterning factor medium comprises;
  • GDNF glial derived neurotrophic factor
  • NGF neurotrophic factor
  • the patterning factor medium comprises;
  • GDNF (5 ng/ml) [glial derived neurotrophic factor]
  • NGF neurotrophic factor
  • the epidermal neural crest stem cells are obtained from the bulge of a hair follicle obtained from a subject.
  • the subject is human.
  • the method further comprises the step of returning the differentiated cells into the subject.
  • Autologous transplantation of this type is advantageous in avoiding graft rejection.
  • cells obtained using the method of the first aspect for the treatment of a disease state.
  • the undifferentiated hEPI-NCSCs could be used for the treatment of a disease state.
  • the cells obtained using the method of the first aspect are for the treatment of Parkinson's disease.
  • the cells obtained using the method of the first aspect are for the treatment of Alzheimer's disease.
  • the cells obtained using the method of the first aspect are for the treatment of autism.
  • this will utilise cholinergic neurons obtained using the above methods.
  • the cells obtained using the method of the first aspect are for the treatment of peripheral neuropathies.
  • the cells obtained using the method of the first aspect are for the treatment of degenerative diseases.
  • the cells obtained using the method of the first aspect are for the treatment of skeletal abnormalities.
  • the cells obtained using the method of the first aspect are for the treatment of bone fractures and fixation of bones.
  • An example of fixation may be during a hip replacement where prosthesis is attached to bone.
  • a graft at this point can help to prevent the prosthetic becoming loose over time.
  • the cells obtained using the method of the first aspect are for the treatment of osteoarthritis or osteoporosis.
  • the cells obtained using the method of the first aspect are for the treatment of stroke.
  • the cells obtained using the method of the first aspect are for the treatment of acute and severely debilitating trauma episodes such as spinal cord injury, head and neck injury.
  • hEPI-NCSC are particularly relevant for treatment in spinal cord injury, as neural crest stem cells are ontologically closely related to spinal cord stem cells.
  • Studies carried out by the inventors in mouse models of spinal cord injury showed that EPI-NCSC grafts caused a significant improvement in sensory connectivity and touch perception, that they can provide neurotrophic support and angiogenic activity, and that they possibly modulate scar formation by synthesis and release of metalloproteases.
  • the inventors believe that hEPI- NCSC have similar properties.
  • the cells obtained using the method of the first aspect are for the treatment of burns.
  • the cells obtained using the method of the first aspect are for the treatment of wounds.
  • the cells obtained using the method of the first aspect are for the treatment of skin conditions.
  • dopaminergic neurons or dopaminergic neuronal progenitors obtained by the method of the first aspect.
  • Expanded but undifferentiated hEPI NCSC could be used in place of the said dopaminergic neurons or dopaminergic neuronal progenitors.
  • a culture medium for the directed differentiation of hEPI-NCSCs into dopaminergic neurons or dopaminergic neuronal progenitors comprising;
  • GDNF glial derived neurotrophic factor
  • NGF neurotrophic factor
  • the culture medium for the directed differentiation of hEPI- NCSCs into dopaminergic neurons or dopaminergic neuronal progenitors comprises;
  • GDNF (5 ng/ml) [glial derived neurotrophic factor]
  • NGF neurotrophic factor
  • a culture medium for the progression of hEPI-NCSCs to a neural stem cell like state comprising;
  • the culture media for the progression of hEPI-NCSCs to a neural stem cell like state comprises;
  • a method of treating Parkinson's disease comprising administering a therapeutically active amount of said dopaminergic neurons or dopaminergic neuronal progenitors.
  • a method of ex-vivo expansion of epidermal neural crest stem cells comprising the step of; culturing an isolated population of epidermal neural crest stem cells in expansion media.
  • the expansion media comprises;
  • ITS+3 contains insulin, transferrin, sodium selenite as well as oleic acid, linoleic acid, and BSA
  • the proliferation media is NeuroCult XF ®
  • the expansion medium comprises antibiotics such as Penicillin and/or Streptomycin.
  • the method of ex-vivo expansion of human epidermal neural crest stem cells is carried out at 5% oxygen.
  • the expansion media comprises;
  • the method of ex-vivo expansion of human epidermal neural crest stem cells also includes the pre-steps of;
  • ITS+3 contains insulin, transferrin, sodium selenite as well as oleic acid, linoleic acid, and BSA
  • the proliferation media is NeuroCult XF ®
  • the proliferation media may be StemPRO MSC SF ®.
  • the expansion media comprises antibiotics such as Penicillin and/or Streptomycin.
  • antibiotics such as Penicillin and/or Streptomycin.
  • Figure 1 Dissection of the bulge from human hair. Hair follicles in the anagen (growth) phase are dissected from full thickness hairy skin. Surrounding fatty tissue and dermis are removed mechanically with a sharpened tungsten needle. The dermal papilla is cut off and discarded. The area of the bulge is cut into 2 or 3 pieces. Bulge explants are pooled placed in culture medium and subsequently placed onto CellStart-coated culture plates, where the bulge explants adhere to the substratum within one hour. Figure 2i hEPI-NCSC emigrating from bulge explant. 2ii. Bulge explant with emigrating cells that have neural crest morphology.
  • FIG. 2ii A Within 6 - 10 days, migratory cells emigrate from about 35 percent of bulge explants. The cells have the typical stellate morphology of neural crest cells. In addition to migrating away from the bulge explant, they proliferate at a rapid rate. Rounded cells in this image are cells that undergo mitosis in order to divide.
  • Figure 2ii B Primary hEPI-NCSC emigrate from adult hair follicle bulge cultures.
  • (iiA-C) Bulge explant with cells emigrating onto the CellStart substratum (arrows) at days six, eight and 10, respectively. Images are from the same bulge explant.
  • FIG. 3 Bulge explant with halo of cells that have putative keratinocyte precursor morphology. Rare bulge explants do not release neural crest cells, but putative keratinocyte progenitor cells. Keratinocytes are easily distinguished from neural crest-derived cells by their typical cobblestone morphology and by the fact that they are non-migratory but remain in close contact to each other and the bulge explant. Bulge explants that contain putative keratinocyte progenitor cells, such as the one shown in this image, are discarded.
  • FIG. 4 Characterization of hEPI-NCSC in primary explants by real-time PCR (qPCR). Data are presented in the black column, left, for each gene.
  • hEPI-NCSC express the neural crest stem cell molecular signature the inventors have defined for mouse EPI-NCSC and mouse embryonic neural crest stem cells (Hu YF, Zhang Z-J and Sieber-Blum, , 2006).
  • Signature genes include VDAC1 , ETS1 , PCbP4, MYO10, H1 FX, THOP1 , MSX2, CRYAB, VARS2, PEG10, CALR, CRMP1 , UBE4B, PYG02, AGPAT6 AND ADAM 12.
  • hEPI-NCSC also express additional neural crest-characteristic genes, including SOX10, SNAI2, TWIST, MS1 ('musashi') AND P75NTR, as well as general stem cell genes, TERT, NES ('nestin') and CD34.
  • hEPI-NCSC express several early lineage genes , including GFAP, MITF DCT, TUBB3, NEFL AND ACTA2, but not COLA2A1.
  • hEPI-NCSC also express the pluripotency genes C-MYC, KLF4, SOX2, LIN28, POU5F1/OCT4 AND NANOG. Data are expressed as percent of average of four house-keeping genes.
  • ETS1 The transcription factor ETS1 (A and A') was expressed in 97.3 ⁇ 2.7% cells and had a nuclear localisation.
  • THOP1 B and B' showed a cytoplasmic location necessary for its roles in protein function and metabolism and was immunoreactive in 100% of cells.
  • MSX2 C and C is an important transcription factor involved in maintaining the balance between survival and apoptosis in neural crest cells and showed 98.2 ⁇ 1.8% expression.
  • CRMP1 (D and D') is located in the cytoplasm in 98.3 ⁇ 1.7% of cells as it is a cytosolic phosphoprotein involved with signal transduction pathways.
  • UBE4B (E and E') is involved in ubiquitination and was seen to have a cytoplasmic localisation in 96.7 ⁇ 1.6% of cells.
  • MYO10 (F and F') was seen to have a cytoplasmic localisation and was expressed in 97.6 ⁇ 2.4% of cells, consistent with its role in cytoskeletal organisation.
  • ADAM12 (G and G') showed a cytoplasmic localisation in 89.0 ⁇ 6.8% of cells as it is a membrane anchored protein and has roles in cell-cell and cell-matrix contacts.
  • CRYAB (H and H') is involved in intracellular structure and subunits act as molecular chaperones and are thus localised to the cytoplasm in 100% of cells.
  • the intermediate filament protein and stem cell marker NESTIN (I and P) was seen to have a cytoplasmic localisation and expressed in 100% of cells.
  • the neural crest marker SOX10 (J and J') has a role as a nucleo-cytoplasmic shuttle protein and was expressed in 98.6 ⁇ 1.4% of cells.
  • hEPI-NCSC in clonal culture differentiate into all major neural crest progeny. All possible permutations of antibody stains have been performed to show that hEPI-NCSC in clonal culture can give rise to many different neural crest derivatives and clone-forming cells were therefore multipotent.
  • Triple stains combining two cell type specific antibodies (black and white) and colored merged images using DyLight 488 (green) or DyLight 594 (red) fluorescence and DAPI (blue) nuclear stain.
  • the top row shows a double stain with antibodies against ACTA2 and COL2A1 , as indicated.
  • the third image, in colour, is the merged image with blue DAPI nuclear stain added.
  • TUBB3-positive neuronal cells coexist with GFAP-positive Schwann cells/glia.
  • GFAP-immunoreactive cells co- exist with COL2A1 -positive bone/cartilage cells.
  • ACTA2-positive smooth muscle cells co-exist with GFAP-positive Schwann cells/glia.
  • Self-renewal is an important part of the definition of a stem cell. It is the ability of a stem cell to give rise to daughter stem cells. Self- renewal was shown here by serial cloning. Cells in primary clones were resuspended by trypsin treatment at 22 days in culture and placed into clonal culture again, where they gave rise to secondary clones. Clone-forming ability was maintained at high levels, as 70.7 ⁇ 7.9% of secondary clones and
  • hEPI-NCSC are multipotent stem cells.
  • Figure 8 Expression of the six essential pluripotency genes at the RNA and protein levels. hEPI-NCSC express transcripts of the six essential pluripotency genes, C-MYC, KLF4, SOX2, LIN28, OCT4/POU5F1 and NANOG. As this was an unexpected finding and in order to calibrate gene expression levels, we compared
  • pluripotency gene expression levels to those in human embryonic stem cells (H9 cell line).
  • H9 cell line Expression of pluripotency genes by hEPI-NCSC was compared to H9 hESC by qPCR and the AACt method used to determine fold differences in expression levels. Three independent donors were assessed (Donor A-C).
  • SOX10 was used as a marker of neural crest stem cells and in all cases was more abundant in hEPI-NCSC than in H9 cells by approximately 100 fold.
  • C- MYC, KLF4 and NANOG were expressed at similar levels in hEPI-NCSC compared to hESC.
  • SOX2, LIN28 and POU5F1/OCT4 were expressed at lower levels. While trends are similar, there are donor-specific variations in expression levels.
  • hEPI-NCSC from donor C were also analyzed for expression of the pluripotency genes at the protein level. Indirect immunocytochemistry was performed using all possible permutations of antibody combinations as labeled ( ⁇ - ). In all cases, hEPI-NCSC were immunoreactive for the relevant markers. The percentage of cells expressing the gene of interest was determined (J); C- MYC, 100 ⁇ 0%, KLF4, 56.5 ⁇ 6.8%, SOX2, 69 ⁇ 4.5%, LIN28, 98.8 ⁇ 0.9%,
  • hEPI-NCSC can be expanded into millions of stem cells without an overall significant loss of stem cell markers.
  • Expression of the neural crest stem cell molecular signature and other neural crest stem cell genes validates the neural crest origin of hEPI-NCSC.
  • the data from primary explants are the average of RNA from three donors; whereas the results from ex vivo expanded cells represent the average of two different donors. Significant differences in expression levels are likely to be due to donor-specific differences. Student's t-test. Asterisk(s) indicate levels of significant difference as follows: * , p ⁇ 0.05, ** *, p ⁇ 0.001. ND, not detected.
  • FIG. 9 B Growth curve of hEPI-NCSC proliferation during ex vivo expansion. On average three million cells per bulge were obtained within 28 days. Notably, at 28 days cell growth has not yet levelled off but is still in the log phase. Further expansion is thus possible. Overall, we show that hEPI-NCSC can be expanded ex vivo efficiently and reproducibly and that they retain sternness. An attractive feature of the expansion protocol is its short duration. Changes in the karyotype are thus less of a concern in hEPI-NCSC than in cell lines that are passaged multiple times. The high numbers of stem cells that can be obtained through ex vivo expansion within a short period of time make testing in animal models of human disease and future applications feasible. (Fig. 9 C).
  • FIG. 10 Protocol for directed differentiation of hEPI-NCSC into dopaminergic neurons.
  • Bulge explants from human hair follicles are prepared as described above. Cultures are maintained at 37 °C in a humidified atmosphere that contains 5% C0 2 and 5% 0 2 . After 7-10 days, bulges are removed and the hEPI-NCSC suspended by trypsin treatment. hEPI-NCSC were then seeded into CellStart-coated 35 mm culture plates at 2,500 cells per plate in expansion medium. Two days later, the cells are switched to "NP" (neural progenitor) medium with the goal to differentiate the multipotent stem cells into neural stem cell-like progenitor cells.
  • NP neural progenitor
  • NP medium changes are done by removing half of the old culture medium and replace it with half of the new medium, in order to acclimate the cells slowly to the new culture condition.
  • Cells are grown in NP medium for 7 - 10 days, depending on cell growth, and are then sub-cultured into "PF" (patterning factor) medium using again 50-50 medium exchanges.
  • PF patterning factor
  • Cells are maintained in PF medium for 2 - 3 weeks at 3% oxygen and 5% CO2.
  • the maturation factor ascorbic acid is added to the PF medium.
  • NP medium cells change morphology from stellate morphology to a more elongated shape and often elaborate short processes (image at lower left).
  • PF medium cells become confluent and change shape again. They elaborate long processes; the soma is first rounded and eventually cells assume the typical shape of midbrain dopaminergic neurons (image, lower right).
  • FIG. 11 In vitro differentiated dopaminergic neurons express markers typical for midbrain dopaminergic neurons. At the RNA level, genes characteristic for midbrain dopaminergic neurons are expressed. Data have been generated by real-time PCR and are expressed as percent of the average of 4 housekeeping genes (HKG). The neuronal marker ⁇ - ⁇ tubulin is expressed. The biosynthetic enzyme, tyrosine hydroxylase, which converts tyrosine to DOPA is expressed, as is the biosynthetic enzyme, DOPA decarboxylase, which converts DOPA into dopamine. The third biosynthetic enzyme in the synthesis of catecholamines, dopamine-beta hydroxylase, is not detectable.
  • Midbrain dopaminergic neuron markers are expressed at the protein level as determined by indirect immunocytochemistry.
  • the dopamine transporter (DAT) is expressed in 79.2 ⁇ 4.6% of cells at 2 weeks in PF medium.
  • the function of DAT is to re-uptake dopamine after release in order to avoid over-stimulation of the post-synaptic neuron.
  • DAT red fluorescence
  • DOPA decarboxylase green fluorescence
  • blue DAPI nuclear stain Cells that express both proteins are yellow fluorescent.
  • Figure 13 Neuronal morphology. At low cell density the neuronal morphology of the in vitro differentiated dopaminergic neurons becomes apparent. A neuron triple stained for DOPA decarboxylase (green fluorescence) and the dopamine transporter (red fluorescence) and DAPI nuclear stain (blue fluorescence) is shown. The cell body (arrow) has elaborated a long process (marked by a parenthesis). Yellow fluorescence is indicative of co-localization of DDC and DAT, and is mostly concentrated in the nerve ending (top).
  • Figure 14 Calcium imaging. Calcium imaging showed that in vitro differentiated dopaminergic neurons are functional neurons in regard to neurotransmitter receptor expression and function. The inventors show that the dopaminergic neurons respond to various agonists, thus indicating that the in vitro differentiated dopaminergic neurons express pertinent neurotransmitter receptors.
  • Fig 14 A Time course. This sequence of images shows the time course of response to ATP with intracellular calcium flux (yellow).
  • Fig. 14 B List of agonists used and their cognate receptors.
  • Fig. 14 C Summary of wave forms in response to agonists used.
  • Figure 15 Expression of GDNF and BDNF by hEPI-NCSC, GDNF (glial derived neurotrophic factor) and BDNF (brain derived neurotrophic factor) are two neurotrophins that are essential for the survival of midbrain dopaminergic neurons. All (100%) ex vivo expanded, undifferentiated, hEPI-NCSC express both neurotrophins at the RNA level and at the protein level, as determined by real-time PCR and indirect immunocytochemistry, respectively. This result indicates that hEPI-NCSC could be useful in delaying the death of midbrain dopaminergic neurons in Parkinson's disease when grafted into the substantia nigra pars compacta of the midbrain.
  • Osteogenic differentiation of hEPI-NCSC shows expression of key markers.
  • hEPI-NCSC were cultured in Ad ance ST EM Osteogenic differentiation medium at 37°C, 5% CO 2 and either 5% or ambient 0 2 for up to 35 days. Cultures were analysed by qPCR or indirect immunocytochemistry for expression of osteogenic markers.
  • A, B, C, D, E, F and G show qPCR data for relative expression levels compared to housekeeping genes (HKG) of the appropriate marker as labelled.
  • Primary culture cells (1° culture) cultured at 5% O 2 as described serve as untreated cells, with experimental cultures treated for up to 35 days.
  • RUNX2 shows an early increase in expression after seven days in both 0 2 conditions to 85.5 ⁇ 0.7% and 83.4 ⁇ 1.6% respectively, which then reduces over the remaining culture period.
  • COL2A1 B
  • Osteopontin (SPP1 ) (C) was seen to show an initial reduction in expression after seven days followed by an increase back to a level similar to untreated cells of 76.2 ⁇ 2.6% in 5% O 2 cultures at day 35. Culturing at ambient O 2 resulted in increasing SPP1 expression over the culture period with 85.9 ⁇ 2.1 % expression at day 35.
  • COL1A1 expression was seen to dramatically increase by day seven from untreated levels of 104.9 ⁇ 0.5% to 125.9 ⁇ 0.2 and 122.8 ⁇ 0.5% for 5% and ambient oxygen tensions respectively. Levels then declined and at day 35 were 1 16.3 ⁇ 0.8 and 108.6 ⁇ 0.4 respectively. A significant effect of oxygen tension was observed on expression levels at each time point. Expression of VDR (G) was seen to remain virtually unchanged over the 35 day culturing period. Untreated expression levels were 94 ⁇ 0.6%, with expression level at day 35 being 96.7 ⁇ 0.4 and 95.7 ⁇ 0.9 respectively. Oxygen tension displayed a significant effect in cultures at days seven and 28. A' shows RUNX2 immunoreactivity in day 14 cultures at ambient O 2 .
