WO2020165615A1 - Method of transformation of hair folicle cells into neurons, method and scale for estimating risk for onset of a particular brain disease - Google Patents

Method of transformation of hair folicle cells into neurons, method and scale for estimating risk for onset of a particular brain disease Download PDF

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WO2020165615A1
WO2020165615A1 PCT/HR2019/000001 HR2019000001W WO2020165615A1 WO 2020165615 A1 WO2020165615 A1 WO 2020165615A1 HR 2019000001 W HR2019000001 W HR 2019000001W WO 2020165615 A1 WO2020165615 A1 WO 2020165615A1
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cells
risk
scale
disease
hair
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PCT/HR2019/000001
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French (fr)
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Dinko MITRECIC
Min Suk Song
Jue Wang
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Omnion Research International D.O.O.
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Definitions

  • This invention provides an in vitro method of transformation of hair follicle cells into neurons, a method and a scale for estimating risk fin ⁇ onset of a particular brain disease.
  • the method according to the present invention comprises the steps of substantially non-invasive taking of a hair sample from a subject, measuring of a level of at least one biomarker of interest, analyzing of said biomaifcers by using the scale of the present invention and estimating with great precision of the possible risk of onset of brain diseases, including, but not limited to Alzheimer’ s disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, dementia with Lewy bodies and other.
  • the originality of this invention arises from obtaining of cells from a hair follicle, transforming said cells into neurons using said method, quantification of selected biomarkers, and then getting the numerical answer of the level of risk for onset of a particular brain disease based on a numerical difference of said biomarkers in at least two subsequent measurements.
  • the present invention also provides a kit to be used to transport the hair sample from the subject to the laboratory where the tests will be performed.
  • Brain diseases represent the largest burden of human society. Expenses of care for patients affected by brain diseases are three times larger than treatment for all patients with all malignant tumors together. Even more, while incidence and prevalence of many diseases decrease and efficiency of their treatment increases, ratio of people affected by incurable brain diseases is growing. Of many brain diseases, the most critical is dementia linked to age, because it is predicted that humans will continue to increase average years of life. This means that number of people affected by various types of dementia will continue to grow almost exponentially. All projections predict that if something will not change, already in 20 to 30 years from now, majority of Western countries will face enormous problem of lack of manpower to take care of large number of people affected by dementia.
  • Alzheimer's is the most common cause of dementia and it accounts for 75% of all dementia cases. It is very strongly connected to the age and it is much more present among old population. On the other hand, there is a visible increase in number of young people affected by this type of dementia. Approximately 200,000 Americans under the age of 65 have early onset Alzheimer’s disease. People affected by Alzheimer’s disease are faced by memory loss and decrease in majority of cognitive abilities which impairs their daily life. As Alzheimer's advances it leads to increasingly complex symptoms, including disorientation, mood and behavior changes. In many cases, there is no clear family risk for Alzheimer's, but every presence of family history increases risk of getting it in the old age.
  • APOE apolipoprotein E
  • This gene has several forms. One of them, APOE e4, increases a person's risk of developing the disease and is also associated with an earlier age of disease onset.
  • amyloid plaques abnormal clumps
  • tau tangled bundles of fibers
  • Those accumulations are made of beta amyloid and tau proteins, which are one of the biomarkers of this disease.
  • pathology of Alzheimer’s is much more complex: it is possible that someone has a large number of visible plaques and clinically early stage of disease and vice versa -there are many reported cases of people which have died in a very advanced stage of disease and post-mortally only few plaques have been found. This highly suggests that levels of those proteins need to be analyzed individually and from the perspective of their change over certain time interval.
  • WO2011144901A1 discloses directed differentiation of epidermal neural crest stem cells, and more specifically human epidermal neural crest stem cells (hEPI- NCSC).
  • US publication US20180140637A1 describes compositions, kits, and methods for generating Schwann cells from epidermal neural crest stem cells (EPI-NCSC), Such EPI-NCSC can be obtained from the bulge of hair follicles.
  • a method of generating Schwann cells of US20180140637A1 comprising: contacting one or more epidermal neural crest stem cells (EPI-NCSC) with a first differentiation culture medium comprising b-mercapto ethanol, SB431542, and a first amount of fetal bovine serum for 1-3 days to generate a first cell population; contacting the first cell population with a second differentiation culture medium comprising all-trans-retinoic acid, SB431542, and the first amount of serum for 1-4 days to generate a second cell population; and contacting the second cell population with a third differentiation culture medium comprising all-trans-retinoic acid, SB431542, fetal bovine serum, fibroblast growth factor-2, a platelet derived growth factor-BB, neuregul
  • WO2011144901 A 1 declares that 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, KJLF4, SOX2, LJN28, OCT-4/POU5F1 and NANOG, cells Isolated from a hair follicle according to an in vitro method of the present invention express NES, SOX2, MSX2, LRIG1, VARS2, ENAH, MYOIO, SORB S3 and PFN1, but does not comprise MYC, KLF4 and POU5F1.
