Expansion and differentiation of neuronal precursor cells

A technology of precursor cells and neurons, applied in the field of preparation of neuron precursor cells, can solve the problems of not being applicable to human skin

Pending Publication Date: 2021-02-19
スキンツーニューロン ピーティーワイ エルティーディー
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, we have previously developed a method on adult canine skin (Valenzuela et al. (2008)), but this method is generally not applicable to human skin due to large inter- and intra-individual differences in hair follicle number and quality

Method used

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  • Expansion and differentiation of neuronal precursor cells
  • Expansion and differentiation of neuronal precursor cells
  • Expansion and differentiation of neuronal precursor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1 - Harvesting of Donor Tissue

[0126] Adult skin was harvested from the occipital scalp as follows: the occiput was shaved, disinfected and anesthetized before harvesting. Using a double-bladed scalpel, samples 10 mm wide by 3 cm long were taken circumferentially in an axial plane around the midline, full thickness up to the fat layer.

Embodiment 2

[0127] Example 2 - Incubation of Donor Tissue with Hair Cycle Regulators

[0128] Conditioning of donor skin tissue with refractory or pro-proliferative hair follicle regulatory factors for up to 100 hours increased the anagen:telogen ratio of the entire skin sample, as well as improved interfollicular synchrony. Thus, pretreatment of donor skin tissue with such factors improves final cell viability, yield and consistency. Incubation of the skin in the environment of supplemented Williams'E medium provides enhanced viability of hair follicles in situ ex vivo. Noggin or SHH as example refractory hair follicle cell cycle factors increases the anagen:telogen ratio and thus the final cell viability, yield and consistency. TGF-β2 as an example pro-proliferative hair follicle cell cycle factor increases the anagen:telogen ratio and thus improves the final cell viability, yield and consistency. The combination of Noggin, SHH and TGF-β2 in supplemented Williams'E medium provides h...

Embodiment 3

[0133] Example 3 - Mechanical treatment of skin and treatment with digestive enzyme reagents.

[0134] Mechanisms for skin dissociation combined with enzymatic digestion to release precursor cells from the extracellular matrix increase cell viability, yield, and consistency. An example combination is the Miltenyi Biotec gentleMACS Dissociator unit used in combination with the gentleMACS Enzymatic Digestion Kit, which together increase cell viability, yield and consistency.

[0135] Custom methods of cell filtration outside of proprietary instructions for use provide high cell viability, yield, and consistency.

[0136] 1. Prepare gentleMACS dissociating enzyme (volumes below are for 4 x 4mm skin fragments).

[0137] 2. Carefully mix buffer L (435ul) and enzyme P (12.5ul)

[0138] 3. Carefully mix Enzyme D (50ul) and Enzyme A (2.5ul)

[0139] 4. Add D / A mixture to L / P mixture in tube C

[0140] 5. Place 4 skin fragments in tube C containing the enzyme and screw on the cap...

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Abstract

The invention relates to preparation of neuronal precursor cells, compositions comprising same and therapeutic uses.

Description

technical field [0001] The present invention relates to the preparation of neuronal precursor cells, compositions comprising them and therapeutic uses. Background technique [0002] Reference to any prior art in the specification is not an acknowledgment or representation that the prior art forms part of the common general knowledge in any jurisdiction or that the prior art could reasonably be expected to be understood, regarded as Partially related and / or combined with other prior art. [0003] Pluripotent stem cells are the usual starting point for the de novo preparation of neurons—the expansion of cells in their original replicative state followed by directed differentiation into a mature neuronal phenotype. Pluripotent stem cells can be isolated from embryonic sources (i.e. embryonic stem cells) or adult stem cell reservoirs (i.e. adult stem cells such as mesenchymal stem cells) or, as recently discovered, by dividing mature differentiated cells (e.g. fibroblasts ) re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/079C12N5/0797
CPCC12N5/0623C12N5/0619A61K35/30C12N2501/41C12N2500/84C12N2501/15C12N2506/092A61K38/4826A61K38/4886A61K38/465A61K38/1841C12N2501/155C12N5/0618C12N5/0622C12N2501/11C12N2501/415C12N2533/52C12N2506/1376C12N2501/40C12N2509/00C12N2509/10
Inventor 迈克尔·巴伦苏埃拉汤姆·邓肯安·长
Owner スキンツーニューロン ピーティーワイ エルティーディー
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