  • B', C and D' show COL2A1 , SPP1 and BGLAP immunoreactivity respectively, from day 35 cultures at ambient O 2 .
  • A", B", C” and D" are quantification of respective antibody staining for each marker over the 35 day culture period. Positive expression is expressed as percentage of total cells.
  • RUNX2 expression in both 5% and ambient O 2 cultures was seen to increase by day 14 to 49.0 ⁇ 2.9% and 58.4 ⁇ 5.0% respectively (A"), followed by a decline and then increase again towards the end of the culture.
  • COL2A1 was not expressed at early time points but increased by day 35 in both O 2 conditions with 68.4 ⁇ 4.3% cells positive from ambient 0 2 cultures (B").
  • hEPI-NCSC Directed differentiation of hEPI-NCSC into melanocytes.
  • hEPI-NCSC were differentiated into pigmented cells in vitro. DOPA reaction analysis of
  • FIG. 1 Cryopreservation of hEPI-NCSC.
  • hEPI-NCSC cultures were frozen in 90% FBS, 10% DMSO using a Nalgene freezing container followed by transfer to liquid nitrogen. Cells were subsequently thawed and trypan blue analysis of cell viability showed 87% cell viability.
  • (A) shows cells before freezing at 24,000 cells/cm 2
  • (B and C) cells in culture on CellStart treated 35mm dishes, one day (5,400 cells/cm 2 ) and three days (14,000 cells/cm 2 ) post thawing, respectively. Scale bars 100 pm.
  • Figure 19 Directed differentiation of hEPI-NCSC into sensory neurons. Expression of markers for sensory neurons at the protein level was determined by indirect immunocytochemsitry.
  • A Beta-Ill tubulin neuronal maker is co- expressed with Bm3a (nuclear localization).
  • B Neurofilament heavy chain (NF200) is co-expressed with Bm3a.
  • C CGRP is co-expressed with Brn3a.
  • IB4 is co-expressed with Brn3a.
  • Isletl is co-expressed with Brn3a.
  • DAPI blue fluorescence
  • Figure 20 Immunocytochemistry showing nuclear localization of osteocalcin expression and cytoplasmic localization of osteopontin expression in cells having primary culture and first subculture in StemPRO® MSC SFM.
  • Figure 21 Time course of Alizarin Red Stain (culture days 0, 7, 14, 28 and 35) from cells having primary culture and first subculture in StemPRO® MSC SFM.
  • One aspect of the current invention is an in vitro methodology to promote the directed differentiation of human epidermal derived-NCSC's (hEPI-NCSC's) into dopaminergic neuronal progenitors, with specific potential significance for the treatment of Parkinson's disease (PD), a neurodegenerative condition caused by the loss of dopaminergic activity in the substantia nigra pars compacta of the human brain.
  • PD Parkinson's disease
  • PD is the second most common neurological disorder globally, affecting approximately 4.1 m people.
  • prevalence rate of PD is set to rise significantly in the coming years, clinical interventions are expected to play a major role in combating the wide spectrum of needs associated with the disease.
  • the methods described herein relate to the directed differentiation of hEPI- NCSC's into dopamine producing neuronal cell lines (dopaminergic neurons or dopaminergic neuronal progenitors). Such cell lines could be used to restore somatic neural function in PD patients when used as part of a cell-based therapeutic approach. It is known in the art to identify the relevant dopamine producing neuronal cell lines by identifying known markers.
  • hEPI-NCSC's migrate onto the collagen or CellStart substratum. Isolated hEPI-NCSC's were then sub-cultured in the same culture medium.
  • TH is considered an early indication of catecholaminergic neurons and dopaminergic activity and identifies the presence of dopaminergic neurons or dopaminergic neural progenitors.
  • hEPI- NCSC's can be manipulated by this method to differentiate into dopamine producing neurons and therefore hold significant potential for the treatment of PD by autologous therapeutic allograft.
  • Hair follicles in anagen phase are isolated from human pubic skin (from elective Caesarean sections with informed consent) or from scalp skin, athough it could be envisaged that hair folilces could be obtained from alternative sources, such as for instance from skin or the arm or axillary skin, or skin from eye lids.
  • the bulge region of hair follicles is micro dissected from epidermis and subdermis. The micro dissected hair follicles are then cleaned mechanically from surrounding tissue and dermal papilla is cut off and discarded collected in Neurocult XF culture medium containing 5nM HEPES at pH7.2.
  • the area of the hair follicle above the bulge is cut off and discarded and the area containing the bulge is cut into two to three pieces and placed into/on coated culture plates such as a collagen or CellStartTM covered culture plate (Fig. 1 ).
  • the bulge explants were incubated at 37°C, 5% CO ⁇ and 5% O2 for 1 hour such that they would attach to the collagen or CellStart substratum.
  • hEPI-NCSC start to emigrate onto the collagen substratum (see Fig. 2i). These cells have the typical stellate morphology of neural crest cells.
  • non-migratory putative keratinocyte precursor cells form a halo around the explant (Fig. 3) and these cultures are discarded.
  • hEPI-NCSC express the neural crest stem cell molecular signature, which has already been defined for mouse EPI-NCSC and mouse embryonic neural crest stem cells (Hu et al, 2006; Fig 4).
  • hEPI-NCSC also express other neural crest stem cell genes, general stem cell genes and, like mouse EPI-NCSC, genes that were used initially used to generate induced pluripotent stem (iPS) cells, as well as some early lineage markers (see Fig. 4 and associated figure legend for details). Some of these are expressed at the RNA, but not the protein level. Signature genes and the six essential pluripotency genes are expressed at the protein level (Fig. 5), as determined by immunocytochemistry. Conclusion: hEPI- NCSC are neural crest derivatives that show sternness.
  • hEPI-NCSC are multipotent, i.e. that one clone-forming cell can give rise to multiple differentiated cell types (Fig 6A and B). All possible permutations of antibody stains have been performed to show that hEPI-NCSC in clonal culture can give rise to multiple different neural crest derivatives and clone forming cells are thus multipotent (Fig 6B).
  • hEPI-NCSC express Nanog both at the RNA and protein levels (Fig 8). Due to te unexpected nature of these results, and in order to calibrate gene expression levels, the inventors compared pluripotentency gene expression levels to those in human embryonic stem cells (Fig 8) (further details provided in Fig 8 figure legend).
  • Ex-vivo expansion of hEPI-NCSC is needed in order to obtain sufficiently large numbers of cells. Larger numbers of cells are typically required for therapeutic use or if required for use in high-throughput screening.
  • the inventors have developed a culture medium that is conducive to the culture of primary explants of bulges and to ex vivo expansion of hEPI-NCSC. The same medium can be used for the initial culturing of the primary explant. There are few significant changes in pertinent sternness gene expression.
  • Fig 9A shows a comparison of gene expression of hEPI-NCSC in primary explants (left; black) versus hEPI-NCSC that have been expanded (right;grey). There are few differences in gene expression.
  • Ex vivo expanded hEPI-NCSC continue to express neural crest stem cell signature genes at the protein level (Fig 9C). Notably greater than 95% of ex vivo expanded cells express the signature genes. On average 1.5 x 10 6 cells can be obtained from one primary explant in 28 days (Fig 9B). It is also notable that at 28 days cell growth is still in the log phase and further expansion is thus still possible.
  • Expansion is preferably carried out at 5% Oxygen (O 2 ).
  • this basal expansion medium is surprisingly effective.
  • the medium differs from that which would be used for other stem cell types, for example Stem Cell Technologies would not suggest the inclusion of;
  • ITS+3 which contains insulin, transferrin and selenium as insulin in particular would usually be expected to cause differentiation which is not required at this stage. Surprisingly the inventors have found that use of this expansion medium does not result in unwanted differentiation.
  • hEPI-NCSCs Directed differentiation of hEPI-NCSCs into dopaminergic neurons or precursors thereof
  • the inventors have now developed culture methods/conditions for the directed differentiation of hEPI-NCSC into dopaminergic neurons. Gene expression at the RNA level is determined by real-time PCR, and confirmation of gene expression at the protein level. Surprisingly the inventors have found that their method results in extremely effective differentiation when compared to other methods of differentiation using different cell types that are described in the prior art.
  • the expanded population of cells is sub-cultured in specific media to direct differentiation as required.
  • the base medium for differentiation is shown in the following table.
  • This medium can be obtained as GMP-compliant culture medium when needed.
  • a preferred method for differentiation into dopaminergic neurons is a two-step sub-culturing method. This method may be useful for directing differentiation into other types of neurons as well.
  • the expanded population is first sub-cultured in a Neural Progenitor (NP) medium for 7 days to differentiate the cells to a neural stem cell like state and then sub-cultured in a Patterning Factor (PF) medium to produce fully differentiated cells.
  • Figure 10 also shows the cell morphology at different stages associated with the directed differentiation of hEPI-NCSCs using the methods secribed below..
  • NP Neural Progenitor
  • Patterning Factors (PF) Medium (starting day 7)
  • GDNF (5 ng/ml) [glial derived neurotrophic factor]
  • NGF neurotrophic factor
  • Results showed that culturing using all three neurotrophins together was best for gene expression at RNA and protein levels and for cell morphology.
  • NT-3 (10 ng/ml) Neural Progenitor (NP) Mix2
  • SCF stem cell factor
  • EGF plus FGF-2 is used to differentiate embryonic stem cells neurospheres into neural progenitors.
  • SCF based on our earlier work on neural crest cells.
  • NT-3 was added based on the inventors understanding that this is important for survival of neural crest cells (Zhang JM et al, 1997). Further experimental work led to preferred media for expansion and directed differentiation of hEPI-NCSCs into DA neurons being as follows;
  • qPCR Real time PCR
  • indirect immuncytochemistry results not shown
  • FIG 13 A neuron triple stained for DOPA decarboxylase (DDC; green fluorescence) and the dopamine transporter (DAT; red fluorescence) and DAPI nuclear stain (blue fluorescence) is shown.
  • DDC DOPA decarboxylase
  • DAT dopamine transporter
  • DAPI nuclear stain blue fluorescence
  • the cell body arrow
  • Yellow fluorescence is indicative of co-localization of DDC and DAT, and is mostly concentrated in the nerve ending (top).
  • Calcium imaging (Fig 14) also showed that cells respond to various agonists (a summary of which is shown in Fig 14B), thus indicating that the in vitro differentiated dopaminergic neurons express pertinent receptors.
  • Fig 14 A shows the time course of response to ATP with intracellular calcium flux. The results show that in vitro differentiated dopaminergic neurons express acetylcholine receptors, purinergic receptors, glutamate receptors and alpha-1 adrenergic receptors. Spontaneous activity was observed as well. Unspecific stimulation resulted in calcium flux also. And CPA emptied all intracellular stores. A summary of wave forms in response to agonists used is shown in Fig 14 C.
  • hEPI-NCSC express both neurotrophins at the RNA level and at the protein level, as determined by real-time PCR and indirect immunocytochemistry, respectively (Fig 15).
  • GDNF glial derived neurotrophic factor
  • BDNF brain derived neurotrophic factor
  • hEPI-NCSC could be useful in delaying the death of midbrain dopaminergic neurons in Parkinson's disease when grafted into the substantia nigra pars compacta of the midbrain.
  • dopaminergic neurons the inventors have also looked to direct differentiation of hEPI-NCSC into sensory neurons using the following methods;
  • NP medium phase 7 days , 5% 0 2 .
  • Brn3a positive cells (%) ⁇ tub positive cells (%) Brn3a/ plll tub positive cells (%)
  • Bm3a is a transcription factor that is specific for sensory neurons. In vivo in adulthood, approximately one third of sensory neurons are large diameter neurons and express neurofilament heavy chain (NF200). They are CGRP negative and IB4 negative. About one third are small diameter nociceptive sensory neurons. They are IB4 positive and CGRP negative. Isletl plays a central role in the transition from sensory neurogenesis to subtype specification (Sun Y, Nature Neurosci, 1 1 : 1283 - 1293 (2008). The data indicate that hEPI- NCSC can be differentiated efficiently into cells that express sensory neuron markers, both for large diameter sensory neurons and small diameter sensory neurons.
  • the inventors are also investigating similar methods to direct differentiation of hEPI-NCSC into sympathetic neurons (useful in the treatment of autism and for modeling neuroblastoma) and cholinergic neurons (useful in the treatment of Alzheimer's disease (AD) as the nucleus basalis of Meinert, which contains cholinergic neurons, degenerates in AD).
  • AD Alzheimer's disease
  • Factors and other additives that can be added to differentiate cholinergic neurons include the following.
  • hEPI-NCSC's human epidermal derived-NCSC's
  • melanocytes pigment cells
  • Melanocytes are cells located in the bottom layer of the epidermis (the stratum basale), the inner ear, the meninges, bone, the heart, and the hair, where they produce the pigment melanin that is responsible for the individual coloration of hair and inner organs and for hearing.
  • One aspect of the present invention is a method for the selective differentiation of human epidermal neural crest stem cells (hEPI-NCSCs) into melanocytes with suggested potential for application in the fields of bio-engineering, and particularly tissue repair, skin replacement, skin rejuvenation, and/or feature augmentation. It is envisaged that melanocytwes obtained using the described methods can be used in artificial skin for the treatment of burn wounds. Melanocytes protect from UV damage from sun light. The presence of mealnocytes in artificial skin is desirable especially for non-white individuals. Isolation and expansion of hEPI-NCSCs
  • bulge explants devoid of the sheath yielded significantly fewer emigrating cells, whereas no cells emigrated from dermal sheath explants (Fig 2).
  • the inventors have therefore developed a reliable and highly reproducible method for the culturing of adherent hEPI-NCSC from dissected adult hair follicle bulge explants. It uses a minimally invasive procedure and represents an abundant source of neural crest-derived stem cells.
  • the bulge of adult human hair follicles were micro-dissected as described previously from pubic hairy skin. De-identified biopsies were obtained with ethical approval from consenting individuals undergoing repeat elective Caesarean sections. The Donor age bracket was 28 - 41 years. Briefly, hair follicles were dissected and mechanically cleaned of dermal and adipose tissues. The dermal papilla and matrix were removed and discarded, the bulge region excised, cut into 2-3 pieces and placed onto CellStart (Invitrogen, Paisley, UK Cat# A10142-01 ) coated 24-well or 35mm plates where they adhered to the substratum within one hour.
  • CellStart Invitrogen, Paisley, UK Cat# A10142-01
  • the explants were incubated in a humidified atmosphere at 37°C, 5% C0 2 and 5% 0 2 .
  • the inventors routinely culture neural crest stem cells at low oxygen tension. It should be noted that 5% oxygen does not constitute hypoxia for hEPI- NCSC, as hypoxia is defined as O2 tension below the normoxic value in a given tissue and oxygen tension in hair follicles ranges between 2.5% and 0.1 % O2.
  • the Culture medium was NeuroCult XF (Stem Cell Technologies, Grenoble, France Cat# 05761) supplemented with 10ng/ml rhFGF2 (R&D Systems, Abingdon, UK Cat# 233-FB), 20ng/ml rhEGF (R&D Systems Cat# 236-EG), 1X ITS+3 (Sigma, Poole, UK Cat# 1-2771 ), 1 % (v/v) FBS (HyClone, Thermo Fisher, Cramlington, UK Cat# SH30070.02), 1X GlutaMAX (Invitrogen, Cat# 35050-038), 1X Penicillin/Streptomycin (Sigma Cat# P0781) and 2.5Mg/ml Amphotericin B (Sigma Cat# A2942).
  • hEPI-NCSC can be expanded into millions of stem cells without an overall significant loss of stem cell markers (Fig 9 A, B).
  • Ex vivo expanded cells did not differentiate spontaneously but continued to express the neural crest stem cell molecular signature as well as SOX10 and NESTIN (Fig 9C).
  • in vitro clonal analysis showed that the majority of in vitro expanded cells remain multipotent and thrive in clonal culture; 53.2 ⁇ 3.6% of clone-forming expanded cells generated clones that contained multiple cells types; 12.3 ⁇ 2.6% died an 34.5 ⁇ 3.0 stopped dividing.
  • Changes in the karyotype are thus of less of a concern in hEPI-NCSC than in cell lines that are passaged multiple times.
  • the high numbers of stem cells that can be obtained through ex vivo expansion make testing in animal models of human disease and future applications feasible.
  • the cells were either fixed with 4% paraformaldehyde (PFA) for indirect immunocytochemistry to allow for identification of, for example, pluripotency genes, or dissolved in TRIzol® (Invitrogen, Cat# 15596-018) for RNA isolation.
  • PFA paraformaldehyde
  • TRIzol® Invitrogen, Cat# 15596-018
  • the inventors had already defined a neural crest stem cell signature that is common and specific to mouse embryonic neural crest stem cells and mouse EPI-NCSC.
  • One rationale for defining the molecular signature was to use it for the characterisation of human equivalent cells.
  • the neural crest stem cell signature is expressed in hEPI-NCSC (Fig. 4).
  • NCSC genes tested and detected include SOX10, SNAI2, TWIST1 , MS1 (Musashi) and p75NTR, thus corroborating the neural crest origin of hEPI-NCSC.
  • Additional stem cell and the pluripotency genes SOX2, C-MYC, KLF4, LIN28, POU5F1/OCT4 and NANOG were expressed as well.
  • the majority of hEPI-NCSC express the molecular signature genes at the protein level as determined by indirect immunocytochemistry (Fig 5).
  • the neural crest stem cell marker SOX10 and the progenitor cell marker NESTIN were both expressed in all cells. Together, these observations confirm expression of pertinent markers and characterises the bulge-derived cells as neural crest-derived cells.
  • Clones were then cultured in NeuroCult NSA (Stem Cell Technologies, Cat# 05752), 1X ITS+3, 1 % (v/v) FBS, 1X Gluta AX, 1X Penicillin/Streptomycin and 2.5Mg/ml Amphotericin B plus addition of specific growth factors for differentiation of multiple cell types for up to 42 days at 37°C, 5% C0 2 , 5% 0 2 .
  • the cell types were typical neural crest derivatives and included cells with immunoreactivity characteristic of myofibroblasts (smooth muscle actin), bone/cartilage cells (collagen type II; COL2A1 ), neurons (neuron-specific ⁇ -tubulin [TUBB3], tyrosine hydroxylase [TH]), and Schwann cells (glial fibrillary acidic protein; GFAP).