  • our two-step in vitro method comprises early and late precursors and finally gives rise to unique homogenous neuronal cell population with 100% cells expressing DCX, TUBB3, MAP2 and SYP.
  • Levels of produced beta amyloid, which is crucial for our scale are, in different parallel clones within 5% of difference.
  • the main object of this invention was to develop a simple, a non- invasive approach which allows anybody to provide between 10 and 20 plucked hairs, send them by a regular post in a kit suitable for transporting plucked hair from the subject to the laboratory where the tests will be performed and to get an answer about the current state of the risk of developing a clinically active brain disease.
  • This includes Alzheimer's, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscle atrophy and other similar neurodegenerative or neurovascular diseases.
  • a method of the present invention is based on obtaining of homogenous population of neurons from a small population of cells between hair root bulge and surface of the skin. Different from some other known methods which use heterogenous phiripotent stem cells from hair, which are giving various neural crest derivatives, a method of the present invention is based on homogenous nestinpositive population with extraordinary efficiency in obtaining neurons. Our approach is simple, robust, non-invasive, faster and most importantly - much more precise (numerically supported) in comparison to other approaches.
  • the present invention provides an in vitro method of transformation of hair follicle cells into DCX, TUBB3, MAP2 and SYP positive neurons which is performed by combination of decreased oxygen (hypoxia), decreased pH and with specific peptides.
  • This altogether forms an innovative in vitro method of transforming hair follicle cells into neurons.
  • One of the major advantages of our method is in bypassing the transformation over a pluripotent stem cell types (IPSC).
  • in vitro method of this invention yields very specific cell sub-populations which allow obtaining homogenous neuronal populations without unwanted cell subpopulations (for example, cells with a strong expression of oncogene or markers for glia or any other unwanted cell populations). In this way we make the method more robust, reproducible and we eliminate the risk of uncontrolled growth of cells, which impairs their application in diagnostic and prognostic purposes.
  • US publication US2013034858A1 provides a method for detecting onset of or the risk of development of, a protein misfolding disease, and a method for predicting the age of onset of a protein misfolding disease using nerve cells derived from iPS cells.
  • the method of US2013034858A1 comprising the steps of establishing iPS cells from somatic cells derived from the test subject; inducing differentiation of said iPS cells into nerve cells; measuring the amount of a causative protein, or the activity or the expression level of an enzyme involved in degradation of the causative protein in said nerve cells; and comparing said measured value with the amount of the causative protein in control cells, or with the activity or the expression level of the enzyme involved in degradation of the causative protein in control cells.
  • the present invention is based on a scale of the risk for certain decade of life which is based on a numerical difference in a level of at least one biomarker detected in two subsequent measurements, thus it is personalized for each subject.
  • the innovative element which is in the core of this invention and which allows precise numerical estimation is detection of the level of at least one biomarker of interest in 1- or 2-years* time interval distance, which then, interpolated to the scale according to the present invention provides precise levels of risk of onset of a particular brain disease.
  • our scale is numerical and gives a precise information of the risk in categories below 20%, 20-40%, 40-60%, 60-80% and above 80%, within 3 years* time interval.
  • the scale of the present invention is relative with time perspective and may be personalized for each tested subject, and more generally the scale is categorized according to the decade of life of the tested subject SUMMARY OF THE INVENTION
  • the present invention relates to an in vitro method of transformation of hair follicle cells into neurons, a method and a scale for estimating of the risk for onset of a particular brain disease.
  • Said methods are not limited only for estimating of the risk for onset of a particular brain disease, but may be used for diagnosing of a particular brain disease or for follow up of the existing brain disease.
  • the present invention provides an in vitro method of transformation of hair follicle cells into neurons comprising the steps of: plucking and sending a hair sample to a laboratory in a transporting medium; isolating of cells from a hair follicle suitable for obtaining an early neural precursors cells; transformation of the early neuronal precursors cells into a late neuronal precursors cells; wherein transformation of the early into the late neuronal precursors cells comprises steps of: cultivating the isolated early neuronal precursor cells in a transformation culturing medium for the first 24 hours in a low ambient oxygen condition followed by applying a transient cycles of hypoxia and acidosis; cultivating the late neural precursors in a proliferation medium in a low ambient oxygen condition; and differentiation of the late neural precursors on Poly-D-lysine and laminin coated surface in a differentiation medium. Neurons generated by the in vitro method are positive for DCX, TUBB3, MAP2 and SYP.
