EP-4281769-B1 - METHODS FOR MANUFACTURE OF NEURONAL PRECURSORS

EP4281769B1EP 4281769 B1EP4281769 B1EP 4281769B1EP-4281769-B1

Inventors

VALENZUELA, Michael
JOHNSON, ADAM

Dates

Publication Date: 20260506
Application Date: 20220121

Claims (15)

An improved method for the generation of neuronal precursor cells from a sample of hair follicles, the method comprising: - treating a sample of hair follicle cells in conditions enabling the transition of hair follicle cells to a growth phase, thereby forming a sample of conditioned follicles; - determining the glucose consumption of the conditioned follicles; - comparing the glucose consumption of the conditioned follicles to a reference glucose consumption profile representative of hair follicles from which viable HFNs were isolated; and/or comparing the glucose consumption of the conditioned follicles to a reference glucose consumption profile representative of hair follicles from which viable HFNs were not successfully isolated; - proceeding to treat the conditioned follicles in conditions enabling enrichment of the number of cells expressing neuronal lineage biomarkers, if the glucose consumption of the conditioned follicles is the same or higher than that of the reference glucose consumption profile representative of hair follicles from which viable HFNs were isolated; or proceeding to treat the conditioned follicles in conditions enabling enrichment of the number of cells containing neuronal lineage biomarkers, if the glucose consumption of the conditioned follicles is higher than that of the reference glucose consumption profile representative of hair follicles from which viable HFNs were not successfully isolated, thereby generating a composition of neuronal precursor cells from a sample of hair follicles.
The method of claim 1, wherein the conditions enabling enrichment of the number of cells expressing neuronal lineage biomarkers comprise a step of contacting the conditioned cells with one or more enzymes or subjecting the conditioned cells to mechanical treatment to dissociate the hair follicles from connective tissue and/or extracellular matrix present in the sample.
The method of claim 1 or claim 2, wherein the conditions enabling enrichment of the number of cells expressing neuronal lineage biomarkers comprise culturing the conditioned cells in culture medium and conditions for promoting the formation and expansion of neurospheres, optionally wherein the culture medium and conditions for promoting the formation and expansion of neurospheres comprises EGF and FGF2.
A method for determining whether source tissue is suitable for providing hair-follicle derived neural precursors (HFNs), the method comprising: - providing a reference glucose consumption profile from intact human hair follicles that have been demonstrated to generate a viable HFN cell line; - providing source tissue in the form of a sample of hair follicle cells; - determining the glucose consumption of the follicles in the sample; - comparing the glucose consumption of the follicles in the sample to the reference glucose consumption profile; - determining that the source tissue is suitable for providing HFNs if the glucose consumption of the follicles is the same or higher than that of the reference glucose consumption profile; or determining that the source tissue is likely not suitable for providing HFNs if the glucose consumption of the follicles is the lower than that of the reference glucose consumption profile.
A method for determining the likelihood that a sample of hair follicles comprises sufficient numbers of HFNs for isolation, the method comprising: - providing a sample of hair follicles; - determining the glucose consumption of the follicles in the sample; - comparing the glucose consumption of the follicles in the sample to a reference glucose consumption profile from intact human hair follicles from which HFNs were successfully isolated; - determining that the sample of hair follicles likely comprises sufficient numbers of HFNs for isolation, if the glucose consumption of the follicles in the sample is the same or higher than that of the reference glucose consumption profile; or determining that the sample of hair follicles likely does not comprise sufficient numbers of HFNs for isolation, if the glucose consumption of the follicles is the lower than that of the reference glucose consumption profile.
The method of any one of claims 1 to 5, wherein the sample of hair follicles comprises a sample of skin comprising hair follicles.
The method of any one of claims 1 to 6, wherein the sample of hair follicles is obtained from human scalp skin, preferably from midline occipital scalp skin.
The method of any one of claims 1 to 7, wherein the glucose consumption of the conditioned cells or cells in the sample is determined from intact (whole) hair follicles.
The method of any one of claims 1 to 8, wherein the glucose consumption of the cells is determined: within about 1 to about 24 hours, preferably between about 4 to 12 hours of collection of isolation or harvesting of the sample from a tissue donor; and/or prior to a step of dissociation of the hair follicles in the sample from connective tissue and extracellular matrix.
The method of any one of claims 1 to 9, wherein the reference glucose consumption profile is derived from one or more samples of hair follicles from which viable HFNs were successfully isolated.
The method of any one of claims 1 to 10, wherein the method further comprises the step of obtaining a reference glucose profile.
The method of any one of claims 1 to 11, wherein the sample comprising the hair follicle is contacted with a medium for supporting hair follicle cells prior to the step of determining the glucose consumption of the cells in the sample.
The method of claim 12, wherein the medium comprises Williams' Medium E; optionally wherein the medium is supplemented with one or more anti-refractory factors and/or one or more pro-growth factors, such as wherein the medium comprises insulin and/or sonic hedgehog.
The method of any one of claims 1 to 13, wherein the glucose consumption is: (i) determined or obtained from directly measuring the rate of glycolysis by the follicles; or (ii) determined indirectly and is inferred from one or more surrogate markers of glucose metabolism, optionally wherein the one or more surrogate markers of glucose metabolism is selected from: pyruvate, NADH, oxygen, lactate and ATP.
The method of any one of claims 1 to 14, wherein the glucose consumption profile is normalised relative to the number of cells in the hair follicle.