  • myofibroblasts smooth muscle actin
  • bone/cartilage cells collagen type II; COL2A1
  • neurons neuroon-specific ⁇ -tubulin [TUBB3], tyrosine hydroxylase [TH]
  • Schwann cells glial fibrillary acidic protein
  • hEPI- NCSC Self-renewal capability of hEPI- NCSC was determined by serial cloning. Primary clones were detached with trypsin and re-seeded at clonal density, which lead to secondary clones. The procedure was repeated to establish tertiary clones. Clone-forming ability was maintained at high levels, as 70.7 ⁇ 7.9% of secondary clones and 54.0 ⁇ 1 1.7% of tertiary clones consisted of fast-growing and motile cells. Double stains with cell- type specific antibodies showed that secondary clones contained multiple cell types as well. The presence of multiple cell types in secondary clones shows that hEPI-NCSC can undergo self-renewal.
  • hEPI-NCSC are multipotent stem cells. Differentiation into melanocytes hEPI-NCSC were differentiated in vitro into melanocytes by adding pertinent growth factors and reagents as described below. Before and after DOPA reaction, pigmented cells were clearly visible, with 65.7 ⁇ 6% of cells positive for melanin (Fig. 17).
  • Real time PCR data (Fig 4) showed that prior to differentiation hEPI-NCSC already express already early lineage markers for melanocytes, including microphthalmia-associated transcription factor (MITF) and dopachrome tautomerase (DCT), which are important for development and function of melanocytes.
  • MITF microphthalmia-associated transcription factor
  • DCT dopachrome tautomerase
  • hEPI-NCSC For in vitro differentiation of hEPI-NCSC into melanocytes, cells were treated with 100nM Endothelin-3 (Sigma Cat# E9137), 20nM Cholera Toxin (Sigma Cat# C8052), 16.2mM 12-0-tetra-decanoylphorbol-13-acetate (TPA) (Sigma Cat# 79346), for up to 17 days at 37°C, 5% CO 2 ,10% 0 2 .
  • the DOPA reaction was performed to enhance the dark melanin hue. Briefly, cultures were fixed with 4% PFA for 20 min at RT, followed by three rinses with PBS and incubated at 37°C with 5mM DOPA (Sigma Cat# D9628) for three hours.
  • hEPI-NCSCs are readily accessible in postnatal hairy skin and can be isolated by a minimally invasive procedure as described above.
  • the cells are then expanded in culture to produce a relatively homogenous population of precursor cells that can then be directed to differentiate into melanocytes via culture in media which includes 16 nM of TPA/PMA (phorbol 12-myristate 13-acetate), 20 nM of cholera toxin, and 100 nM of endothelin-3. Phenotype is subsequently confirmed by melanin granule visualization after DOPA reaction under the microscope following 4 day incubation in vitro.
  • a preferred culture medium for differentiating of human epidermal neural crest stem cells (hEPI-NCSCs) into melanocytes is;
  • Pigment Cell Medium - 85% a-MEM, 10% FBS, 5% Chick Embryo Extract, 100nM Endothelin-3, 20nM Cholera Toxin, 16.2mM PMA/TPA, GlutaMAX, Amphotericin, Gentamicin
  • hEPI-NCSC's human epidermal derived-NCSC's
  • the inventors have also directed differentiation of the isolated and expanded hEPI-NCSCs into sialoprotein and osteocalcin producing osteoblasts using the following culture medium. Again, a two-step sub culturing was not required, however 3D scaffolding was used to provide a support structure for the differentiated cells to grow. Neural crest cells give rise to various craniofacial bones. Developing protocols for the directed differentiation of hEPI-NCSC into bone cells is thus a step towards future applications of hEPI-NCSC in cell-based therapies.
  • Cells were isolated by trypsinisation and seeded onto CellStart-treated 35mm plates at 2.5X10 3 cells per plate. Cultures were grown in AdvanceSTE osteogenic differentiation medium (HyClone; Thermo Fisher Cat# SH30877.KT), 1X GlutaMAX, 1X Penicillin/Streptomycin and 2.5pg/ml Amphotericin B, with 50/50 medium exchanges on alternate days. Cultures were incubated in a humidified atmosphere at 37°C, 5% CO 2 and either 5% O 2 or ambient air for up to 35 days. Alizarin Red S staining of fixed cultures was performed to detect deposition of calcium.
  • AdvanceSTE osteogenic differentiation medium HyClone; Thermo Fisher Cat# SH30877.KT
  • 1X GlutaMAX 1X Penicillin/Streptomycin
  • Amphotericin B 2.5pg/ml Amphotericin B
  • Figure 16 shows that bone cells are present after the culturing step by using 'Alizarin Red S' stain (marker for calcifying bone cells) to confirm the phenotype.
  • RNA level was determined by qPCR and at the protein level by indirect immunocytochemistry (Fig 16). Calcification was shown by histological stain with Alizarin Red S (Fig 16). Interestingly, transcripts of many osteogenic early lineage markers were already expressed by stem cells in primary explants.
  • COL2A1 immunoreactivity was not detected until day 14 with the expected punctuate staining pattern within the cytoplasm (Fig 16 B').
  • COL2A1 expression levels were similar in both 5% and ambient O2 concentrations with 66.7 ⁇ 8.7% and 68.4 ⁇ 4.3% positive cells respectively (Fig 16 B").
  • Transcripts for osteopontin (SPP1), the main phosphorylated glycoprotein of bone were already present in primary explants and increased overtime in differentiation medium (Fig 16 C). At the protein level, osteopontin was significantly higher expressed in cells that were cultured at ambient oxygen compared to cells grown at 5% oxygen (Fig 16 C").
  • Osteocalcin is a member of the Gla protein family and contributes to the non-collagenous matrix in bone. Like other bone-characteristic genes, osteocalcin transcripts were detected already in primary explants and expression levels increased with progressing time in differentiation culture (Fig 16 D). There was no significant difference in the percentage of cells with osteocalcin immunoreactivity between the 5% oxygen and ambient air culture conditions (Fig 16 D"). Core-binding factor subunit beta (CBFB) transcripts were already present at high levels in primary explants and remained relatively unchanged throughout the culture period at both oxygen tensions (Fig 16 E).
  • CBFB Core-binding factor subunit beta
  • qPCR for collagen type 1 alpha 1 (COL1A1) (Fig 16 F) showed expression at low levels in primary explants, but increased significantly to high expression levels by day 7 in both oxygen tensions. A significant difference in expression due to oxygen tension was seen at each time point. At the end point, expression levels were significantly higher in cells that were cultured at 5% oxygen. Unexpectedly, vitamin D receptor (VDR) transcripts were already present at detectable levels in primary explants (Fig 16 G) and remained relatively unchanged throughout the culture period with no significant difference in expression levels at 5% and ambient oxygen at the end of the culture period. Alizarin Red S staining, which is an indicator of calcium deposits, was intense in cultures at both 0 2 tensions (Fig 16 H' and H"). Overall, we show that hEPI-NCSC can be differentiated efficiently into bone cells in adherent culture. Whereas individual gene expression varied between 5% and ambient oxygen levels and often differed significantly, the overall trend favours culturing at ambient oxygen tension.
  • the bar charts below illustrate a comparison of RUNX2 and CBFB expression observed in experiments using primary culture and first subculture in NeuroCult XF® (EXP 1 ) or StemPRO® MSC SFM (EXP 2).
  • the bar charts show Real-time PCR (RUNX2 relative expression, CBFB relative expression, VDR relative expression) and Immunocytochemistry (RUNX2 ICC, Osteopontin ICC and Osteocalcin ICC)
  • Vitamin D receptor is also more abundant at the RNA level In this medium.
  • More cells express RUNX2, osteopontin and osteocalcin as determined by indirect immunocytochemsitry.
  • osteopontin As collagen type II is also regarded as a marker for chondrocytes, this reduced expression at the protein level is an important observation.
  • osteopontin was expressed at high levels from early in culture. A significantly higher level of expression was seen at all time points of the culture. Osteocalcin expression in experiment 2 was also seen earlier and to a higher level in experiment 2. Importantly, osteocalcin expression was also seen in the nucleus in experiment 2 whereas it had only been cytoplasmic in experiment 1 ( Figure 20 shows culture day 7 stain).
  • Alizarin Red S stained cells were dissolved overnight in 0.01 % Triton X-100; absorption measured at 492 nm.
  • Figure 21 shows the time course of Alizarin Red stain (Culture days 0, 7, 1 , 28, 35).
  • the rationale for using the different medium was to see whether using a mesenchymal stem cell medium would improve resultant differentiation into osteocytes compared to NeuroCult® XF medium. It seems that it may even be the case that the MSC medium primes the cells to a more progenitor-like state before differentiation.
  • hEPI-NCSC are plated at clonal density into 350 mm dishes coated with or without fibronectin. Cells are maintained in a humidified incubator at 37°C with 5% C0 2 and 5% 0 2
  • culture medium will be change to standard culture medium supplemented with:
  • ex vivo expanded hEPI-NCSC can be frozen, stored frozen, thawed and subsequently grown in culture again.
  • Cells were frozen in 90% FBS, 10% DMSO in a Nalgene freezing container (Sigma Cat# C1562) to -80°C overnight and then transferred to liquid Nitrogen. Subsequently, cells were thawed and re- cultured. Trypan blue stain showed 87% cell viability after thawing and the cells continued to proliferate (Figure 18).
  • hEPI-NCSC can be frozen by inexpensive means and thawed again with high yield.
  • hEPI-NCSC have been shown by the inventors to have many desirable features, the sum of which makes them a highly attractive type of somatic stem cell.
  • hEPI-NCSC As remnants of an embryonic tissue, the neural crest, hEPI-NCSC have the well- recognized physiological ability to generate a wide array of cell types and tissues.
  • This innate high level of multipotency, combined with the expression of pluripotency genes and efficient ex vivo expansion render this stem cell type conducive not only to autologous transplantation but potentially also to efficient reprogramming into iPS cells.
  • hEPI-NCSC are readily accessible in the hairy skin by minimal invasive procedure. The patient's own hEPI-NCSC could therefore be harvested, expanded ex vivo and then used for autologous transplantation.
  • the present invention provides benefits over the prior art by providing a range of effective and high yield culture methods for the differentiation of adult hEPI- NCSCs in vitro.
  • Such differentiated hEPI-NCSCs hold significant therapeutic potential for the treatment of various neoplastic and non-neoplastic diseases including Parkinson's disease, multiple sclerosis, amyotropic lateral sclerosis (ALS), osteoarthritis, osteoporosis, stroke, and cerebellar degeneration, and various acute and severely debilitating trauma episodes such as spinal cord injury, head and neck injury, and severe burn and/or wound repair.
  • hEPI-NCSCs are also very useful candidates for the study of and intervention in the ageing process, and hold significant potential for use as both genetic delivery vehicles and high throughput drug screening and/or drug discovery techniques, with the former being of specific relevance to the treatment of neurogenetic diseases.
  • Rat mesenchymal stem cells increase tyrosine hydroxylase expression and dopamine content in ventral mesencephalic cells in vitro.
  • Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Wernig M, Zhao J, Pruszak J, Hedlund E, Fu D, Soldner F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch R.

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Abstract

The invention relates to the directed differentiation of epidermal neural crest stem cells, and more specifically human epidermal neural crest stem cells (hEPI-NCSC), for example to their differentiation into dopaminergic neurons, dopaminergic neuronal progenitors, sensory neurons, sympathetic neurons, cholinergic neurons, melanocytes, Schwann cells, smooth muscle, osteogenic differentiation into osteocytes, bone and cartilage and adrenergic differentiation.

Description

Expansion and Directed differentiation of epidermal neural crest stem cells.
The present invention relates to directed differentiation of epidermal neural crest stem cells, and more specifically human epidermal neural crest stem cells (hEPI- NCSC), for example to their differentiation into dopaminergic neurons, dopaminergic neuronal progenitors, sensory neurons, sympathetic neurons, cholinergic neurons, melanocytes, Schwann cells, smooth muscle, osteogenic differentiation into osteocytes, bone and cartilage and adrenergic differentiation. Embryonic neural crest stem cells (NCSC's) physiologically give rise to a wide array of different cell types and tissues. Neural crest cells give rise to the autonomic and enteric nervous systems, most primary sensory neurons and their glia, craniofacial bone/cartilage, smooth musculature of the cardiac outflow tract and great vessels, endocrine cells such as the adrenal medulla and C-cells of the thyroid , and melanocytes if the skin and internal organs. This is especially significant for the purposes of cell-based therapies and biomedical engineering of tissues. Neural crest-derived multipotent stem cells persist in the periphery during embryogenesis and in the adult organism. One such location is the bulge of whisker follicles, as has been shown by the inventors in the Wnt1-cre::R26R mouse model in which all neural crest cells and dorsal neural tube cells specifically and indelibly express β-galactosidase. According to their location in the bulge by the epidermal outer root sheath, the stem cells were thus termed epidermal neural crest stem cells (EPI-NCSC). A neural crest stem cell molecular signature comprising 19 genes that are abundantly and uniquely expressed in mouse embryonic neural crest stem cells and EPI-NCSC, but not in epidermal stem cells or other skin-resident stem cells/progenitors was subsequently defined. The potential of EPI-NCSC has begun to be realized in mouse models of spinal cord injury, where mouse EPI-NCSC grafts led to an improvement in sensory connectivity and touch perception. Others have also reported hair follicle derived progenitor cells, both from within and outside the bulge region, the latter having shown promise in a model of sciatic nerve injury. Furthermore, multipotent progenitor cells of various ontological origins within skin and hair follicles have been identified. Cells from the dermal papilla and other locations in the skin, such as foreskin, have been termed skin-derived precursors (SKPs). Similarly, Yu et a/ (2010) reported that other human hair follicle-derived neural crest-like cells formed spheres in culture following enzymatic digestion of intact hair follicles, and they expressed neuronal markers when injected into the mouse brain. However, significantly, these cells differ from hEPI-NCSC, as they are isolated from entire hair follicles, do not show any migratory behaviour, and express non-crest markers. It is therefore questionable as to whether Yu et al describe a substantially pure population of neural crest derived stem cells. hEPI- NCSC have the distinct advantage that due to their migratory ability they can be isolated with ease as a highly pure population of multipotent stem cells of neural crest origin. The neural crest is a transient embryonic structure that arises during neurulation at the boundary of the neural plate and the somatic ectoderm. Neural crest cells subsequently delaminate from the forming neural tube and migrate to various locations in the embryo to generate a wide array of progeny. Neural crest derivatives include craniofacial bone/cartilage, meninges, tooth papillae, the autonomic and enteric nervous systems, most primary sensory ganglia, endocrine cells such as the adrenal medulla, smooth musculature of the cardiac outflow tract and great vessels and pigment cells (melanoyctes) of the skin and internal organs. Human epidermal neural crest stem cells (hEPI-NCSC) are particularly attractive candidates for cell-based therapy and regenerative medicine as they combine several desirable features. hEPI-NCSCs are adult stem cells but are of embryonic origin, the neural crest. The cells reside in a postnatal location, the bulge of the hair follicle. They are multipotent and able to generate all major neural crest derivatives, including neurons, nerve supporting cells, bone/cartilage cells, smooth muscle cells, and melanocytes. They are readily accessible in the hairy skin, for example from pubic skin, scalp skin or other suitable areas, by minimally invasive procedures. By virtue of their migratory ability the inventors have already shown that hEPI-NCSCs can be isolated as a highly pure population of stem cells, and they can be expanded ex vivo into millions of cells, without losing stem cell markers. They express the neural crest stem cell molecular signature as well as other neural crest and global stem cell genes. The inventors have previously been involved in providing methods for isolating cultures of hEPI-NCSCs, and these are discussed in detail in US Application Number 1 1/376498.
The inventors have determined in the Wnt1-cre::R26R compound transgenic mouse, in which all neural crest cells and their derivatives are specifically and indelibly labelled by expression of beta-galactosidase, that the bulge of hair follicles contains neural crest stem cells, and they have developed a technique for the explantation and culture of the bulge of human hair.
Wataya et al (2008) discusses the efficacy of cell replacement therapy in Parkinson's disease, concluding that several studies on foetal tissue (mouse and human) have already established the quantity and purity of transplanted cells necessary for meaningful therapeutic application. The same paper also highlights the various groups that have already succeeded in differentiating various types of neurons including telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons, from human embryonic stem cells. However, this paper acknowledges a number of problems inherent within the existing methods for differentiation on top of the known general concerns in relation to the use of embryonic stem cells. Nadri et al (2008) also recently demonstrated the potential of stem cell approaches to PD by taking mesenchymal stem cells ( SCs) from the eye conjunctiva (cjMSCs) and demonstrating differentiation into dopaminergic precursors. Successful differentiation was confirmed by RT-PCR, immunocytochemistry and flow cytometry expression analysis of genes TH, Ptx3, and Nurr1. These results suggest that cjMSCs may also be a suitable and available source of autologous stem cells for the cell based therapy of PD. However, the methods of obtaining this stem cell population (which differs from the population of the present invention) are inherently problematic as it is difficult, and often painful, to extract stem cell biopsies from the conjunctiva of the eye. Jin et al (2008) has similarly shown that brain-derived neurotrophic factor (BDNF) can up-regulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells, and that co-culturing rat ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow up-regulates TH and dopamine (DA) expression. Park et al (2008) have also described the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases. Deierborg et al (2008) provide an excellent review of several exciting regenerative approaches to PD developed over the last 20 years, and Levy et al (2008) specifically assesses the developmental potential of h SC to replace the midbrain dopamine neurons selectively lost in PD. To date, such approaches have typically only involved embryonic and/or foetal derived neural stem cells, thereby severely restricting successful human application.
When considering the general state of the art it is noted that US20070254281 A1 claims the uses of LMX 1 a as an indicator of early dopaminergic neuronal activity, (however it does not specifically refer to hEPI-NCSCs or a method of directed differentiation of the same). Similarly, WO05030240A2 refers to the use of VEGF-C or VEGF-D to stimulate neural stem cell growth and differentiation for use in cellular therapy, but does not describe methods for differentiating hEPI- NCSCs. Neither of these patents are useful when considering the differentiation of hEPI-NCSCs.
Skeletal abnormalities, bone trauma, osteoarthritis and deteriorating joints are examples of conditions, which severely compromise an individual's quality of life and ability to perform daily tasks of living. Derivation of replacement bone and cartilage is thus of particular interest. Osteogenic differentiation by various methods has been reported for mesenchymal stem cells, adipose cells and bone marrow stem cells with others also reporting osteogenic differentiation of NCSC derived from human embryonic stem cells (hESC), SKPs, mouse ESC and cord blood. The advantage of hEPI-NCSC is that the neural crest has the physiological ability to generate bone, as a subset of cranial bones are of neural crest origin. Likewise, melanoyctes are established neural crest derivatives. In hairy skin, they give hair its colour by injecting melanin granules into keratinocytes that form new hair. hEPI-NCSCs are identified as being cells which express, to variable degrees and in a donor dependent manner, the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28 , OCT-4/POU5F1 and NANOG.