  • the present invention provides a method of estimating of the risk for onset of a particular brain disease, diagnosing of particular brain disease or for follow up of the existing brain disease by using neurons obtained according to the in vitro method comprising the steps of: plucking and sending a hair sample to a laboratory in a vial containing a transporting medium; measuring of a level of at least one biomarker of interest, wherein said measuring is performed at least two times in a given time interval; and applying a scale, the scale is based on two subsequent measurements of the level of at least one biomarker, wherein the scale is function of a numerical difference between measured levels of at least one biomarker in said two subsequent measurements, wherein if more than one biomarker is measured, the numerical difference relates to the same biomarker for which a separate scale is established.
  • the scale is available to a very wide human population, and more importantly, the idea is to perform testing of a subject, in at least two subsequent measurements in a time interval ranging from 1 to 2 years, which results will detect levels or features of biomarkers and then provide in a form of the scale an information with great precision about the current risk of developing active brain disease.
  • methods and the scale of the present invention may be used for diagnosing of a particular brain disease or for follow up of the existing brain disease by using neurons obtained according to the in vitro method of the present invention.
  • the present disclosure provides an in vitro method of transformation of hair follicle cells into neurons.
  • the present disclosure also provides an early neuronal precursors cells obtained by the in vitro method which are positive for NES, SOX2, MSX2, LRIG1, VARS2, ENAH, MYOIO, SORBS3 and FFN1, but does not comprise MYC, KLF4 and POU5F1.
  • the present invention provides a late neuronal precursors cells which are positive fin ⁇ NES and TBR1.
  • the present disclosure also provides a method and a scale for estimating of the risk for onset of a particular brain disease, diagnosing of particular brain disease or for follow up of the existing brain disease by using neurons obtained according to the in vitro method.
  • the main problem with brain diseases is that train is not accessible using usual diagnostic procedures.
  • the brain tissue is hidden behind the blood brain barrier.
  • the major problem which present invention solves is to provide the in vitro method of transformation of hair follicle cells into neurons which mirror neurons in the brain of a tested subject.
  • our method uses cell population obtained from a small region in the hair, which differs from other reported cell populations.
  • the most innovative element of this invention is the scale based on detection and measurement of levels of beta amyloid proteins in human population which was analyzed separately in their 5th, 6th, 7th and 8th decade of life. More than a decade of meticulous follow of up of levels of those proteins, based on following families with high risk of onset of Alzheimer’s disease (familial disease forms), but also based on genetically non-peculiar families and discovering sporadic spontaneous onset of disease, allowed us to create the scale.
  • the scale is based on an incremental increase or decrease of a level of at least one biomarker of interest, wherein the incremental increase or decrease is a numerical difference of the measured level of at least one biomarker between two subsequent measurements.
  • the numerical difference is expressed in the scale as a percentage change in relation to the preceding measured level of biomarker, wherein the increase or decrease of the subsequent measured level of biomarkers expressed in percentages is said percentage change.
  • the scale gives a precise insight in the current status in the terms of a change of at least one biomarker between two subsequent measurements.
  • Subsequent measurements may be measurements between a first and a second measurement, the second and a third measurement and so on.
  • the first measurement refers to the measurement of the level of at least one biomarker that occurred for the first time.
  • the scale is relative with a time perspective and is categorized according to the decade of life of the tested subject
  • the invention described here is based on detection of a time interval in which a significant increase or decrease is occurring, thus giving a numerical value of the risk for onset of a particular brain disease.