Description

Field of the invention The present invention is directed to methods for determining the likelihood that source tissue will be suitable for the generation of viable precursor cells. Related application This application claims priority from Australian provisional application AU 2021900142, Background of the invention A major challenge in the practical development of cell-based therapies is cellular variability. Such variability can arise at multiple levels and from multiple sources: biological variability within and between source donor tissue, variability in transfection methods or even basic reagents, batch-to-batch variability (from the same cellular motherstock), line-to-line variability (between donors), clone-to-clone variability (within a line from the same donor), and of course even cell-to-cell variability within a cell population or within the same cell over time. Theoretically, all such observed variance can be deconstructed into biological variability (which is intrinsic and hence cannot be eliminated), manufacture variability (which is by design and can be controlled and optimised) and technical error (which is also unavoidable but can be minimized). Regardless of the cell type or manufacture process, source- or donor start-tissue variability is a major and unaddressed challenge. This, in effect, introduces a systematic component of variation into the manufacturing process at the outset and contributes to variance in the final cellular product. Moreover, current cell manufacture methods do not sufficiently understand this form of variance in order to introduce quality controls over it. In other words, current technology does not understand what biological feature to measure in the source tissue, in order to quantify how different is this donor tissue to the usual or ideal, nor govern the process with a control procedure over the final product ("does this donor source tissue meet the relevant criteria?"). There is a need for the identification of biomarkers which are predictive of the suitability of source tissue for use in the generation of viable precursor cells. Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art. Summary of the invention The present invention is based on the discovery by the inventors of a predictive biomarker for the manufacture of human hair-follicle derived neuronal precursors based on source tissue. Accordingly, in a first aspect, the present invention provides a method for determining whether source tissue is suitable for providing hair-follicle derived neural precursors (HFNs), the method comprising: providing a reference glucose consumption profile from intact human hair follicles that have been demonstrated to generate a viable HFN cell line;providing source tissue in the form of a sample of hair follicle cells;determining the glucose consumption of the follicles in the sample;comparing the glucose consumption of the follicles in the sample to the reference glucose consumption profile;determining that the source tissue is suitable for providing hair-follicle derived neural precursors (HFNs) if the glucose consumption of the follicles is the same or higher than that of the reference glucose consumption profile. Further, the present invention provides a method for determining whether source tissue is suitable for providing hair-follicle derived neural precursors (HFNs), the method comprising: providing a reference glucose consumption profile from intact human hair follicles that have been demonstrated to generate a viable HFN cell line;providing source tissue in the form of a sample of hair follicle cells;determining the glucose consumption of the follicles in the sample;comparing the glucose consumption of the follicles in the sample to the reference glucose consumption profile;determining that the source tissue is likely not suitable for providing hair-follicle derived neural precursors (HFNs) if the glucose consumption of the follicles is lower than that of the reference glucose consumption profile. It will also be appreciated that the reference glucose consumption profile used may be from one or more samples of intact hair follicles which were shown to not be suitable for the generation of viable HFNs. Accordingly, in a further aspect, the invention provides a method for determining whether source tissue is suitable for providing hair-follicle derived neural precursors (HFNs), the method comprising: providing a reference glucose consumption profile from intact human hair follicles that did not generate a viable HFN cell line;providing source tissue in the form of a sample of hair follicle cells;determining the glucose consumption of the follicles in the sample;comparing the glucose co