According to the present invention there is provided a method for inducing or stimulating differentiation of a population of stem cells, which includes the step of; culturing a population of epidermal neural crest stem cells in the presence of added reagents, said reagents selected to induce or stimulate differentiation of the cells into one or more pre-determined, identifiable cell type or cell types.
Preferably the predetermined, identifiable cell type is a neural crest derivative.
By taking advantage of their migratory ability, hEPI-NCSC can be harvested as a highly pure population of stem cells without an overt need for further purification, as non-stem cells are not proliferative and within a short time are outgrown by the stem cells and left behind upon subculture. Furthermore, optimization of culture conditions provided bulge explants that produce emigrating hEPI-NCSC with high yield. Ease of isolation, high yield, and rapid ex vivo expansion, along with high purity of the cell population are highly desirable features of hEPI-NCSC and important considerations for cost-effective stem cell production. Neural crest derivatives include craniofacial bone/cartilage, meninges, tooth papillae, the autonomic and enteric nervous systems, most primary sensory and their nerve-supporting glia, endocrine cells such as the adrenal medulla, smooth musculature of the cardiac outflow tract and great vessels and pigment cells (melanoyctes) of the skin and internal organs.
There are many reasons why such differentiated populations of cells would be useful. For example, they could be used in various therapies to replace or supplement a patient's own cells (such as the use of replacement supplementary dopaminergic neurons in the treatment of Parkinson's disease). The differentiated cell populations/differentiated hEPI-NCSCs could also be used in high throughput drug screening. Predetermined cell populations, which can be substantially pure, could be produced and used for specific testing or screening purposes/ drug discovery programmes.
Preferably the method is for inducing or stimulating differentiation of epidermal neural crest stem cells into one or more of dopaminergic neurons, dopaminergic neuronal progenitors, melanocytes, Schwann cells, smooth muscle, and osteocytes, bone and/or cartilage, sensory neurons, sympathetic neurons or cholinergic neurons.
Preferably the epidermal neural crest stem cells are a substantially pure population of epidermal neural crest stem cells. Preferably the epidermal neural crest stem cells are a substantially pure, expanded population of epidermal neural crest stem cells.
Preferably the method is for inducing or stimulating differentiation of human epidermal neural crest stem cells.
Preferably the method comprises the steps of; - isolating anagen phase hair follicles from a subject
- isolating the bulge area of hair follicles and placing it into adherent culture
- isolating epidermal neural crest stem cells from the bulge explants
- sub-culturing the isolated cells in the presence of added reagents selected to induce or stimulate differentiation of the cells into one or more predetermined, identifiable cell types.
Preferably anagen phase hair follicles are in early or late anagen phase. Preferably the added reagents are selected from the groups;
(a) dibutyryl cyclic AMP (db c-AMP) whereby they become immune- fluorescent for neuron-specific beta-Ill tubulin and tyrosine hydroxylase (TH); or
(b) TPA (12-0- tetra-decanoylphorbol-133-acteate (PMA), (phorbol 12- myristate 13-acetate), cholera toxin and endothelin-3;
(c) BMP2;
(d) NGF, TGF-P2 and forskolin; or
(e) Neuregulin-1 and CNTF. Where the added reagent is db c-AMP, combinations of neurotrophic factors (NGF, BDNF, NT-3) may also be included.
Notably, the use of different added reagents will result in the EPI-NCSCs differentiating into different cell types, for example, BMP2 (10 ng/ml) for bone differentiation; NGF (20 ng/ml), TGF- 2 (1 ng/ml) and forskolin (10μΜ) for neural differentiation; and Neuregulin-1 (10nM) plus CNTF (10ng/ml) for Schwann cell differentiation.
Preferably, the method includes the step of expanding the population of isolated cells prior to the sub-culturing step. The expansion step is necessary in cases where the resulting cells are to be used for therapeutic applications. It has been acknowledged in the art that there are significant difficulties associated with expanding stem cell populations of any type whilst retaining their essential "sternness" and multipotency.
Expansion is typically obtained by culturing the isolated epidermal neural crest stem cells. The culture media for expansion may comprise proliferation media such as NeuroCult XF ®, StemPRO® SC SFM, FGF2, EGF, FBS (foetal bovine serum), ITS+3 (contains insulin, transferrin, sodium selenite as well as oleic acid, linoleic acid, and BSA), GlutaMAX ®, Amphotericin, Penicillin/Streptomycin.
In certain embodiments the isolated cells undergo a 2-step sub-culturing process, the first sub-culturing step being culturing the cells in a Neural Progenitor (NP) medium and the second sub-culturing step being culturing the cells in a patterning factor (PF) medium.
Preferably, in order to obtain dopaminergic neurons or dopaminergic neuronal progenitors, the isolated cells are sub-cultured in a PF medium comprising SHH, FGF-8, GDNF and TGF-beta2.
Preferably the NP medium comprises;
FGF-2
EGF SCF NT-3
Most preferably the NP medium comprises;
FGF-2 (10 ng/ml)
EGF (20 ng/ml)
SCF (5 ng/ml)
NT-3 (10 ng/ml) Sub-culturing the cells in a neural progenitor medium effectively converts the multipotent stem cells to a neural stem cell like state. Further culturing in a patterning factor medium then directs differentiation of said cells. This is particularly useful when the aim is to produce a population of dopaminergic neurons or dopaminergic neuronal progenitors.
Preferably the patterning factor medium comprises;
SHH ["sonic hedgehog"]
FGF-8
TGF-p2
GDNF [glial derived neurotrophic factor]
db c-A P
combinations of neurotrophic factors (NGF, BDNF, NT-3)
Most preferably the patterning factor medium comprises;
SHH (100 ng/ml) ["sonic hedgehog"]
FGF-8 (50 ng/ml)
TGF-P2 (1 ng/ml)
GDNF (5 ng/ml) [glial derived neurotrophic factor]
db c-AMP(100uM),
combinations of neurotrophic factors (NGF, BDNF, NT-3)
Preferably the epidermal neural crest stem cells are obtained from the bulge of a hair follicle obtained from a subject.
Preferably the subject is human.
Optionally the method further comprises the step of returning the differentiated cells into the subject. Autologous transplantation of this type is advantageous in avoiding graft rejection.
According to a further aspect of the present invention, there is provided a population of differentiated cells obtained by the abovementioned method.
According to a further aspect of the present invention there is provided cells obtained using the method of the first aspect for the treatment of a disease state. Alternatively, the undifferentiated hEPI-NCSCs could be used for the treatment of a disease state.
Optionally, the cells obtained using the method of the first aspect are for the treatment of Parkinson's disease.
Optionally, the cells obtained using the method of the first aspect are for the treatment of Alzheimer's disease.
In particular this will utilise sympathetic neurons obtained using the above methods.
Optionally, the cells obtained using the method of the first aspect are for the treatment of autism. In particular this will utilise cholinergic neurons obtained using the above methods.
Optionally, the cells obtained using the method of the first aspect are for the treatment of peripheral neuropathies. Optionally, the cells obtained using the method of the first aspect are for the treatment of degenerative diseases.
Optionally, the cells obtained using the method of the first aspect are for the treatment of skeletal abnormalities.
Optionally, the cells obtained using the method of the first aspect are for the treatment of bone fractures and fixation of bones. An example of fixation may be during a hip replacement where prosthesis is attached to bone. A graft at this point can help to prevent the prosthetic becoming loose over time.
Optionally, the cells obtained using the method of the first aspect are for the treatment of osteoarthritis or osteoporosis.
Optionally, the cells obtained using the method of the first aspect are for the treatment of stroke. Optionally, the cells obtained using the method of the first aspect are for the treatment of acute and severely debilitating trauma episodes such as spinal cord injury, head and neck injury. hEPI-NCSC are particularly relevant for treatment in spinal cord injury, as neural crest stem cells are ontologically closely related to spinal cord stem cells. Studies carried out by the inventors in mouse models of spinal cord injury showed that EPI-NCSC grafts caused a significant improvement in sensory connectivity and touch perception, that they can provide neurotrophic support and angiogenic activity, and that they possibly modulate scar formation by synthesis and release of metalloproteases. The inventors believe that hEPI- NCSC have similar properties. Optionally, the cells obtained using the method of the first aspect are for the treatment of burns. Optionally, the cells obtained using the method of the first aspect are for the treatment of wounds.
Optionally, the cells obtained using the method of the first aspect are for the treatment of skin conditions.
For the above treatment uses the cells obtained using the method of the first aspect could be replaced with undifferentiated hEPI-NCSCs.
Artificial skin comprising melanocytes cells obtained using the method of the first aspect.
According to a further aspect of the present invention there is provided dopaminergic neurons or dopaminergic neuronal progenitors obtained by the method of the first aspect.
Expanded but undifferentiated hEPI NCSC could be used in place of the said dopaminergic neurons or dopaminergic neuronal progenitors.
According to a further aspect of the present invention there is provided a culture medium for the directed differentiation of hEPI-NCSCs into dopaminergic neurons or dopaminergic neuronal progenitors comprising;
SHH ["sonic hedgehog"]
FGF-8
TGF-p2
GDNF [glial derived neurotrophic factor]
db c-AMP combinations of neurotrophic factors (NGF, BDNF, NT-3)
Most preferably the culture medium for the directed differentiation of hEPI- NCSCs into dopaminergic neurons or dopaminergic neuronal progenitors comprises;
SHH (100 ng/ml) ["sonic hedgehog"]
FGF-8 (50 ng/ml)
TGF-p2 (1 ng/ml)
GDNF (5 ng/ml) [glial derived neurotrophic factor]
db c-AMP(100uM),
combinations of neurotrophic factors (NGF, BDNF, NT-3)
According to a further aspect of the present invention there is provided a culture medium for the progression of hEPI-NCSCs to a neural stem cell like state comprising;
FGF-2
EGF SCF NT-3
Most preferably the culture media for the progression of hEPI-NCSCs to a neural stem cell like state comprises;
FGF-2 (10 ng/ml)
EGF (20 ng/ml)
SCF (5 ng/ml)
NT-3 (10 ng/ml)
According to a further aspect of the present invention there is provided a method of treating Parkinson's disease comprising administering a therapeutically active amount of said dopaminergic neurons or dopaminergic neuronal progenitors. According to a further aspect of the present invention there is provided a method of ex-vivo expansion of epidermal neural crest stem cells comprising the step of; culturing an isolated population of epidermal neural crest stem cells in expansion media.
Preferably the expansion media comprises;
proliferation media, FGF2, EGF, FBS (fetal bovine serum), ITS+3, GlutaMAX ®, Amphotericin. ITS+3 contains insulin, transferrin, sodium selenite as well as oleic acid, linoleic acid, and BSA
Optionally the proliferation media is NeuroCult XF ® Preferably the expansion medium comprises antibiotics such as Penicillin and/or Streptomycin.
Preferably the method of ex-vivo expansion of human epidermal neural crest stem cells is carried out at 5% oxygen.
Preferably the expansion media comprises;
proliferation media, FGF2, EGF, FBS (fetal bovine serum), ITS+3, GlutaMAX ®, Amphotericin. Preferably the method of ex-vivo expansion of human epidermal neural crest stem cells also includes the pre-steps of;
isolating early anagen hair follicles from a subject;
isolating the bulge area of hair follicles and placing it into adherent culture;
isolating epidermal neural crest stem cells from the bulge explants. According to a further aspect there is provided a method of producing a stable cell line of human epidermal neural crest stem cells in vitro by culturing an isolated population of epidermal neural crest stem cells in expansion media. ITS+3 contains insulin, transferrin, sodium selenite as well as oleic acid, linoleic acid, and BSA
Optionally the proliferation media is NeuroCult XF ® The proliferation media may be StemPRO MSC SF ®.
Preferably the expansion media comprises antibiotics such as Penicillin and/or Streptomycin. A cell line of human epidermal neural crest stem cells produced by the abovementioned method wherein the cells retain multipotency.
In order to provide a better understanding of the present invention, examples will now be described with reference to the materials and methods used. And with reference to the following figures;
Figure 1. Dissection of the bulge from human hair. Hair follicles in the anagen (growth) phase are dissected from full thickness hairy skin. Surrounding fatty tissue and dermis are removed mechanically with a sharpened tungsten needle. The dermal papilla is cut off and discarded. The area of the bulge is cut into 2 or 3 pieces. Bulge explants are pooled placed in culture medium and subsequently placed onto CellStart-coated culture plates, where the bulge explants adhere to the substratum within one hour. Figure 2i hEPI-NCSC emigrating from bulge explant. 2ii. Bulge explant with emigrating cells that have neural crest morphology. (Figure 2ii A) Within 6 - 10 days, migratory cells emigrate from about 35 percent of bulge explants. The cells have the typical stellate morphology of neural crest cells. In addition to migrating away from the bulge explant, they proliferate at a rapid rate. Rounded cells in this image are cells that undergo mitosis in order to divide. (Figure 2ii B). Primary hEPI-NCSC emigrate from adult hair follicle bulge cultures. (iiA-C) Bulge explant with cells emigrating onto the CellStart substratum (arrows) at days six, eight and 10, respectively. Images are from the same bulge explant. (iiD) The dermal sheath was removed from the bulge and cultured. (iiE) Bulge with the dermal sheath removed in culture. No cell emigration or proliferation was seen in iiD and iiE after 12 days in culture. (iiF) Percentage cell emigration by day 12 post explanting from bulges cultured with (45.3±13.0%, n=68) and without (6.3±6.3%, n=46) the dermal sheath and the dermal sheath alone (0±0%, n=29). (iiG) Percentage cell emigration from bulges of early anagen (stage lll-IV) follicles (58.9±12.8%, n=41 ) and late anagen (stages V-VI) follicles (54.4±4.3%, n=697) shows no significant difference in cell emigration from anagen stage hair follicle bulges. Student's t-test. Asterisk(s) indicate levels of significant difference as follows: *, p<0.05. Scale bars = 100pm.
Figure 3. Bulge explant with halo of cells that have putative keratinocyte precursor morphology. Rare bulge explants do not release neural crest cells, but putative keratinocyte progenitor cells. Keratinocytes are easily distinguished from neural crest-derived cells by their typical cobblestone morphology and by the fact that they are non-migratory but remain in close contact to each other and the bulge explant. Bulge explants that contain putative keratinocyte progenitor cells, such as the one shown in this image, are discarded.
Figure 4. Characterization of hEPI-NCSC in primary explants by real-time PCR (qPCR). Data are presented in the black column, left, for each gene. hEPI-NCSC express the neural crest stem cell molecular signature the inventors have defined for mouse EPI-NCSC and mouse embryonic neural crest stem cells (Hu YF, Zhang Z-J and Sieber-Blum, , 2006). Signature genes include VDAC1 , ETS1 , PCbP4, MYO10, H1 FX, THOP1 , MSX2, CRYAB, VARS2, PEG10, CALR, CRMP1 , UBE4B, PYG02, AGPAT6 AND ADAM 12. hEPI-NCSC also express additional neural crest-characteristic genes, including SOX10, SNAI2, TWIST, MS1 ('musashi') AND P75NTR, as well as general stem cell genes, TERT, NES ('nestin') and CD34. As the inventors and others have shown previously for other types of stem cell, hEPI-NCSC express several early lineage genes , including GFAP, MITF DCT, TUBB3, NEFL AND ACTA2, but not COLA2A1. hEPI-NCSC also express the pluripotency genes C-MYC, KLF4, SOX2, LIN28, POU5F1/OCT4 AND NANOG. Data are expressed as percent of average of four house-keeping genes.
Figure 5. Signature genes are expressed by hEPI-NCSC in primary explants at the protein level as shown by indirect immunocytochemistry.
Images shown for corresponding stains are the antibody specific black and white image plus the colour merged image, with DyLight 488 or DyLight 594 fluorescence and DAPI nuclear stain (blue fluorescence). The transcription factor ETS1 (A and A') was expressed in 97.3±2.7% cells and had a nuclear localisation. THOP1 (B and B') showed a cytoplasmic location necessary for its roles in protein function and metabolism and was immunoreactive in 100% of cells. MSX2 (C and C) is an important transcription factor involved in maintaining the balance between survival and apoptosis in neural crest cells and showed 98.2±1.8% expression. CRMP1 (D and D') is located in the cytoplasm in 98.3±1.7% of cells as it is a cytosolic phosphoprotein involved with signal transduction pathways. UBE4B (E and E') is involved in ubiquitination and was seen to have a cytoplasmic localisation in 96.7±1.6% of cells. MYO10 (F and F') was seen to have a cytoplasmic localisation and was expressed in 97.6±2.4% of cells, consistent with its role in cytoskeletal organisation. ADAM12 (G and G') showed a cytoplasmic localisation in 89.0±6.8% of cells as it is a membrane anchored protein and has roles in cell-cell and cell-matrix contacts. CRYAB (H and H') is involved in intracellular structure and subunits act as molecular chaperones and are thus localised to the cytoplasm in 100% of cells. The intermediate filament protein and stem cell marker NESTIN (I and P) was seen to have a cytoplasmic localisation and expressed in 100% of cells. The neural crest marker SOX10 (J and J') has a role as a nucleo-cytoplasmic shuttle protein and was expressed in 98.6±1.4% of cells. DAPI (4',6-diamidine-2-phenylidole- dihydrochloride) is blue nuclear counter stain. Scale bars = 50 pm.
Figure 6. Proof of multipotency of hEPI-NCSC.
(A) In vitro clonal analysis. One clone-forming cell gives rise to a colony that contains multiple cells types. Phase contrast images of a clone at day 1 (18 hrs after seeding) to day seven. Single hEPI-NCSC in clonal cultures proliferate and form clones. hEPI-NCSC were isolated by trypsin isation from primary explants (bulge explants) and seeded at clonal density (60 cells per 35 mm culture plate). Single cells were then identified and circled with a diamond tipped circle scribe. (Day 1) Single circled cell (rectangle) and inset (enlarged image of rectangle), (Day 2) four daughter cells (rectangle) and inset (enlarged image of rectangle), following division of the founder cell from Day 1. (Day 3, 4, 5 and 7) shows proliferation of the clone after three, four, five and seven days respectively such that after seven days the clone consists of many cells. The dish has been scratched with a tungsten needle (arrow head in Day 1) and orientated so that the field of vision is identical for each image and to show that the clone has derived from a single cell and as a proof that subsequent images have been taken from the same clone. Arrows in Day 5 and Day 7 point to dividing cells. Scale bars = 100pm.