  • Fig. 1 illustrates a tip of the plucked human hair - hair follicle
  • Fig. 2 illustrates a part of a hair root from which the cells from a hair follicle are selected, namely an upper portion of a hair follicle, above a main bulb, near the connection with a sebaceous gland;
  • Fig. 3 is a higher magnification of a portion of a hair follicle which demarcates the upper border of the region from where we isolate cells;
  • Fig. 4 illustrates a hair follicle after treatment with enzymes Collagenase and Dispase for separation of an epithelial layer (root sheath) from the major portion of the hair;
  • Fig. 5 illustrates an elongated cell which migrate out the isolated (upper) portion of the hair follicle
  • Fig. 6 illustrates typical thick colonies formed 5-7 days after migration of a hair follicle cells
  • Fig. 7 illustrates cells isolated from the upper portion of the hair follicle where (A)-; are native cells, (B)-nuclei; and (C)- nestin positive signal of those cells;
  • Fig. 8 is a graph illustrating comparison of efficiency in obtaining neuronal precursors from 6 subjects, 3 males (age 23, 36 and 59) and 3 females (age 23, 35, 61);
  • Fig. 9 illustrates cells three days after they have been exposed to the transformation culturing medium
  • Fig. 10 illustrates Map2 expression in the protocol which includes two steps of neural precursors (A) and in the one which includes three steps of neural precursors (B);
  • Fig. 11 illustrates a vial filled with a transporting medium for sending hair samples
  • Fig. 12 is a graph illustrating comparison in efficiency of obtaining hair follicle cells after sending different hair samples in a vial, and processing some hair samples after three days when compared to samples which were processed after five days;
  • Fig. 13 is a graph illustrating a correlation of concentration of beta amyloid 42 and symptomatic stage of Alzheimer's disease
  • Figs. 14 to 16 are graphs illustrating comparison of history of follow up of 3 subjects with confirmed mutations and the history of familial Alzheimer's disease.
  • Fig. 17 is a graph illustrating an increasing risk scale in the 50s and 60 years of life for entering into a symptomatic stage of Alzheimer's disease.
  • Fig. 18 is a graph illustrating an increasing risk scale in the 70s and 80 years of life for entering into a symptomatic stage of Alzheimer's disease.
  • neural stem cell refers to an undifferentiated cell that can proliferate, self-renew, and differentiate into the adult neural cells of the brain. NSCs are capable of selfmaintenance (self-renewal), meaning that with each cell division, one daughter cell will also be a stem cell.
  • the non-stem cell progeny of NSCs are termed neural progenitor cells. Neural progenitors cells generated from a single multi potent NSC are capable of differentiating into neurons, astrocytes (type I and type P), and oligodendrocytes. Hence, NSCs are "multipotent” because their progeny has multiple neural cell stumps.
  • neural progenitor cell or “neural precursor cell” refer to a cell that can generate progeny such as neuronal cells (c.g., neuronal precursors or mature neurons), glial precursors, mature astrocytes, or mature oligodendrocytes. Typically, the cells express some of the phenotypic markers that are characteristic of the neural lineage.
  • a “neuronal progenitor cell” or “neuronal precursor cell” is a cell that can generate progeny that are mature neurons.
  • the term "isolated" with reference to a cell refers to a cell that is in an environment different from that in which the cell naturally occurs, e.g., where the cell naturally occurs in a multicellular organism, and the cell is removed from the multicellular organism, the cell is "isolated.” This usually means that the cell is“isolated” from its original natural environment (organism) and placed into an artificial environment.
  • the artificial environment usually means a medium for cell growth and/or differentiation. This is usually referred as“in vitro cell cultivation”.
  • murines rats, mice
  • felines felines
  • ungulates e.g., equines, bovines, ovines, porcines, caprines
  • the term“level of biomarket” refers to a, quantity or status of molecule which can be protein, peptide, lipid, metabolite, nucleic-acid based molecule (for example, RNA or DNA), then marker based on physiological functions like cellular action potential, influx and efflux of certain molecules, status of mitochondria, status of microtubules, status of ribosomes or combinations thereof.
  • the biomarker of interest refers to a molecule such as a protein, a peptide, derivative of tyrosine, derivative of cholesterol that is deposited outside the cells or a molecule that accumulates inside the cells.
  • examples of the causative protein include, but are not limited to, amyloid beta and tau.
  • the present disclosure provides an in vitro method of transformation of hair follicle cells into neurons from human subjects in a very simple, fast, efficient and non-invasive way.
  • Neurons obtained by said method are used to get a quantified, precise estimation of the risk to enter a symptomatic phase of brain disease, for example Alzheimer’s disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy and other neurodegenerative or neuroinflammatory disease within the 3 years* time interval from the moment of taking a very first hair sample.
  • the in vitro method generally comprises steps of; plucking and sending a hair sample to a laboratory in a transporting medium; isolating of cells from the hair sample suitable for obtaining of an early neural precursors cells; transformation of the early neural precursors cells into a late neuronal precursors cells, wherein transformation of an isolated early neuronal precursors cells into a late neuronal precursor cells comprises steps of: cultivating the isolated early neuronal precursor cells in a transformation culturing medium for the first 24 hours in a low ambient oxygen condition followed by applying a transient cycles of hypoxia and acidosis; cultivating of the late neural precursors cells in a proliferation medium in an low ambient oxygen condition; and differentiation of neural precursors cells into neurons on Poly-D-lysine and laminin coated surface in a differentiation medium.