(B) hEPI-NCSC in clonal culture differentiate into all major neural crest progeny. All possible permutations of antibody stains have been performed to show that hEPI-NCSC in clonal culture can give rise to many different neural crest derivatives and clone-forming cells were therefore multipotent. Triple stains combining two cell type specific antibodies (black and white) and colored merged images using DyLight 488 (green) or DyLight 594 (red) fluorescence and DAPI (blue) nuclear stain. The top row shows a double stain with antibodies against ACTA2 and COL2A1 , as indicated. The third image, in colour, is the merged image with blue DAPI nuclear stain added. A green ACTA2-positive (arrowhead) and a red COL2 A 1 -positive bone/cartilage cell (arrow) co-exist in the same clone. Second row, ACTA2 and TUBB3 double stain to show that smooth muscle cells can co-exist in the same clone with neuronal cells. Third row, some TU BBS- positive neuronal cells express tyrosine hydroxylase (TH), identifying them as catecholaminergic neurons. Fourth row, TUBB3-positive neuronal cells coexist with GFAP-positive Schwann cells/glia. Fifth row, GFAP-immunoreactive cells co- exist with COL2A1 -positive bone/cartilage cells. Sixth row, ACTA2-positive smooth muscle cells co-exist with GFAP-positive Schwann cells/glia. To elicit cell differentiation in clonal cultures, clones were treated with relevant growth factors; BMP2 for bone differentiation, NGF, TGF-beta2 and forskolin for neural differentiation, and neuregulin plus CNTF for Schwann cell differentiation. Scale bars = 50 pm.
Figure 7. Self-renewal. Self-renewal is an important part of the definition of a stem cell. It is the ability of a stem cell to give rise to daughter stem cells. Self- renewal was shown here by serial cloning. Cells in primary clones were resuspended by trypsin treatment at 22 days in culture and placed into clonal culture again, where they gave rise to secondary clones. Clone-forming ability was maintained at high levels, as 70.7±7.9% of secondary clones and
54.0±11.7% of tertiary clones consisted of fast-growing motile cells. Double stains with cell-type specific antibodies showed that these secondary clones contained multiple cell types as well. The presence of multiple cell types in secondary clones shows that hEPI-NCSC can undergo self-renewal. Taken together, it has thus been shown that hEPI-NCSC are multipotent stem cells. Figure 8. Expression of the six essential pluripotency genes at the RNA and protein levels. hEPI-NCSC express transcripts of the six essential pluripotency genes, C-MYC, KLF4, SOX2, LIN28, OCT4/POU5F1 and NANOG. As this was an unexpected finding and in order to calibrate gene expression levels, we compared
pluripotency gene expression levels to those in human embryonic stem cells (H9 cell line). Expression of pluripotency genes by hEPI-NCSC was compared to H9 hESC by qPCR and the AACt method used to determine fold differences in expression levels. Three independent donors were assessed (Donor A-C).
SOX10 was used as a marker of neural crest stem cells and in all cases was more abundant in hEPI-NCSC than in H9 cells by approximately 100 fold. C- MYC, KLF4 and NANOG were expressed at similar levels in hEPI-NCSC compared to hESC. SOX2, LIN28 and POU5F1/OCT4 were expressed at lower levels. While trends are similar, there are donor-specific variations in expression levels. hEPI-NCSC from donor C were also analyzed for expression of the pluripotency genes at the protein level. Indirect immunocytochemistry was performed using all possible permutations of antibody combinations as labeled (Α- ). In all cases, hEPI-NCSC were immunoreactive for the relevant markers. The percentage of cells expressing the gene of interest was determined (J); C- MYC, 100±0%, KLF4, 56.5±6.8%, SOX2, 69±4.5%, LIN28, 98.8±0.9%,
POU5F1/OCT4, 95.7±1.7% and NANOG, 92.6±3.5%. It needs to be noted that distribution of immunoreactivity is often cytoplasmic, not nuclear. This indicates that the genes are expressed but that most likely some are not functionally active. Scale bars=50 μιτι Figure 9. Ex vivo expansion of hEPI-NCSC.
(Fig 9 A) hEPI-NCSC can be expanded into millions of stem cells without an overall significant loss of stem cell markers. Analysis by qPCR of expression levels of the neural crest stem cell molecular signature, pluripotency genes, other neural crest stem cell genes, general stem cell genes and early lineage genes in primary explants (left; black) and after ex vivo expansion (right, grey). Expression of the neural crest stem cell molecular signature and other neural crest stem cell genes validates the neural crest origin of hEPI-NCSC. The data from primary explants are the average of RNA from three donors; whereas the results from ex vivo expanded cells represent the average of two different donors. Significant differences in expression levels are likely to be due to donor-specific differences. Student's t-test. Asterisk(s) indicate levels of significant difference as follows: *, p<0.05, ***, p<0.001. ND, not detected.
(Fig. 9 B). Growth curve of hEPI-NCSC proliferation during ex vivo expansion. On average three million cells per bulge were obtained within 28 days. Notably, at 28 days cell growth has not yet levelled off but is still in the log phase. Further expansion is thus possible. Overall, we show that hEPI-NCSC can be expanded ex vivo efficiently and reproducibly and that they retain sternness. An attractive feature of the expansion protocol is its short duration. Changes in the karyotype are thus less of a concern in hEPI-NCSC than in cell lines that are passaged multiple times. The high numbers of stem cells that can be obtained through ex vivo expansion within a short period of time make testing in animal models of human disease and future applications feasible. (Fig. 9 C). Expression of signature genes and other stem cell genes by ex vivo expanded hEPI-NCSC is maintained at the protein level. Indirect immunocytochemistry shows that greater than 95% of ex vivo expanded cells express the signature genes (Α-Η' as labelled). I, Γ; all cells express NESTIN. J, J', all cells express the neural crest stem cell marker SOX10. Scale bars = 50 Mm.
Figure 10. Protocol for directed differentiation of hEPI-NCSC into dopaminergic neurons. Bulge explants from human hair follicles are prepared as described above. Cultures are maintained at 37 °C in a humidified atmosphere that contains 5% C02 and 5% 02. After 7-10 days, bulges are removed and the hEPI-NCSC suspended by trypsin treatment. hEPI-NCSC were then seeded into CellStart-coated 35 mm culture plates at 2,500 cells per plate in expansion medium. Two days later, the cells are switched to "NP" (neural progenitor) medium with the goal to differentiate the multipotent stem cells into neural stem cell-like progenitor cells. Medium changes are done by removing half of the old culture medium and replace it with half of the new medium, in order to acclimate the cells slowly to the new culture condition. Cells are grown in NP medium for 7 - 10 days, depending on cell growth, and are then sub-cultured into "PF" (patterning factor) medium using again 50-50 medium exchanges. Cells are maintained in PF medium for 2 - 3 weeks at 3% oxygen and 5% CO2. During the last week of culture, the maturation factor ascorbic acid is added to the PF medium. While in NP medium, cells change morphology from stellate morphology to a more elongated shape and often elaborate short processes (image at lower left). While in PF medium, cells become confluent and change shape again. They elaborate long processes; the soma is first rounded and eventually cells assume the typical shape of midbrain dopaminergic neurons (image, lower right).
Figure 11. In vitro differentiated dopaminergic neurons express markers typical for midbrain dopaminergic neurons. At the RNA level, genes characteristic for midbrain dopaminergic neurons are expressed. Data have been generated by real-time PCR and are expressed as percent of the average of 4 housekeeping genes (HKG). The neuronal marker β-ΙΙΙ tubulin is expressed. The biosynthetic enzyme, tyrosine hydroxylase, which converts tyrosine to DOPA is expressed, as is the biosynthetic enzyme, DOPA decarboxylase, which converts DOPA into dopamine. The third biosynthetic enzyme in the synthesis of catecholamines, dopamine-beta hydroxylase, is not detectable. This is a desirable result, as it shows that the neurotransmitter, dopamine, is not converted into norepinephrine. Markers expressed by midbrain dopaminergic neurons are also expressed by in vitro differentiated hEPI-NCSC. They include NURR1 , PITX3, EN1 , LMX1 b, VMAT2, GIRK2, FOXA2 and DAT (dopamine transporter). Figure 12. Midbrain dopaminergic neuron markers are expressed at the protein level as determined by indirect immunocytochemistry.
Upper left; Virtually all cells in the culture are neuronal, as 99.4±0.7% of cells express the neuron-specific gene, beta-Ill tubulin. Up to 95% of all cells express the biosynthestic enzymes tyrosine hydroxylase (93.4±0.7%) and DOPA decarboxylase (92.7±1.8%), as well as the neurotransmitter, dopamine (95.1±1.9%). Differences between the three percentage values are statistically not significant (p=0.4). NURR1 , which is an early marker of midbrain dopamine neuron differentiation is expressed also at the end point of the experiment, but in fewer cells (76.4±33.4%). The dopamine transporter (DAT) is expressed in 79.2±4.6% of cells at 2 weeks in PF medium. The function of DAT is to re-uptake dopamine after release in order to avoid over-stimulation of the post-synaptic neuron.
Upper right. Merged images of a double stain with antibodies against NURR1 (red fluorescence) and dopamine (green fluorescence) and blue DAPI nuclear stain. Note that NURR1 has a nuclear localization, indicating that it is functionally active. Note also that the expression pattern of dopamine is punctuate, indicating that the neurotransmitter is packaged into synaptic vesicles.
Lower left, beta-Ill tubulin and tyrosine hydroxylase double stain combined with blue DAPI nuclear stain. This image confirms that most cells that express the neuron-specific marker beta-Ill tubulin also express tyrosine hydroxylase (TH), a biosynthetic enzyme that converts tyrosine to L-DOPA. Note the punctuate pattern of TH immunoreactivity, indicating that the neurotransmitter is packaged into synaptic vesicles.
Lower right, dopamine transporter (DAT; red fluorescence) antibody stain combined with DOPA decarboxylase (green fluorescence) stain and blue DAPI nuclear stain. Cells that express both proteins are yellow fluorescent. Figure 13: Neuronal morphology. At low cell density the neuronal morphology of the in vitro differentiated dopaminergic neurons becomes apparent. A neuron triple stained for DOPA decarboxylase (green fluorescence) and the dopamine transporter (red fluorescence) and DAPI nuclear stain (blue fluorescence) is shown. The cell body (arrow) has elaborated a long process (marked by a parenthesis). Yellow fluorescence is indicative of co-localization of DDC and DAT, and is mostly concentrated in the nerve ending (top).
Figure 14: Calcium imaging. Calcium imaging showed that in vitro differentiated dopaminergic neurons are functional neurons in regard to neurotransmitter receptor expression and function. The inventors show that the dopaminergic neurons respond to various agonists, thus indicating that the in vitro differentiated dopaminergic neurons express pertinent neurotransmitter receptors.
Fig 14 A: Time course. This sequence of images shows the time course of response to ATP with intracellular calcium flux (yellow).
Fig. 14 B: List of agonists used and their cognate receptors. Thus the results show that in vitro differentiated dopaminergic neurons express acetylcholine receptors, purinergic receptors, glutamate receptors and alpha-1 adrenergic receptors. Spontaneous activity was observed as well. Unspecific stimulation resulted in calcium flux also. And CPA emptied all intracellular stores.
Fig. 14 C: Summary of wave forms in response to agonists used. Figure 15: Expression of GDNF and BDNF by hEPI-NCSC, GDNF (glial derived neurotrophic factor) and BDNF (brain derived neurotrophic factor) are two neurotrophins that are essential for the survival of midbrain dopaminergic neurons. All (100%) ex vivo expanded, undifferentiated, hEPI-NCSC express both neurotrophins at the RNA level and at the protein level, as determined by real-time PCR and indirect immunocytochemistry, respectively. This result indicates that hEPI-NCSC could be useful in delaying the death of midbrain dopaminergic neurons in Parkinson's disease when grafted into the substantia nigra pars compacta of the midbrain.
Figure 16. Osteogenic differentiation of hEPI-NCSC shows expression of key markers. hEPI-NCSC were cultured in Ad ance ST EM Osteogenic differentiation medium at 37°C, 5% CO2 and either 5% or ambient 02 for up to 35 days. Cultures were analysed by qPCR or indirect immunocytochemistry for expression of osteogenic markers. A, B, C, D, E, F and G show qPCR data for relative expression levels compared to housekeeping genes (HKG) of the appropriate marker as labelled. Primary culture cells (1° culture) cultured at 5% O2 as described serve as untreated cells, with experimental cultures treated for up to 35 days. RUNX2 (A) shows an early increase in expression after seven days in both 02 conditions to 85.5±0.7% and 83.4±1.6% respectively, which then reduces over the remaining culture period. COL2A1 (B) was seen to increase in expression with time in both O2 concentrations to a comparable final level at day 35 of 59.9±2.3% and 61.7±0.8% respectively with no expression detected in primary culture cells. Osteopontin (SPP1 ) (C) was seen to show an initial reduction in expression after seven days followed by an increase back to a level similar to untreated cells of 76.2±2.6% in 5% O2 cultures at day 35. Culturing at ambient O2 resulted in increasing SPP1 expression over the culture period with 85.9±2.1 % expression at day 35. A statistically significant difference was seen in SPP1 expression levels between C½ concentrations at days seven and 35. Expression of osteocalcin (BGLAP) (D) was shown to increase in both O2 conditions by day seven to 81.7±0.6% and 81.4±0.3% respectively and to remain relatively unchanged in both O2 concentrations for the remainder of the culture. Core-binding factor subunit beta (E) was seen to show little change in expression throughout the culture. At day 35 expression levels of 88.8±0.64% and 90.4±0.2% were seen for 5% and ambient oxygen cultures respectively. The oxygen tension affected expression levels significantly at day seven. COL1A1 expression (F) was seen to dramatically increase by day seven from untreated levels of 104.9±0.5% to 125.9±0.2 and 122.8±0.5% for 5% and ambient oxygen tensions respectively. Levels then declined and at day 35 were 1 16.3±0.8 and 108.6±0.4 respectively. A significant effect of oxygen tension was observed on expression levels at each time point. Expression of VDR (G) was seen to remain virtually unchanged over the 35 day culturing period. Untreated expression levels were 94±0.6%, with expression level at day 35 being 96.7±0.4 and 95.7±0.9 respectively. Oxygen tension displayed a significant effect in cultures at days seven and 28. A' shows RUNX2 immunoreactivity in day 14 cultures at ambient O2. B', C and D' show COL2A1 , SPP1 and BGLAP immunoreactivity respectively, from day 35 cultures at ambient O2. A", B", C" and D" are quantification of respective antibody staining for each marker over the 35 day culture period. Positive expression is expressed as percentage of total cells. RUNX2 expression in both 5% and ambient O2 cultures was seen to increase by day 14 to 49.0±2.9% and 58.4±5.0% respectively (A"), followed by a decline and then increase again towards the end of the culture. COL2A1 was not expressed at early time points but increased by day 35 in both O2 conditions with 68.4±4.3% cells positive from ambient 02 cultures (B"). SPP1 was also seen to increase in expression throughout the culture period with highest expression seen at day 35 in ambient O2 cultures, with 81.1 ±3.0% cells positive (C"). A significant difference between C¾ concentrations on SPP1 immunoreactivity was seen at days 14 and 35. BGLAP expression was also seen to increase between days 28 and 35 in both oxygen conditions, and was significant at the different oxygen conditions at days 14 and 28. By day 35, 70.7±7.6% and 81.1 ±3.1 % cells were immunoreactive from 5% and ambient O2 cultures respectively (D"). Student's t- test. Asterisk(s) indicate levels of significant difference as follows: *, p<0.05, ** p<0.005, *** p<0.001. ND = not detected. Scale bars = 20 pm. H, H', H" and H'" show Alizarin Red S staining of day 35 control and experimental cultures from both 5% and ambient 02 tensions as labelled. Arrows show regions of positive Alizarin Red S staining in treated cultures, thus indicating calcium deposition, an essential feature of osteogenic differentiation. Scale bars = 100 Mm. Figure 17. Directed differentiation of hEPI-NCSC into melanocytes.
Directed differentiation of hEPI-NCSC into melanocytes. hEPI-NCSC were differentiated into pigmented cells in vitro. DOPA reaction analysis of
differentiated cells confirmed expression of pigment in 65.7±6% of cells as viewed by bright field microscopy (see, e.g., arrows). Scale bar=100 μιη
Figure 18. Cryopreservation of hEPI-NCSC. hEPI-NCSC cultures were frozen in 90% FBS, 10% DMSO using a Nalgene freezing container followed by transfer to liquid nitrogen. Cells were subsequently thawed and trypan blue analysis of cell viability showed 87% cell viability. (A) shows cells before freezing at 24,000 cells/cm2, (B and C) cells in culture on CellStart treated 35mm dishes, one day (5,400 cells/cm2) and three days (14,000 cells/cm2) post thawing, respectively. Scale bars = 100 pm.
Figure 19: Directed differentiation of hEPI-NCSC into sensory neurons. Expression of markers for sensory neurons at the protein level was determined by indirect immunocytochemsitry. (A) Beta-Ill tubulin neuronal maker is co- expressed with Bm3a (nuclear localization). (B) Neurofilament heavy chain (NF200) is co-expressed with Bm3a. (C) CGRP is co-expressed with Brn3a. (D) IB4 is co-expressed with Brn3a. (E) Isletl is co-expressed with Brn3a. DAPI (blue fluorescence), nuclear stain.
Figure 20: Immunocytochemistry showing nuclear localization of osteocalcin expression and cytoplasmic localization of osteopontin expression in cells having primary culture and first subculture in StemPRO® MSC SFM.
Figure 21 : Time course of Alizarin Red Stain (culture days 0, 7, 14, 28 and 35) from cells having primary culture and first subculture in StemPRO® MSC SFM.
Directed differentiation of human epidermal derived-NCSC's fhEPI-NCSC's) into dopaminergic neuronal oroaenitors/dopaminergic neurons
One aspect of the current invention is an in vitro methodology to promote the directed differentiation of human epidermal derived-NCSC's (hEPI-NCSC's) into dopaminergic neuronal progenitors, with specific potential significance for the treatment of Parkinson's disease (PD), a neurodegenerative condition caused by the loss of dopaminergic activity in the substantia nigra pars compacta of the human brain.
PD is the second most common neurological disorder globally, affecting approximately 4.1 m people. As the prevalence rate of PD is set to rise significantly in the coming years, clinical interventions are expected to play a major role in combating the wide spectrum of needs associated with the disease.
The methods described herein relate to the directed differentiation of hEPI- NCSC's into dopamine producing neuronal cell lines (dopaminergic neurons or dopaminergic neuronal progenitors). Such cell lines could be used to restore somatic neural function in PD patients when used as part of a cell-based therapeutic approach. It is known in the art to identify the relevant dopamine producing neuronal cell lines by identifying known markers.
Initial experiments allowed for the identification and isolation of anagen phase hair follicles from human pubic skin, collected during elective Caesarean section and following informed patient consent. Similar experiments have also been performed with human hair follicles from the scalp and it should be understood that it would be possible to isolate the bulge region from other hairy skin areas. The bulge region of the hair follicles were then micro-dissected and placed into collagen-coated culture plates, where after 6 - 10 days of culture in medium consisting of, Neurocult XF (Stem Cell Technologies), FGF-2 (10 ng/ml; R&D Systems), EGF (20 ng/ml; R&D Systems), FBS (1 %; Thermo Fisher). ITS+3 (Sigma), Gluta AX (Invitrogen), Amphotericin (Sigma), Penicillin/Streptomycin (Sigma), the hEPI-NCSC's migrate onto the collagen or CellStart substratum. Isolated hEPI-NCSC's were then sub-cultured in the same culture medium. In a second sub-culture in the presence of first Neural Progenitor medium (FGF-2, EGF, NT-3) for 7 to 10 days at 5% oxygen and then in the presence of patterning factors (SHH, FGF-8, GDNF, TGF-beta2) at 3% oxygen, whereby they became immune-fluorescent for neuron-specific beta-Ill tubulin and tyrosine hydroxylase (TH) the principle enzyme involved in the conversion of the amino acid l-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA), the precursor of the human neurotransmitter dopamine. It is widely acknowledged within the literature that such expression of TH is considered an early indication of catecholaminergic neurons and dopaminergic activity and identifies the presence of dopaminergic neurons or dopaminergic neural progenitors. The results suggest that hEPI- NCSC's can be manipulated by this method to differentiate into dopamine producing neurons and therefore hold significant potential for the treatment of PD by autologous therapeutic allograft.
Further experiments were carried out to quantify TH gene expression by real-time PCR, and investigate the expression of amino acid decarboxylase, (the enzyme responsible for the conversion of DOPA to dopamine), the expression of nuclear receptor related protein 1 (NURR1 - an intracellular transcription factor associated with dopaminergic activity in the brain), PNMT, PITX3, EN1 AND LMX1 B. DBH (dopamine-beta-hydroxylase), which converts dopamine to noradrenaline, was not expressed as determined by real-time PCR.
The following experimental work was carried out.
Method for obtaining primary exolants and hEPI-NCSC isolation Hair follicles in anagen phase are isolated from human pubic skin (from elective Caesarean sections with informed consent) or from scalp skin, athough it could be envisaged that hair folilces could be obtained from alternative sources, such as for instance from skin or the arm or axillary skin, or skin from eye lids. The bulge region of hair follicles is micro dissected from epidermis and subdermis. The micro dissected hair follicles are then cleaned mechanically from surrounding tissue and dermal papilla is cut off and discarded collected in Neurocult XF culture medium containing 5nM HEPES at pH7.2. The area of the hair follicle above the bulge is cut off and discarded and the area containing the bulge is cut into two to three pieces and placed into/on coated culture plates such as a collagen or CellStart™ covered culture plate (Fig. 1 ). The bulge explants were incubated at 37°C, 5% CO∑ and 5% O2 for 1 hour such that they would attach to the collagen or CellStart substratum. After 6 - 10 days, hEPI-NCSC start to emigrate onto the collagen substratum (see Fig. 2i). These cells have the typical stellate morphology of neural crest cells.
In some rare explants non-migratory putative keratinocyte precursor cells form a halo around the explant (Fig. 3) and these cultures are discarded.
It was found that it is important to retain the dermal sheath over the bulge. In the absence of the dermal sheath, significantly fewer bulge explants yielded hEPI- NCSC. Conversely, the dermal sheath explants did not yield emigrating hEPI- NCSC (Fig 2(H) D and E). hEPI-NCSC express the neural crest stem cell molecular signature, which has already been defined for mouse EPI-NCSC and mouse embryonic neural crest stem cells (Hu et al, 2006; Fig 4). hEPI-NCSC also express other neural crest stem cell genes, general stem cell genes and, like mouse EPI-NCSC, genes that were used initially used to generate induced pluripotent stem (iPS) cells, as well as some early lineage markers (see Fig. 4 and associated figure legend for details). Some of these are expressed at the RNA, but not the protein level. Signature genes and the six essential pluripotency genes are expressed at the protein level (Fig. 5), as determined by immunocytochemistry. Conclusion: hEPI- NCSC are neural crest derivatives that show sternness.
In vitro clonal analysis shows that hEPI-NCSC are multipotent, i.e. that one clone-forming cell can give rise to multiple differentiated cell types (Fig 6A and B). All possible permutations of antibody stains have been performed to show that hEPI-NCSC in clonal culture can give rise to multiple different neural crest derivatives and clone forming cells are thus multipotent (Fig 6B).
Sub-cloning of clones showed that primary, secondary and tertiary clones contain clone-forming cells (Fig 7). Cells in primary clones were resuspended by trypsin treatment at 22 days in culture and placed into culture again where they give rise to secondary clones. Immunocytochemistry of secondary clones shows that secondary clones are capable of self renewal and contain multiple cell types. This leads to the conclusion that hEPI-NCSC can undergo self-renewal and are thus multipotent stem cells. Unexpectedly, human EPI-NCSC express the six essential pluripotency genes, SOX2, C-MYC, KLF4, LIN28, OCT-4/POU5F1 and NANOG (Fig 8). Unlike mouse EPI-NCSC, hEPI-NCSC express Nanog both at the RNA and protein levels (Fig 8). Due to te unexpected nature of these results, and in order to calibrate gene expression levels, the inventors compared pluripotentency gene expression levels to those in human embryonic stem cells (Fig 8) (further details provided in Fig 8 figure legend).
Ex-vivo expansion of hEPI-NCSC Ex-vivo expansion of hEPI-NCSC is needed in order to obtain sufficiently large numbers of cells. Larger numbers of cells are typically required for therapeutic use or if required for use in high-throughput screening. The inventors have developed a culture medium that is conducive to the culture of primary explants of bulges and to ex vivo expansion of hEPI-NCSC. The same medium can be used for the initial culturing of the primary explant. There are few significant changes in pertinent sternness gene expression. Fig 9A shows a comparison of gene expression of hEPI-NCSC in primary explants (left; black) versus hEPI-NCSC that have been expanded (right;grey). There are few differences in gene expression. Differences observed are likely donor-specific. Ex vivo expanded hEPI-NCSC continue to express neural crest stem cell signature genes at the protein level (Fig 9C). Notably greater than 95% of ex vivo expanded cells express the signature genes. On average 1.5 x 106 cells can be obtained from one primary explant in 28 days (Fig 9B). It is also notable that at 28 days cell growth is still in the log phase and further expansion is thus still possible.
Details of the media used in the expansion step are given below. Expansion is preferably carried out at 5% Oxygen (O2).
Expansion medium (Neurocult expression medium components)
Component Supplier Cat#
NeuroCult XF Stem Cell Technologies 05761
FGF2 R&D Systems 233-FB/CF
EGF R&D Systems 236-EG-200
FBS Thermo Fisher SH30070.02
ITS+3 Sigma 1-2771
GlutaMAX Invitrogen 35050-038
Amphotericin Sigma A2942
Penicillin/Streptomycin Sigma P0781 Culture Medium for primary bulge explants and ex vivo expansion NeuroCult XF (formerly ACF), r FGF2 (10ng/ml), rhEGF (20ng/ml), 1 % (v/v) FBS, ITS+3, GlutaMAX, Amphotericin, Penicillin/Streptomycin. Basal Medium
NeuroCult-XF Basal medium with proliferation supplement
StemCell Technologies, Catalogue number 05761
FGF-2
R&D Systems, rhFGF-basic, 233-FB/CF,
Working concentration, 10 nanogram/ml
EGF
R&D Systems, rhEGF, 236-EG,
Working concentration, 20 nanogram/ml
GlutaMax™
Invitrogen, 35050-038 (100X solution at 200mM)
Working concentration, 1X at 2mM
Penicillin/Streptomycin
Sigma, P0781 (100X solution)
Working concentration, 1X
Amphotericin
Sigma, A2942 (100X solution at 250 microgram/ml)
Working concentration, 1X at 2.5 microgram/ml
Fetal bovine serum
SH30070.02 (HyClone catalogue number)
[sold through Thermo Fisher, # HYC-001 -326W]
Working concentration, 1 %
ITS+3
1-2771 Sigma (100X solution)
Working concentration, 1X The inventors have found that this basal expansion medium is surprisingly effective. Notably, the medium differs from that which would be used for other stem cell types, for example Stem Cell Technologies would not suggest the inclusion of;
Figure imgf000035_0001
It is particularly surprising as typically one would expect to avoid fetal bovine serum (FBS) as it is non-human. Equally surprising is the incorporation of ITS+3 which contains insulin, transferrin and selenium as insulin in particular would usually be expected to cause differentiation which is not required at this stage. Surprisingly the inventors have found that use of this expansion medium does not result in unwanted differentiation.
Directed differentiation of hEPI-NCSC The inventors are the first to identify the potential for differentiating EPI-NCSC and in particular human EPI-NCSC. It is extremely surprising to find that this cell type can be differentiated with the levels of efficiency identified by the inventors herein using the methods and associated culture medium that have been developed. We describe below a number of experiments that have been carried out to exemplify the differentiation step this invention.
Directed differentiation of hEPI-NCSCs into dopaminergic neurons or precursors thereof The inventors have now developed culture methods/conditions for the directed differentiation of hEPI-NCSC into dopaminergic neurons. Gene expression at the RNA level is determined by real-time PCR, and confirmation of gene expression at the protein level. Surprisingly the inventors have found that their method results in extremely effective differentiation when compared to other methods of differentiation using different cell types that are described in the prior art.
The expanded population of cells is sub-cultured in specific media to direct differentiation as required. The base medium for differentiation is shown in the following table.
Figure imgf000036_0001
Medium - NeuroCult NSA, 1 % FBS, ITS+3, GlutaMAX, Amphotericin, Penicillin/Streptomycin
This medium can be obtained as GMP-compliant culture medium when needed. As is shown in figure 10, a preferred method for differentiation into dopaminergic neurons is a two-step sub-culturing method. This method may be useful for directing differentiation into other types of neurons as well. The expanded population is first sub-cultured in a Neural Progenitor (NP) medium for 7 days to differentiate the cells to a neural stem cell like state and then sub-cultured in a Patterning Factor (PF) medium to produce fully differentiated cells. Figure 10 also shows the cell morphology at different stages associated with the directed differentiation of hEPI-NCSCs using the methods secribed below..
Initial experiments were carried out to test the use of different combinations of Neurotrophins in the differentiation stage. Neural Progenitor (NP) Medium (first 7 days of second subculture) 5% 02 rhFGF-2 (10 ng/ml)
EGF (20 ng/ml)
SCF (5 ng/ml)
NT-3 (10 ng/ml)
This is a unique medium, which essentially converts the multipotent stem cells to a neural stem cell like state prior to then using a patterning factor medium culture to then fully differentiate the cells. Patterning Factors (PF) Medium (starting day 7)
SHH (100 ng/ml) ["sonic hedgehog"]
FGF-8 (50 ng/ml)
TGF-p2 (1 ng/ml)
GDNF (5 ng/ml) [glial derived neurotrophic factor]
Plus additional factors :
db c-A P(IOOuM), starting day 7
combinations of neurotrophic factors (NGF, BDNF, NT-3)
It is the additional factors that are thought to specifically direct differentiation.
Culturing was carried out on the following groups with the following culture conditions;
Group 1
NP medium, first 6 days of second subculture
PF, starting day 7
db c-AMP(100uM), starting day 7
Group 2
NP medium, first 6 days of second subculture
PF, starting day 7 db c-AMP(IOOuM), BDNF (20 ng/ml) starting day 7 Group 3
NP medium, first 6 days of second subculture
PF, starting day 7
db c-AMP(-IOOuM), NGF (20 ng/ml) starting day 7
Group 4
NP medium, first 6 days of second subculture
PF, starting day 7
db c-AMP( OOuM), NT-3 (10 ng/ml)
starting day 7
Group 5
NP medium, first 6 days of second subculture
PF, starting day 7
db c-AMP(100u ),BDNF (20 ng/ml), NGF (20 ng/ml) starting day 7 Group 6
NP medium, first 6 days of second subculture
PF, starting day 7
db c-AMP(IOOu ), BDNF (20 ng/ml), NGF (20 ng/ml), NT-3 (10 ng/ml) starting day 7 Group 7
NP medium, first 6 days of second subculture
PF, starting day 7
db c-AMP(IOOuM), BDNF (20 ng/ml), NT-3 (10 ng/ml)
starting day 7
Group 8 NP medium, first 6 days of second subculture
PF, starting day 7
db c-A P(IOOuM), NGF (20 ng/ml),NT-3 (10 ng/ml)
starting day 7
Results showed that culturing using all three neurotrophins together was best for gene expression at RNA and protein levels and for cell morphology.
Further Experiments were carried out to determine whether SCF and ascorbic acid are required.
The following media were considered;
Neural Progenitor (NP) Mix1
FGF-2 (10 ng/ml)
EGF (20 ng/ml)
SCF (5 ng/ml)
NT-3 (10 ng/ml) Neural Progenitor (NP) Mix2
FGF-2 (10 ng/ml)
EGF (20 ng/ml)
NT-3 (10 ng/ml) Patterning Factors (PF) Mix
SHH (100 ng/ml)
FGF-8 (50 ng/ml)
GDNF (5 ng/ml)
TGF-P2 (1 ng/ml)
Experimental Design Group 1
NP medium 2, first 7 days of second subculture
PF medium, starting day 7, plus:
db c-AMP(100u )
BDNF (20 ng/ml)
NGF (20 ng/ml)
NT-3 (10 ng/ml), starting day 7 Group 2
NP medium 1 , first 7 days of second subculture
PF medium, starting day 7, plus:
db c-AMP(100u )
BDNF (20 ng/ml)
NGF (20 ng/ml)
NT-3 (10 ng/ml)
starting day 7
Group 3
NP medium 1 , first 7 days of second subculture
PF, starting day 7, plus
db c-AMP(I OOuM),
BDNF (20 ng/ml),
NGF (20 ng/ml),
NT-3 (10 ng/ml), starting day 7,
Ascorbic Acid 200uM, starting day 10
It was determined that SCF is needed for neural progenitor formation and ascorbic acid supports neuronal maturation. Mix1 was found to be better than Mix2 because stem cell factor (SCF) is necessary for neuroprogenitor formation. EGF plus FGF-2 is used to differentiate embryonic stem cells neurospheres into neural progenitors. SCF based on our earlier work on neural crest cells. NT-3 was added based on the inventors understanding that this is important for survival of neural crest cells (Zhang JM et al, 1997). Further experimental work led to preferred media for expansion and directed differentiation of hEPI-NCSCs into DA neurons being as follows;
Primary culture & expansion.
5% 02
Medium NeuroCult XF (Stem Cell Technologies, Cat# 05761 ) supplemented with 10 ng/ml rhFGF2 (R&DSystems, Cat# 233-FB), 20 ng/ml rhEGF (R&D Systems Cat# 236-EG), 1X ITS+3 (Sigma, Cat# 1-2771), 1% (v/v) FBS (HyClone, Thermo Fisher,Cat# SH30070.02), 1X GlutaMAX
(Invitrogen, Cat# 35050-038), 1X Penicillin/Streptomycin (Sigma Cat# P0781) and 2.5 g/ml Amphotericin B (Sigma, Cat# A2942).
Real time PCR (qPCR) (results below) and indirect immuncytochemistry (results not shown) experiments were carried out which showed that GDNF and BDNF were expressed, at both RNA and protein levels, in substantially all of the expanded hEPI-NCSC population. More specifically, qPCR experiments showed the following RNA expression levels (as a % of average of 4 housekeeping genes ± S.E.M);
GDNF 76.710.3
BDNF 79.8±0.1
Differentiation
NP medium phase
7 days , 5% O2,
Medium NeuroCult N-SA (Stem Cell Technologies, Cat# 05753) supplemented with 10 ng/ml rhFGF2 (R&D Systems, Cat# 233-FB), 20 ng/ml rhEGF (R&D Systems Cat# 236-EG), 5 ng/ml rhSCF (R&D Systems, Cat# 255-SC-010/CF), 10 ng/ml rhNT-3 (R&D Systems Cat# 267-N3-005/CF) 1X ITS+3 (Sigma, Cat# I- 2771), 1 % (v/v) FBS (HyClone, Thermo Fisher,Cat# SH30070.02), 1X GlutaMAX (Invitrogen, Cat# 35050-038), 1X Penicillin/Streptomycin (Sigma Cat# P0781) and 2.5 pg/ml Amphotericin B (Sigma, Cat# A2942).
Patterning and maturation phase from day 8
-18 days, 3%02
Medium NeuroCult N-SA (Stem Cell Technologies, Cat# 05753) supplemented with 100 ng/ml rhSHH-N (R&D Systems, Cat# 1314-SH-025/CF), 50 ng/ml rhFGF8f (R&D Systems, Cat# 5027-FF-025/CF), 5 ng/ml rhGDNF (R&D Systems, Cat# 212-GD-010/CF), 1 ng/ml rhTGF-p2 (R&D Systems, Cat# 302- B2-002/CF), 10 ng/ml rhNT-3 (R&D Systems Cat# 267-N3-005/CF), 20 ng/ml rhBDNF (R&D Systems, Cat# 248-BD-005/CF), 20 ng/ml rhNGF (R&D Systems Cat# 256-GF-100/CF), 100uM db c-AMP (Sigma, Cat# D0260), 1X ITS+3 (Sigma, Cat# 1-2771 ), 1 % (v/v) FBS (HyClone, Thermo Fisher, Cat# SH30070.02), 1X GlutaMAX
(Invitrogen, Cat# 35050-038), 1X Penicillin/Streptomycin (Sigma Cat# P0781) and 2.5 μg/ml Amphotericin B (Sigma, Cat# A2942).
From day 15 200 mM Ascorbic Acid (Sigma Ca# A4544). qPCR was earned out on the differentiated population to show that at the RNA level, genes characteristic for midbrain dopaminergic neurons are expressed (Fig 11 ), confirming that the hEPI-NCSC population had differentiated into dopaminergic neurons. The qPCR results indicated the expression levels of genes known to be dopaminergic markers (as % of average of 4 housekeeping genes). Indirect immunochemistry was also carried out and Figure 12 provides evidence that dopaminergic neuron genes are also expressed at the protein level. It is notable that the present methods result in unparalleled levels of differentiation. Immunocytochemistry data indicates that 99% of all of the cells in the differentiated population are neuronal and 95% are dopaminergic. More specifically, according to immunocytochemistry experiments, the percentage of cells expressing dopaminergic markers was as follows;
13-111 tubulin 99.4±0.7%
tyrosine hydroxylase 93.4±3.2%
Dopa decarboxylase* 92.7±1.8%
Dopamine* 95.1 ±1.9%
NURR1 76.4±3.4%
Dopamine transporter 79.2±4.6%
* = p=0.4
Prior art methods (such as those disclosed in Cho, 2007; Perrier, 2004; Shimada, 2008; Wernig, 2008; Yan, 2009; which discuss the potential of differentiation of various cell types (notably none of these suggest using hEPI-NCSCs) show significantly lower efficiency of differentiation. For example, in Yan Y only -30% of cells are TH positive (with 50-60% of B-lll tubulin positive cells also being positive for TH); in Wernig only 5% of cells are differentiated; in Shimada 29% of colonies contained TH and or B-lll tubulin and only 10% of B-lll tubulin positive were also TH positive; in Perrier only 30-50% of cells were b-lll tubulin positive (of those only 64-70% were also TH positive; and in Cho only 77% were B-ll l tubulin positive and of those only 86% were also TH positive; 50.9% dopaminergic neurons of total cells).