  • Isolating of cells from the hair sample comprises steps of immersing the hair sample in a culture dish comprising Collagenase and Dispase enzymes for 12 hours at 4 °C, followed by rinsing in Phosphate buffered saline and isolating of upper portion of the hair root, above the main bulb, near the connection with sebaceous gland.
  • the transporting medium comprises DMEM supplemented with Penicillin/Streptomycin, anti-fungal nystatin and anti-mycoplasma.
  • An isolated cells are immersed in DMEM supplemented with Penicillin/Streptomycin and anti-fungal nystatin and adhering the isolated cells to a culture dish treated by fibronectin.
  • the transformation culturing medium comprises Opti-MEM, B27, N2, BDNF (all Gibco), Penicillin/Streptomycin, and peptides Forskolin, DNP-derived active peptide, NAP, CHIR99021, LIF, and FGF2. It has been found that applying of low ambient oxygen condition for cultivating the isolated cells provides better efficiency m their proliferation and survival. In addition, applying of transient cycles of hypoxia and acidosis for cultivating the isolated cells resulted in higher rates of transformation from early to late neuronal precursors. Therefore, the in vitro method comprises cultivating the isolated cells in the transformation culturing medium for the first 24 hours in an ambient oxygen condition of 10% oxygen followed by applying a transient cycles of hypoxia and acidosis.
  • the proliferation medium comprises DMEM/F-12, B-27 Supplement, N-2 Supplement, Penicillin/Streptomycin, FGFb and EGF.
  • the neural precursors cells are differentiated into neurons on Poly-D-lysine and laminin coated surface in the differentiation medium, the differentiation medium comprises Neurobasal and Penicillin/Streptomycin with glutamine.
  • a time frame within which occurs transformation of hair follicle cells into neurons according to said in vitro method is 20 days.
  • a subject neurons generated by the in vitro method are positive for DCX, TUBB3, MAP2 and SYP.
  • Said neurons transformed from a hair follicle cell of a subject are used in a method of estimating the risk for the onset of a particular brain disease, diagnosing of a particular brain disease and for following up on an existing brain disease.
  • the subject is a human subject
  • the brain disease is selected from the group consisting of, including but not limited to, Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, and dementia with Lewy bodies and other neurodegenerative or neuroinflammatory diseases.
  • a late neuronal precursors cells generated by the in vitro method are positive for NES and TBR1.
  • present invention provides a vial of 2 ml comprising a transporting medium (see fig. 11).
  • a simple drawing is provided in which it is shown that forceps is needed in order to wrap a hair (one by one) around forceps and then pull it out applying a sudden movement
  • the tip of the hair which was pulled out of the subject’s scalp has to be immersed in the transporting medium, while the opposite end can be cut (depending on the length of the individual hair) or, which is also possible, can be let to protrude outside when the vial is closed by complete insertion of the screw cap.
  • Number of pulled hairs is preferably in a range between 10 and 20.
  • the transporting medium comprises DMEM (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA) supplemented with Penicillin/Streptomycin (Gibco by Life Technologies), anti-fungal nystatin (Gibco by Life Technologies) and anti-mycoplasma (Gibco by Life Technologies).
  • PBS Phosphate buffered saline
  • DMEM Phosphate buffered saline
  • Penicillin/Streptomycin Gibco by Life Technologies
  • anti-fungal nystatin Gibco by Life Technologies
  • Small pieces of the isolated tissue adhere to the surface treated by fibronectin within 1 h.
  • Preferred incubation parameters are 37 °C, 5 % CQi and 10 % (O 2 .
  • the early neuronal precursors cells are positive for NES, SOX2, MSX2, LRIG1, VARS2, ENAH, MYO10, SORBS3 and PFN1, but does not comprise MYC, KLF4 and POU5F1.
  • Table 1 bellow shows expression of genes which define a population of early neuronal precursors cells isolated from the upper portion of the hair bulb.
  • the late neural precursors cells are cultured in a proliferation medium in an ambient oxygen condition of 5% oxygen.
  • a time frame for cultivation said cells depends on required number of cells, preferably between 3-7 days.
  • the late neuronal precursor cells are positive for NES and TBR1.