It was further shown that at low cell density the neuronal morphology of the in vitro differentiated dopaminergic neurons is clearly apparent (Fig 13). A neuron triple stained for DOPA decarboxylase (DDC; green fluorescence) and the dopamine transporter (DAT; red fluorescence) and DAPI nuclear stain (blue fluorescence) is shown. The cell body (arrow) has elaborated a long process (marked parenthesis). Yellow fluorescence is indicative of co-localization of DDC and DAT, and is mostly concentrated in the nerve ending (top). Calcium imaging (Fig 14) also showed that cells respond to various agonists (a summary of which is shown in Fig 14B), thus indicating that the in vitro differentiated dopaminergic neurons express pertinent receptors. Fig 14 A shows the time course of response to ATP with intracellular calcium flux.The results show that in vitro differentiated dopaminergic neurons express acetylcholine receptors, purinergic receptors, glutamate receptors and alpha-1 adrenergic receptors. Spontaneous activity was observed as well. Unspecific stimulation resulted in calcium flux also. And CPA emptied all intracellular stores. A summary of wave forms in response to agonists used is shown in Fig 14 C.
The inventors also show that all ex vivo expanded, undifferentiated, hEPI-NCSC express both neurotrophins at the RNA level and at the protein level, as determined by real-time PCR and indirect immunocytochemistry, respectively (Fig 15). GDNF (glial derived neurotrophic factor) and BDNF (brain derived neurotrophic factor) are two neurotrophins that are essential for the survival of midbrain dopaminergic neurons. This result indicates that hEPI-NCSC could be useful in delaying the death of midbrain dopaminergic neurons in Parkinson's disease when grafted into the substantia nigra pars compacta of the midbrain. As well as dopaminergic neurons the inventors have also looked to direct differentiation of hEPI-NCSC into sensory neurons using the following methods;
1 Primary culture (isolation and expansion).
5% 02
Medium NeuroCult XF (Stem Cell Technologies, Cat# 05761 ) supplemented with 10 ng/ml rhFGF2 (R&DSystems, Cat# 233-FB), 20 ng/ml rhEGF (R&D Systems Cat# 236-EG), 1X ITS+3 (Sigma, Cat# 1-2771 ), 1 % (v/v) FBS (HyClone, Thermo Fisher,Cat# SH30070.02), 1X GlutaMAX (Invitrogen, Cat# 35050-038), 1X Penicillin/Streptomycin (Sigma Cat# P0781) and 2.5 pg/ml Amphotericin B (Sigma, Cat# A2942). 2. Differentiation
NP medium phase, 7 days , 5% 02.
Medium NeuroCult N-SA (Stem Cell Technologies, Cat# 05753) supplemented with 10 ng/ml rhFGF2 (R&D Systems, Cat# 233-FB), 20 ng/ml rhEGF (R&D Systems Cat# 236-EG), 5 ng/ml rhSCF (R&D Systems, Cat# 255-SC-010/CF), 10 ng/ml rhNT-3 (R&D Systems Cat# 267-N3-005/CF), 1X ITS+3 (Sigma, Cat# I- 2771), 1 % (v/v) FBS (HyClone, Thermo Fisher,Cat# SH30070.02), 1X GlutaMAX (Invitrogen, Cat# 35050-038), 1X Penicillin/Streptomycin (Sigma Cat# P0781 ) and 2.5 pg/ml Amphotericin B (Sigma, Cat# A2942).
Sensory neuron medium phase, -18 days, 5% 02.
Medium NeuroCult N-SA (Stem Cell Technologies, Cat# 05753) supplemented with 10 ng/ml rhGDNF (R&D Systems, Cat# 212-GD-010/CF),10 ng/ml rhNT-3 (R&D Systems Cat# 267-N3-005/CF), 20 ng/ml rhBDNF (R&D Systems, Cat# 248-BD-005/CF), 20 ng/ml rhNGF (R&D Systems Cat# 256-GF-100/CF), 1 mM db c-AMP (Sigma, Cat# D0260), 1X ITS+3 (Sigma, Cat# 1-2771), 1 % (v v) FBS (HyClone, Thermo Fisher,Cat# SH30070.02), 1 X GlutaMAX (Invitrogen, Cat# 35050-038), 1X Penicillin/Streptomycin (Sigma Cat# P0781) and 2.5 pg/ml Amphotericin B (Sigma, Cat# A2942).
Results are shown in Figure 19 and shown in the following table:
ICC results
Figure imgf000045_0001
Brn3/CGRP
Brn3a positive cells (%) CGRP positive cells (%) Bm3a/CGRP positive cells (%)
99.6±0.4 97.2±0.4 97.2±0.4
Brn3/IB4
Brn3a positive cells (%) IB4 positive cells (%) Brn3a/IB4 positive cells (%)
98.1±1.1 93.4±1.1 93.4±1.1
Β(·η3/βΙΙΙ tubulin
Brn3a positive cells (%) βΙΙΙ tub positive cells (%) Brn3a/ plll tub positive cells (%)
98.1±1.1 98. 1.1 98.1 ±1.1
Figure imgf000046_0001
Bm3a is a transcription factor that is specific for sensory neurons. In vivo in adulthood, approximately one third of sensory neurons are large diameter neurons and express neurofilament heavy chain (NF200). They are CGRP negative and IB4 negative. About one third are small diameter nociceptive sensory neurons. They are IB4 positive and CGRP negative. Isletl plays a central role in the transition from sensory neurogenesis to subtype specification (Sun Y, Nature Neurosci, 1 1 : 1283 - 1293 (2008). The data indicate that hEPI- NCSC can be differentiated efficiently into cells that express sensory neuron markers, both for large diameter sensory neurons and small diameter sensory neurons.
Sensory neurons obtained in this way could be used in the treatment of peripheral neuropathies.
The inventors are also investigating similar methods to direct differentiation of hEPI-NCSC into sympathetic neurons (useful in the treatment of autism and for modeling neuroblastoma) and cholinergic neurons (useful in the treatment of Alzheimer's disease (AD) as the nucleus basalis of Meinert, which contains cholinergic neurons, degenerates in AD). Factors and other additives that can be added to differentiate cholinergic neurons include the following.
NGF BDNF CNTF NT-3
B-27 supplement
N2 supplement
bFGF
BMP9
Retinoic Acid
Forskolin
Directed differentiation of human epidermal derived-NCSC's (hEPI-NCSC's) into melanocytes (pigment cells)
Melanocytes are cells located in the bottom layer of the epidermis (the stratum basale), the inner ear, the meninges, bone, the heart, and the hair, where they produce the pigment melanin that is responsible for the individual coloration of hair and inner organs and for hearing. One aspect of the present invention is a method for the selective differentiation of human epidermal neural crest stem cells (hEPI-NCSCs) into melanocytes with suggested potential for application in the fields of bio-engineering, and particularly tissue repair, skin replacement, skin rejuvenation, and/or feature augmentation. It is envisaged that melanocytwes obtained using the described methods can be used in artificial skin for the treatment of burn wounds. Melanocytes protect from UV damage from sun light. The presence of mealnocytes in artificial skin is desirable especially for non-white individuals. Isolation and expansion of hEPI-NCSCs
Isolation and expansion methods were carried out substantially as described previously, however are described again here for completeness.
In order to obtain highly pure populations of hEPI-NCSC the inventors took advantage of their predictable migratory behaviour. Cells with stellate morphology emigrated from bulge explants 6 - 10 days post explantation (Fig 2). Bulges from early and late anagen phase provided a high percentage of explants that released emigrating cells (Fig 2). Interestingly, the presence of the dermal sheath was essential for a high yield of bulge explants with emigrating cells. Bulge explants with dermal sheath resulted in hEPI-NCSC emigration from an average of 45.3±13.0% of cultured explants (Fig 2 A-C, F). In contrast, bulge explants devoid of the sheath yielded significantly fewer emigrating cells, whereas no cells emigrated from dermal sheath explants (Fig 2). The inventors have therefore developed a reliable and highly reproducible method for the culturing of adherent hEPI-NCSC from dissected adult hair follicle bulge explants. It uses a minimally invasive procedure and represents an abundant source of neural crest-derived stem cells.
Specifically, the bulge of adult human hair follicles were micro-dissected as described previously from pubic hairy skin. De-identified biopsies were obtained with ethical approval from consenting individuals undergoing repeat elective Caesarean sections. The Donor age bracket was 28 - 41 years. Briefly, hair follicles were dissected and mechanically cleaned of dermal and adipose tissues. The dermal papilla and matrix were removed and discarded, the bulge region excised, cut into 2-3 pieces and placed onto CellStart (Invitrogen, Paisley, UK Cat# A10142-01 ) coated 24-well or 35mm plates where they adhered to the substratum within one hour. The explants were incubated in a humidified atmosphere at 37°C, 5% C02 and 5% 02. To protect cells against oxidative stress, the inventors routinely culture neural crest stem cells at low oxygen tension. It should be noted that 5% oxygen does not constitute hypoxia for hEPI- NCSC, as hypoxia is defined as O2 tension below the normoxic value in a given tissue and oxygen tension in hair follicles ranges between 2.5% and 0.1 % O2. The Culture medium was NeuroCult XF (Stem Cell Technologies, Grenoble, France Cat# 05761) supplemented with 10ng/ml rhFGF2 (R&D Systems, Abingdon, UK Cat# 233-FB), 20ng/ml rhEGF (R&D Systems Cat# 236-EG), 1X ITS+3 (Sigma, Poole, UK Cat# 1-2771 ), 1 % (v/v) FBS (HyClone, Thermo Fisher, Cramlington, UK Cat# SH30070.02), 1X GlutaMAX (Invitrogen, Cat# 35050-038), 1X Penicillin/Streptomycin (Sigma Cat# P0781) and 2.5Mg/ml Amphotericin B (Sigma Cat# A2942). Four days post onset of emigration of hEPI-NCSC, bulges were removed with a bent tungsten needle and isolated by trypsinisation for sub- culturing. Briefly, for trypsinisation, cultures were rinsed with PBS-EDTA before addition of trypsin (Worthington Biochemical Corporation, Lakewood, NJ, USA. Cat# LS003703) at 500 pg/ml in PBS-EDTA. Trypsin treatment was stopped with trypsin inhibitor (Sigma Cat# T6522; 1 mg ml) that was dissolved in culture medium and the cell suspension collected.
The same culture medium was used for ex vivo expansion of hEPI-NCSC. The inventors have shown that hEPI-NCSC can be expanded into millions of stem cells without an overall significant loss of stem cell markers (Fig 9 A, B). Ex vivo expanded cells did not differentiate spontaneously but continued to express the neural crest stem cell molecular signature as well as SOX10 and NESTIN (Fig 9C). Notably, in vitro clonal analysis showed that the majority of in vitro expanded cells remain multipotent and thrive in clonal culture; 53.2±3.6% of clone-forming expanded cells generated clones that contained multiple cells types; 12.3±2.6% died an 34.5±3.0 stopped dividing. While early lineage markers were expressed in both cells in primary explants and in ex vivo expanded cells, they were not expressed at the protein level (see e.g., dopachrome tautomerase. The changes in gene expression levels observed in expansion culture are likely due to donor-specific differences, as due to technical issues the data for primary explants and ex vivo expanded cells were derived from tissue of different donors. On average, three million cells per bulge explant were obtained within 28 days. As illustrated in Fig 9B, expansion did not level off at 28 days and therefore, if desirable, could be continued for longer periods of time. Overall, the inventors show that hEPI-NCSC can be expanded ex vivo efficiently and reproducibly and that they retain sternness. An attractive feature of the expansion protocol is its short duration. Changes in the karyotype are thus of less of a concern in hEPI-NCSC than in cell lines that are passaged multiple times. The high numbers of stem cells that can be obtained through ex vivo expansion make testing in animal models of human disease and future applications feasible.
In some cases rather than subculturing the cells to expand them, the cells were either fixed with 4% paraformaldehyde (PFA) for indirect immunocytochemistry to allow for identification of, for example, pluripotency genes, or dissolved in TRIzol® (Invitrogen, Cat# 15596-018) for RNA isolation. The inventors had already defined a neural crest stem cell signature that is common and specific to mouse embryonic neural crest stem cells and mouse EPI-NCSC. One rationale for defining the molecular signature was to use it for the characterisation of human equivalent cells. The neural crest stem cell signature is expressed in hEPI-NCSC (Fig. 4). Other NCSC genes tested and detected include SOX10, SNAI2, TWIST1 , MS1 (Musashi) and p75NTR, thus corroborating the neural crest origin of hEPI-NCSC. Additional stem cell and the pluripotency genes SOX2, C-MYC, KLF4, LIN28, POU5F1/OCT4 and NANOG were expressed as well. Equally, the majority of hEPI-NCSC express the molecular signature genes at the protein level as determined by indirect immunocytochemistry (Fig 5). The neural crest stem cell marker SOX10 and the progenitor cell marker NESTIN were both expressed in all cells. Together, these observations confirm expression of pertinent markers and characterises the bulge-derived cells as neural crest-derived cells.
Clonal cultures Cells from primary explants were detached by trypsin treatment, seeded at clonal density (60 cells per 35mm plate) onto CellStart treated 35mm dishes and allowed to attach overnight. The next morning, single cells were identified and circled using a diamond tipped circle scribe (circle diameter 4mm). Single cells in circles that overlapped with another circle were excluded from analysis. Greater than 90% were single cells and circles overlapped rarely. Clones were then cultured in NeuroCult NSA (Stem Cell Technologies, Cat# 05752), 1X ITS+3, 1 % (v/v) FBS, 1X Gluta AX, 1X Penicillin/Streptomycin and 2.5Mg/ml Amphotericin B plus addition of specific growth factors for differentiation of multiple cell types for up to 42 days at 37°C, 5% C02, 5% 02.
In clonal culture, 67.1 ±3.2% of single cells formed a clone that consisted of highly proliferative cells. The remaining cells either died or formed clones consisting of 4-6 non-migratory cells with the flattened morphology of myofibroblasts. Figure 19 shows a primary clone. A single cell is shown that generated four daughter cells within 24 hours. By day 7 the clone consisted of hundreds of migratory cells, as seen by the changing shape of the clone. Clones were probed with cell type-specific antibodies in all possible permutations to show that they contained multiple cell types and thus represented multipotent cells (Fig 6B). The cell types were typical neural crest derivatives and included cells with immunoreactivity characteristic of myofibroblasts (smooth muscle actin), bone/cartilage cells (collagen type II; COL2A1 ), neurons (neuron-specific βΙΙΙ-tubulin [TUBB3], tyrosine hydroxylase [TH]), and Schwann cells (glial fibrillary acidic protein; GFAP). These observations show that multiple cell types can co-exist in the same clone and therefore provide proof that hEPI-NCSC are multipotent. Moreover, the data shows that under suitable culture conditions hEPI-NCSC can generate all major neural crest derivatives. Self-renewal is an important aspect of sternness. Self-renewal capability of hEPI- NCSC was determined by serial cloning. Primary clones were detached with trypsin and re-seeded at clonal density, which lead to secondary clones. The procedure was repeated to establish tertiary clones. Clone-forming ability was maintained at high levels, as 70.7±7.9% of secondary clones and 54.0±1 1.7% of tertiary clones consisted of fast-growing and motile cells. Double stains with cell- type specific antibodies showed that secondary clones contained multiple cell types as well. The presence of multiple cell types in secondary clones shows that hEPI-NCSC can undergo self-renewal. Taken together, the inventors have thus shown that hEPI-NCSC are multipotent stem cells. Differentiation into melanocytes hEPI-NCSC were differentiated in vitro into melanocytes by adding pertinent growth factors and reagents as described below. Before and after DOPA reaction, pigmented cells were clearly visible, with 65.7±6% of cells positive for melanin (Fig. 17). Real time PCR data (Fig 4) showed that prior to differentiation hEPI-NCSC already express already early lineage markers for melanocytes, including microphthalmia-associated transcription factor (MITF) and dopachrome tautomerase (DCT), which are important for development and function of melanocytes.
For in vitro differentiation of hEPI-NCSC into melanocytes, cells were treated with 100nM Endothelin-3 (Sigma Cat# E9137), 20nM Cholera Toxin (Sigma Cat# C8052), 16.2mM 12-0-tetra-decanoylphorbol-13-acetate (TPA) (Sigma Cat# 79346), for up to 17 days at 37°C, 5% CO2,10% 02. The DOPA reaction was performed to enhance the dark melanin hue. Briefly, cultures were fixed with 4% PFA for 20 min at RT, followed by three rinses with PBS and incubated at 37°C with 5mM DOPA (Sigma Cat# D9628) for three hours. Cells were post-fixed with 4% PFA for 20 min at RT, rinsed with PBS and visualised with bright field light microscopy. In summary, it is advantageous that hEPI-NCSCs are readily accessible in postnatal hairy skin and can be isolated by a minimally invasive procedure as described above. The cells are then expanded in culture to produce a relatively homogenous population of precursor cells that can then be directed to differentiate into melanocytes via culture in media which includes 16 nM of TPA/PMA (phorbol 12-myristate 13-acetate), 20 nM of cholera toxin, and 100 nM of endothelin-3. Phenotype is subsequently confirmed by melanin granule visualization after DOPA reaction under the microscope following 4 day incubation in vitro.
A preferred culture medium for differentiating of human epidermal neural crest stem cells (hEPI-NCSCs) into melanocytes is;
Pigment Cell Medium - 85% a-MEM, 10% FBS, 5% Chick Embryo Extract, 100nM Endothelin-3, 20nM Cholera Toxin, 16.2mM PMA/TPA, GlutaMAX, Amphotericin, Gentamicin
Figure imgf000053_0001
Notably a two-step sub culturing was not required and expanded cells can simply be cultured in the above media (usually for 7 days) to effect differentiation. Furthermore, the inventors have found that an appropriate amount of Went3a can replace the choleratoxin component.
Directed differentiation of human epidermal derived-NCSC's (hEPI-NCSC's) into osteoblasts/osteocvtes/bone cells.
The inventors have also directed differentiation of the isolated and expanded hEPI-NCSCs into sialoprotein and osteocalcin producing osteoblasts using the following culture medium. Again, a two-step sub culturing was not required, however 3D scaffolding was used to provide a support structure for the differentiated cells to grow. Neural crest cells give rise to various craniofacial bones. Developing protocols for the directed differentiation of hEPI-NCSC into bone cells is thus a step towards future applications of hEPI-NCSC in cell-based therapies.
Isolation and expansion of hEPI-NCSCs
This was carried out in the same manner as the isolation step used for differentiation of human epidermal derived-NCSC's (hEPI-NCSC's) described above.
Osteogenic Differentiation into bone cells
Cells were isolated by trypsinisation and seeded onto CellStart-treated 35mm plates at 2.5X103 cells per plate. Cultures were grown in AdvanceSTE osteogenic differentiation medium (HyClone; Thermo Fisher Cat# SH30877.KT), 1X GlutaMAX, 1X Penicillin/Streptomycin and 2.5pg/ml Amphotericin B, with 50/50 medium exchanges on alternate days. Cultures were incubated in a humidified atmosphere at 37°C, 5% CO2 and either 5% O2 or ambient air for up to 35 days. Alizarin Red S staining of fixed cultures was performed to detect deposition of calcium. Briefly, cultures were washed twice with PBS at RT, stained for two hours at RT with 2% (w/v) Alizarin Red S, pH 4.2 (Sigma Cat# A5533), followed by three PBS washes and then visualised with an inverted microscope. Medium - Thermo AdvanceSTEM Osteogenic Differentiation Medium, GlutaMAX, Amphotericin, Penicillin/Streptomycin
Figure imgf000055_0001
Figure 16 shows that bone cells are present after the culturing step by using 'Alizarin Red S' stain (marker for calcifying bone cells) to confirm the phenotype.
Low oxygen tension is the preferred way to expand neural crest stem cells and is currently an accepted culture technique for stem cells. By contrast, in published protocols other types of stem cell are differentiated into bone cells at ambient oxygen tension. The inventors therefore compared osteogenic differentiation at two different oxygen tensions, 5% 02 and ambient air. Cultures were analyzed as primary explants and at 7, 28 and 35 days of in vitro differentiation. Gene expression at the RNA level was determined by qPCR and at the protein level by indirect immunocytochemistry (Fig 16). Calcification was shown by histological stain with Alizarin Red S (Fig 16). Interestingly, transcripts of many osteogenic early lineage markers were already expressed by stem cells in primary explants. These included the essential early lineage genes RUNX2 and CBFB as well as osteocalcin, osteopontin, collagen type 1 and vitamin D receptor (Fig 16). Indirect immunocytochemistry showed intense perinuclear RUNX2 immunoreactivity by day 14 (Fig 16 A') with 49.0±2.9% and 58.4±5.0% cells positive in cultures at 5% O2 and at ambient oxygen tension, respectively (Fig 16 A"). Similar to the qPCR results, early high RUNX2 immunoreactivity at day 14 was followed by a decrease with progressive cell differentiation. Both qPCR and antibody staining for collagen type II alpha 1 (COL2A1 ), a marker of bone/cartilage differentiation showed an increase in expression at both 02 conditions over the culture period (Fig 16 B). Similarly, COL2A1 immunoreactivity was not detected until day 14 with the expected punctuate staining pattern within the cytoplasm (Fig 16 B'). By day 35, COL2A1 expression levels were similar in both 5% and ambient O2 concentrations with 66.7±8.7% and 68.4±4.3% positive cells respectively (Fig 16 B"). Transcripts for osteopontin (SPP1), the main phosphorylated glycoprotein of bone were already present in primary explants and increased overtime in differentiation medium (Fig 16 C). At the protein level, osteopontin was significantly higher expressed in cells that were cultured at ambient oxygen compared to cells grown at 5% oxygen (Fig 16 C"). Osteocalcin (BGLAP) is a member of the Gla protein family and contributes to the non-collagenous matrix in bone. Like other bone-characteristic genes, osteocalcin transcripts were detected already in primary explants and expression levels increased with progressing time in differentiation culture (Fig 16 D). There was no significant difference in the percentage of cells with osteocalcin immunoreactivity between the 5% oxygen and ambient air culture conditions (Fig 16 D"). Core-binding factor subunit beta (CBFB) transcripts were already present at high levels in primary explants and remained relatively unchanged throughout the culture period at both oxygen tensions (Fig 16 E). qPCR for collagen type 1 alpha 1 (COL1A1) (Fig 16 F) showed expression at low levels in primary explants, but increased significantly to high expression levels by day 7 in both oxygen tensions. A significant difference in expression due to oxygen tension was seen at each time point. At the end point, expression levels were significantly higher in cells that were cultured at 5% oxygen. Unexpectedly, vitamin D receptor (VDR) transcripts were already present at detectable levels in primary explants (Fig 16 G) and remained relatively unchanged throughout the culture period with no significant difference in expression levels at 5% and ambient oxygen at the end of the culture period. Alizarin Red S staining, which is an indicator of calcium deposits, was intense in cultures at both 02 tensions (Fig 16 H' and H"). Overall, we show that hEPI-NCSC can be differentiated efficiently into bone cells in adherent culture. Whereas individual gene expression varied between 5% and ambient oxygen levels and often differed significantly, the overall trend favours culturing at ambient oxygen tension.
Improved differentiation of hEPI-NCSCs into bone cells was observed following primary culture and first subculture of cells in StemPRO® MSC SFM medium (Invitrogen). The bar charts below illustrate a comparison of RUNX2 and CBFB expression observed in experiments using primary culture and first subculture in NeuroCult XF® (EXP 1 ) or StemPRO® MSC SFM (EXP 2). The bar charts show Real-time PCR (RUNX2 relative expression, CBFB relative expression, VDR relative expression) and Immunocytochemistry (RUNX2 ICC, Osteopontin ICC and Osteocalcin ICC)
Real-time PCT results show RUNX2 and CBFB, two transcription factors essential for bone differentiation and expressed early in differentiation are more abundant in StemPRO MSC SFM medium.
Vitamin D receptor (VDR) is also more abundant at the RNA level In this medium.
Previous method (= EXP 1 )
Primary culture and 1st subcultures in "NeuroCult XF" medium; differentiation in Advance STEM medium
Improved) method(= EXP 2)
Primary culture and 1st subcultures in "StemPRO MSC SFM" medium; differentiation in AdvanceSTEM medium
Figure imgf000058_0001
Figure imgf000058_0002
Figure imgf000059_0001
Figure imgf000059_0002
OSTEOPONTIN ICC
Figure imgf000060_0001
DAY O DAY 7 DAY 14 DAY 28 DAY 35
OSTEOCALCIN ICC
Figure imgf000060_0002
AY O DAY 7 DAY 14 DAY 28 DAY 35
More cells express RUNX2, osteopontin and osteocalcin as determined by indirect immunocytochemsitry.
Analysis at the protein level showed that the key transcription factor RUNX2 was expressed at significantly higher levels than in experiment 1. It was also faintly detected in day 0 samples which may be attributable to the MSC medium, which may be "priming" the cells and maintaining them towards a progenitor like state. By day 7 of differentiation RUNX2 expression was seen in greater than 95% of cells. This RUNX2 expression was also seen in the nucleus in addition to cytoplasm throughout the culture period in experiment 2, thus showing the protein to be active. In experiment 1 , RUNX2 was only seen in the cytoplasm and surrounding the nucleus. Collagen type II expression was seen to have a delayed expression until day 14, similar to that seen in experiment 1 , which then declined between days 28 and 35. As collagen type II is also regarded as a marker for chondrocytes, this reduced expression at the protein level is an important observation. In contrast to experiment 1 , osteopontin was expressed at high levels from early in culture. A significantly higher level of expression was seen at all time points of the culture. Osteocalcin expression in experiment 2 was also seen earlier and to a higher level in experiment 2. Importantly, osteocalcin expression was also seen in the nucleus in experiment 2 whereas it had only been cytoplasmic in experiment 1 (Figure 20 shows culture day 7 stain).
Quantification of Alizarin Red S stain
(indicates calcification) Culture conditions EXP 2 in "StemPRO MSC SFM" medium
Figure imgf000061_0001
Alizarin Red S stained cells were dissolved overnight in 0.01 % Triton X-100; absorption measured at 492 nm. Figure 21 shows the time course of Alizarin Red stain (Culture days 0, 7, 1 , 28, 35). Overall, the increased expression of markers at RNA and protein levels suggest that the modified culturing of primary explants and initial sub cultures may have a beneficial effect. The rationale for using the different medium was to see whether using a mesenchymal stem cell medium would improve resultant differentiation into osteocytes compared to NeuroCult® XF medium. It seems that it may even be the case that the MSC medium primes the cells to a more progenitor-like state before differentiation.
Noradrenergic Differentiation of hEPI-NCSC
The inventors have also carried out work in relation to noradrenerg differentiation
After a growth period in NP medium, hEPI-NCSC are plated at clonal density into 350 mm dishes coated with or without fibronectin. Cells are maintained in a humidified incubator at 37°C with 5% C02 and 5% 02
Cells will be cultured for 6 days with "standard culture medium" containing:
• Neurocult-XF supplemented with
• 20 ng/ml bFGF,
· 2% B27 supplement,
• 35 ng/ml retinoic acid and
• 50μΜ 2-Mercaptoethanol
Then, standard culture medium will be supplemented with:
• 5μΜ Dibutyryl-cAMP / Forskolin
• 1 ng/ml BMP4 for 6 days more to promote sympathetic differentiation
Finally, culture medium will be change to standard culture medium supplemented with:
« 50 ng/ml NGF and
. 50 ng/ml NT-3
for the last 6 days to differentiate into noradrenergic neurons.
Cryopreservation
For future applications, it needs to be shown that ex vivo expanded hEPI-NCSC can be frozen, stored frozen, thawed and subsequently grown in culture again. We show here that this is indeed the case. Cells were frozen in 90% FBS, 10% DMSO in a Nalgene freezing container (Sigma Cat# C1562) to -80°C overnight and then transferred to liquid Nitrogen. Subsequently, cells were thawed and re- cultured. Trypan blue stain showed 87% cell viability after thawing and the cells continued to proliferate (Figure 18). We thus show that hEPI-NCSC can be frozen by inexpensive means and thawed again with high yield.
In Summary hEPI-NCSC have been shown by the inventors to have many desirable features, the sum of which makes them a highly attractive type of somatic stem cell. As remnants of an embryonic tissue, the neural crest, hEPI-NCSC have the well- recognized physiological ability to generate a wide array of cell types and tissues. This innate high level of multipotency, combined with the expression of pluripotency genes and efficient ex vivo expansion render this stem cell type conducive not only to autologous transplantation but potentially also to efficient reprogramming into iPS cells. hEPI-NCSC are readily accessible in the hairy skin by minimal invasive procedure. The patient's own hEPI-NCSC could therefore be harvested, expanded ex vivo and then used for autologous transplantation. The present invention provides benefits over the prior art by providing a range of effective and high yield culture methods for the differentiation of adult hEPI- NCSCs in vitro. Such differentiated hEPI-NCSCs hold significant therapeutic potential for the treatment of various neoplastic and non-neoplastic diseases including Parkinson's disease, multiple sclerosis, amyotropic lateral sclerosis (ALS), osteoarthritis, osteoporosis, stroke, and cerebellar degeneration, and various acute and severely debilitating trauma episodes such as spinal cord injury, head and neck injury, and severe burn and/or wound repair. hEPI-NCSCs are also very useful candidates for the study of and intervention in the ageing process, and hold significant potential for use as both genetic delivery vehicles and high throughput drug screening and/or drug discovery techniques, with the former being of specific relevance to the treatment of neurogenetic diseases.
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Stem Cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.
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Claims

Claims
1. A method for inducing or stimulating differentiation of a population of cells which includes the step of;
culturing a population of epidermal neural crest stem cells (EPI- NCSCs) in the presence of added reagents, said reagents selected to induce or stimulate differentiation of the cells into one or more predetermined, identifiable cell types.
2. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in Claim 1 wherein the predetermined, identifiable cell type is a neural crest derivative.
3. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in Claim 2 wherein the method is for inducing or stimulating differentiation of epidermal neural crest stem cells into one or more of dopaminergic neurons, dopaminergic neuronal progenitors, sympathetic neurons, sensory neurons, cholinergic neurons, melanocytes, Schwann cells, smooth muscle, and osteocytes, bone and/or cartilage.
4. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of the previous Claims wherein the epidermal neural crest stem cells are a substantially pure population of epidermal neural crest stem cells.
5. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of the previous Claims wherein the method is for inducing or stimulating differentiation of human epidermal neural crest stem cells.
6. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of the previous Claims wherein the method comprises the steps of; isolating anagen phase hair follicles from a subject
isolating the bulge area of hair follicles and placing it into adherent culture
isolating epidermal neural crest stem cells from the bulge explants sub-culturing the isolated cells in the presence of added reagents selected to induce or stimulate differentiation of the cells into one or more pre-determined, identifiable cell types.
7. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of the previous Claims wherein the added reagents are selected from the following groups;
(a) dibutyryl cyclic AMP (db c-AMP) whereby they become
immunoreactive for neuron-specific beta-Ill tubulin and tyrosine hydroxylase (TH); or
(b) TPA (12-0- tetra-decanoylphorbol-133-acteate) and/or PMA (phorbol 12-myristate 13-acetate), cholera toxin and endothelin- 3;
(c) BMP2; or
(d) NGF, TGF-P2 and forskolin; or
(e) Neuregulin-1 and CNTF.
8. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of the previous Claims wherein the method includes the step of expanding the population of isolated cells prior to the sub-culturing step.
9. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in Claim 8 wherein the expansion step comprises culturing the isolated epidermal neural crest stem cells in expansion media.
10. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in Claim 9 wherein expansion media comprises; proliferation media, FGF2, EGF, FBS, ITS+3, GlutaMAX ®,
Amphotericin, Penicillin/Streptomycin.
11.A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of the previous Claims wherein the isolated cells undergo a 2-step sub-culturing process, the first sub-culturing step being culturing the cells in a Neural Progenitor (NP) Mix and the second sub-culturing step being culturing the cells in a patterning factor mix.
12. A method as in Claim 1 for inducing or stimulating differentiation of epidermal neural crest stem cells, to obtain dopaminergic neurons or dopaminergic neuronal progenitors, wherein, the isolated cells are sub- cultured in a patterning factor mix.
13. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in Claims 1 1 or 12 wherein the Neural Progenitor (NP) Mix comprises;
FGF-2
EGF SCF NT-3
14. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of Claims 1 1 to 13 wherein the patterning factor mix comprises;
SHH ["sonic hedgehog"]
FGF-8
TGF-p2
GDNF [glial derived neurotrophic factor]
15. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in Claim 4 wherein the patterning factor mix further comprises;
db c-AMP
combinations of neurotrophic factors (NGF, BDNF, NT-3)
16. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of the previous Claims wherein the epidermal neural crest stem cells are obtained from the bulge of a hair follicle obtained from a subject.
17. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in Claim 16 wherein the subject is mammalian.
18. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of the previous Claims wherein the subject is human.
19. A method for inducing or stimulating differentiation of epidermal neural crest stem cells as in any of the previous Claims wherein the method further comprises the step of returning the differentiated cells into the subject.
20. A population of differentiated cells obtained by the method of any of Claims 1 to 19.
21. Cells obtained by the method of any of Claims 1 to 19 for the treatment of a disease state.
22. Cells obtained by the method of any of Claims 1 to 19 for the treatment of Parkinson's disease.
23. Cells obtained by the method of any of Claims 1 to 19 for the treatment of Alzheimer's disease.
24. Cells obtained by the method of any of Claims 1 to 19 for the treatment of autism.
25. Cells obtained by the method of any of Claims 1 to 19 for the treatment of peripheral neuropathies.
26. Cells obtained by the method of any of Claims 1 to 19 for the treatment of degenerative diseases.
27. Cells obtained by the method of any of Claims 1 to 19 for the treatment of skeletal abnormalities.
28. Cells obtained by the method of any of Claims 1 to 19 for the treatment of orthopaedics or bone fractures.
29. Cells obtained by the method of any of Claims 1 to 19 for the treatment of osteoarthritis or osteoporosis.
30. Cells obtained by the method of any of Claims 1 to 19 for the treatment of stroke.
31. Cells obtained by the method of any of Claims 1 to 9 for the treatment of acute and severely debilitating trauma episodes such as spinal cord injury, head and neck injury.
32. Cells obtained by the method of any of Claims 1 to 19 for the treatment of burns.
33. Cells obtained by the method of any of Claims 1 to 9 for the treatment of burns or wounds.
34. Cells obtained by the method of any of Claims 1 to 19 for the treatment of skin conditions.
35. Human epidermal neural crest stem cells (hEPI-NCSC) for use in the treatment of any of the diseases referred to in Claims 21 to 34.
36. Dopaminergic neurons or dopaminergic neuronal progenitors obtained by the method of any of Claims 1 to 19.
37. Artificial skin comprising melanocytes obtained by the method of any of Claims 1 to 19.
38. A culture media for the directed differentiation of hEPI-NCSCs into dopaminergic neurons or dopaminergic neuronal progenitors comprising;
SHH ["sonic hedgehog"]
FGF-8
TGF-P2
GDNF [glial derived neurotrophic factor]
39. A culture media for the directed differentiation of hEPI-NCSCs as in Claim 38, further comprising
db c-AMP; and
combinations of neurotrophic factors (NGF, BDNF, NT-3)
40. A culture media for the progression of hEPI-NCSCs to a neural stem cell like state comprising;
FGF-2
EGF SCF NT-3
41. A method of treating Parkinson's disease comprising administering a therapeutically active amount of said dopaminergic neurons or dopaminergic neuronal progenitors.
42. A method of treating Parkinson's disease comprising administering a therapeutically active amount of human epidermal neural crest stem cells.
43. A method of ex-vivo expansion of human epidermal neural crest stem cells comprising the step of;
culturing an isolated population of epidermal neural crest stem cells in expansion medium.
44. A method of ex-vivo expansion of human epidermal neural crest stem cells as in Claim 43 wherein the expansion medium comprises;
proliferation medium, FGF2, EGF, FBS (fetal bovine serum), ITS+3, GlutaMAX ®, Amphotericin.
45. A method of ex-vivo expansion of human epidermal neural crest stem cells as in Claim 44 wherein the expansion medium comprises antibiotics such as Penicillin and/or Streptomycin.
46. A method of ex-vivo expansion of human epidermal neural crest stem cells as in any of Claims 43 to 45 wherein the method is carried out at 5% oxygen.
47. A method of ex-vivo expansion of human epidermal neural crest stem cells as in any of Claims 43 to 46 wherein the method also includes the pre-steps of;
isolating early anagen hair follicles from a subject;
isolating the bulge area of hair follicles and placing it into adherent culture;
isolating epidermal neural crest stem cells from the bulge explants.
48. A method of producing a stable cell line of human epidermal neural crest stem cells in vitro by culturing an isolated population of epidermal neural crest stem cells in expansion media.
49. Expansion media for use in the methods of claims 43 to 48, comprising; proliferation media, FGF2, EGF, FBS (fetal bovine serum), ITS+3, GlutaMAX ®, Amphotericin.
50. Expansion media as in Claim 49 wherein the proliferation media is
NeuroCult XF ®.
51 . Expansion media as in any of Claims 49 or 50 comprising antibiotics such as Penicillin and/or Streptomycin.
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