  • Isolated late neuronal precursor cells are cultivated in a proliferation medium comprising DMEM/F- 12 (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA), B-27 Supplement (Gibco by Life Technologies), N-2 Supplement (Gibco by Life Technologies), Penicillin/Streptomycin (Gibco by Life Technologies), 20 ng/mL Recombinant Mouse Fibroblast Growth Factor-basic (FGFb, ThermoFisher Scientific, Pittsburgh, PA, USA) and 20 ng/mL Recombinant Mouse Epidermal Growth Factor (EGF, ThermoFisher Scientific).
  • the late neuronal precursor cells are cultivated in suspension, and after two days neurospheres are formed.
  • neurospheres are dissociated and then obtained single late neuronal precursors cells are plated on 12-mm coverslips (200 to 250 000 cells/coverslip) as well as on 6-well plates (1 000 000 cells/well). Coverslips and wells were previously coated with Poly-D-lysine (500 mg/mL, 24h at 37°C, Sigma) and laminin (10 mg/mL, 24h at 37°C, Sigma). 24 hours after plating, the medium is changed into a differentiation medium comprising Neurobasal (Gibco by Life Technologies) which supports neurons in vitro and antibiotic with glutamine (Pen Strep Glutamine, Gibco by Life Technologies).
  • Table 3 below shows expression of genes which define differentiated neurons obtained by the in vitro method of fee present invention.
  • Amphotericin B (Thermo Fisher Scientific, catalog number: 15290026)
  • Dulbecco s modified Eagle medium (DMEM) high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11960044)
  • the claimed method can be used to obtain early neuronal precursors cells from a human hair follicle.
  • Fig. 1 illustrates a tip of the plucked human hair - hair follicle. If pulled out by the fast movement, it regularly contains a whole follicle which ends with the hair bulb. In this figure, the hair root sheath is also well visible.
  • the length of here-shown follicle is 2 mm.
  • Fig. 2 illustrates an upper portion of the hair follicle which we isolate during the claimed protocol. Tip of the arrow is showing the region from which the majority of our cells are originating.
  • the length of the here-shown part of the follicle is 350 mm.
  • Fig. 3 is a higher magnification of a portion of a hair follicle which demarcates the upper border of the region from where we isolate cells claimed in this protocol.
  • Tip of the arrow is marking a junction point of the smooth muscle.
  • a region rich with cells we are isolating is just beneath this segment
  • an upper portion of the hair root, above the main bulb, near the connection with sebaceous gland was cut and then immersed in Collagenase and Dispase (Roche, Fisher Scientific), using 35 -mm cell culture dish (Thermo Fisher Scientific) and left at 4°C for 12 hours (overnights).
  • Fig. 4 illustrates a hair follicle after treatment with enzymes Collagenase and Dispase for separation of an epithelial layer (root sheath) from a major portion of a hair sample.
  • the region rich with cells used in subject method is marked by circle.
  • FIG. 5 illustrates an elongated cell which migrate out the isolated (upper) portion of the hair follicle. They very strongly attach to the fibronectin-coated surface and migrate around the pieces of follicle.
  • human recombinant epidermal growth factor Merck, catalog number GF144
  • Fig. 6 shows typical thick colonies formed 5-7 days after migration of the hair follicle cells. Cells are not anymore so elongated and spindle shaped, but slowly became more rounded. They exhibit high stability in the cultivation medium and very low cell death, less than 10%. Pieces of hair tissue were removed, cells were detached from the surface using trypsin and then counted using counting chambers.
  • Fig.7-(A) illustrates cells isolated from the upper portion of the hair follicle.
  • Fig. 7- (B) illustrates cells obtained five days after start of the cultivation.
  • Fig. 7 (C) illustrates stem cells which are nestin positive. It revealed that isolated early neuronal precursors cells express nestin - early marker of neuronal precursors.
  • Fig. 8 is a graph illustrating comparison of efficiency in obtaining neuronal precursors from 6 volunteers - 3 males (age 23, 36 and 59) and 3 females (age 23, 35, 61) obtained from 5 hairs after 5 days. Although no statistically significant difference was observed in regard to age or sex, there was a slight tendency to obtain more cells in volunteers of younger age. Average number of obtained early neuronal precursors cells from total of 5 hairs after 5 days was 51.000ti.200.
  • the claimed method may be used to obtain early neuronal precursors cells from human hair root
  • 6 volunteers, 3 males and 3 females we have shown show that a sufficient number of early neuronal precursors cells is obtained regardless of the age and sex.
  • Example 2 Comparison of efficiency of transformation of early neuronal precursors cells obtained from hair follicle cells into late neuronal precursors cells bv using hvpoxia and normoxia
  • Isolation of cells from different body regions can yield different results.
  • different protocols can differ in efficiency to obtain a sufficient number of cells. Protocols usually include description of specific surfaces needed for cell attachment, specific media and specific components which help to obtain needed type of cells.
  • Our method is based on transformation medium which contains specific components needed to transform selected hair follicle cells into neurons.
  • cells are exposed to cycles of hypoxia and acidosis. We have shown that combination of hypoxia and acidosis support transformation of cells into neurons.
  • Hair follicle cells obtained according to our protocol for isolation of cells were cultivated in a low ambient oxygen conditions (10% oxygen) for 24 h in DMEM (Gibco by Life Technologies, Thermo Fisher Scientific, Pittsburgh, PA) and Penicillin/Streptomycin (Gibco by Life Technolopes). Then there were transferred into a transformation culturing medium.
  • the transformation culturing medium is modified cultivating medium comprising Opti-MEM (Gibco by Life Technologies, Thermo Fisher Scientific, Pittsburgh, PA) with components B27, N2, BDNF (all Gibco), Peniciliin/Streptomycin (Gibco by Life Technologies) in which following transformation components have been added: Forskolin, 14 mM; DNP-derived active peptide, NAP, 1 mM, CHIR99021, 3mM, LIF,
  • hypoxia low ambient oxygen conditions
  • the transformation medium was exchanged with neuronal medium in both cell groups (normoxia and hypoxia).
  • the proliferation medium was DMEMZF-12 (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA), 20 ng/mL Recombinant Mouse Fibroblast Growth Factor- basic (FGFb, ThermoFisher Scientific, Pittsburgh, PA, USA) and 20 ng/mL Recombinant Mouse Epidermal Growth Factor (EOF, ThermoFisher Scientific) with B-27 Supplement (Gibco by Life Technologies), N-2 Supplement (Gibco by Life Technologies) and Penicillin/Streptomycin (Gibco by Life Technologies). 24 hours after plating, the medium was changed into a differentiation medium comprising Neurobasal (Gibco by Life Technologies) which supports neurons in vitro and antibiotic with glutamine (Pen Strep Glutamine, Gibco by Life Technologies).
  • Fig. 9 illustrates cells three days after they have been exposed to the transformation culturing medium. It can be seen that cells continue to form a shape between elongated spindles and rounded cells.
  • FIG. 10 illustrates comparison of Map2 expression, marker of differentiated neurons in the protocol where nestin precursors were directly transferred into the differentiation medium (A) and when additional step, with transformation using the transformation medium, hypoxia and acidosis is used (B).
  • A the differentiation medium
  • B hypoxia and acidosis
  • Example 3 Testing the potential to isolate hair follicle stem cells after sending them in a transporting medium
  • Fig. 11 illustrates the vial filled with the transporting medium. Every subject received a simple drawing in which it was shown feat they need to take forceps, wrap a hair (one by one) around it and then pull it out applying a sudden movement.
  • the tip of the hair which was pulled out of the scalp has to be immersed in the transport medium, while the opposite end can be cut (depending on the length of the individual hair) or, which is also possible, can be let to protrude outside when the vial is closed by complete insertion of the screw cap.
  • Subjects have been asked to pull out 10 hairs from their scalp and immerse them one by one into the vial filled with the transporting medium. We instructed them that after they collect 10 hairs, they can decide either to cut all ten hairs by scissors above the opening of the vial or just to close the vial by careful inserting the screw cap. We also suggested if the person has problems with fine coordination that this procedure should be carried out by a more skillful member of the household, a neighbor or a friend.
  • Fig. 12 is graph showing comparison in efficiency of obtaining hair follicle cells after sending them in the vial, with isolation occurring 3 and 5 days after sampling the hair.
  • Graph is showing efficiency in 10 subjects of different age (20-88), number of hairs they sent to us (4-12) and number of cells obtained.
  • One of the most important observations here is that the number of cells obtained 5 days after sampling is significantly lower than in the case when delay was 3 days.
  • a significantly larger number of cells was obtained in samples which were processed after three, when compared to samples which were processed after five days.
  • no significant differences were observed in expression of nestin, which was in both groups of samples rather homogenously expressed.
  • This invention comprises elements of measurement of levels of molecules for which we or others have shown that can be used as biomarkers. More precisely, in this invention we show in which way one needs to analyze levels of molecules (e.g. proteins) to get a reliable result
  • the quantity of tiie protein is measured using a lysate of cells cultured in above defined conditions.
  • tiie molecule of interest is the protein which can be detected only or preferably outside of cells, the total quantity of the protein found in the total medium is measured. Quantity of the protein of interest is normalized to the total quantity of detected proteins.
  • the biomarker is human beta amyloid 1-42 (Ab42). This is the fragment produced by cleavage of amyloid precursor protein (APP) by B- and y-secretase. This peptide is composed of 42 amino acid residues (AB42).
  • the biomarker of interest may be level of cellular excitability.
  • the biomarker of interest may be diffuse cytoplasmic accumulations of a-synuclein and Lewy body precursor inclusions.
  • assessment of the concentration of any biomarker from the medium can be obtained by methods routinely used, like ELISA, Western blotting, immunoprecipitation, quantitative immunocytochemistry, radioimmunoassay or similar methods. Concentration of the protein needs to be assessed in comparison to some of the house keeping genes, used as reference (e.g. GAPDH, HSP90, beta tubulin).
  • Neurons obtained by the present in vitro method are left 2 weeks to reach a mature stage. For all this time period, supernatant is constantly collected (3 times per week), centrifuged at 4 °C at 2,500 rpm for 7 minutes to remove the precipitate before use. To prevent protein degradation, 4-(2-aminomethyl) benzene-sulfonyl fluoride hydrochloride is added to the medium, which is stored at -80 °C till the use.
  • ELISA test For the purpose of detection of concentration of beta amyloid, our own antibody raised against amino acids 33-42 of Human beta Amyloid 42 is used. To measure levels of proteins, ELISA test can be applied. ..Sandwich ELISA“measures concentration of antigen between two layers of antibodies (usually called - capture and detection antibody). The antigen of interest typically contains two antigenic sites capable of binding two different antibodies.
  • the first step is composed of coating the 96 well plate with the capture antibody (cone 5 -20 mg/mL), in carbonate buffer pH 9.6. 12 hours, 4 C. After washing in phosphate buffer, blocking step using 5% non-fat dry milk, 2 hours in PBS is applied. After another washing round, the plate is ready to accept the peptide.
  • the goal of this experiment was to use method we developed and to test if it can be used to detect levels of beta amyloid in patients affected by Alzheimer’s disease.
  • the molecule of interest which is here declared as the biomarker is human beta amyloid 1-42 (Ab42). This is the fragment produced by -leavage of amyloid precursor protein (APP) by B- and y-secretase. This peptide is composed of 42 amino acid residues (AB42).
  • Alzheimer's disease For this purpose, we selected two patients clinically diagnosed is Alzheimer's disease. Subject 1, a female, age 75, with history of Alzheimer's dementia lasting for 4 years, with mini mental state score of 15 (moderate dementia). No other peculiar health problems. Subject 2, a male, age 77, with history of Alzheimer's dementia lasting for 5 years, with mini mental state score of 14 (moderate dementia). History of two heart infarctions and diabetes type 2.
  • the transformation culturing medium is comprising modified cultivation medium (Opti-Mem based) in which following components have been added: Forskolin, 14 mM; DNP-derived active peptide, NAP, I mM, CHIR99021, 3mM, LIF, 20 ngZml and FGF2, 20 ng/ml.
  • Neurons are left 2 weeks to reach a mature stage. For all this time period, supernatant is constantly collected (3 times per week), centrifuged at 4 °C at 2,500 rpm for 7 minutes to remove the precipitate before use. To prevent protein degradation, 4-(2-aminomethyl) benzene-sulfonyl fluoride hydrochloride is added to the medium, which is stored at -80°C till the use.
  • the first step is composed of coating the 96 well plate with the capture antibody (cone 5 -20 ng/mL), in carbonate buffer pH 9.6. 12 hours, 4 C. After washing in phosphate buffer, blocking step using 5% non-frit dry milk, 2 hours in PBS is applied. After another washing round, the plate is ready to accept the peptide. 100 mL of medium from the cell cultures is added, quadriplicates, 120 min, 37 °C. Washing step with phosphate buffer, 50 mL of detection antibody is added 2 h incubation at room temperature, Wash the well with phosphate buffer.
  • Fig. 13 shows that applying clamed in vitro method of transformation of hair follicle cells into neurons we can clearly distinguish between subjects affected by Alzheimer’s disease and healthy subjects.
  • Average concentration of beta amyloid 42 for two patients was 68 and 79 fM, while for healthy subjects of the same age was 24 and 16 fM.
  • the present invention further provides a method of estimating of the risk for onset of a particular brain disease by using neurons obtained according to the in vitro method previously described.
  • the method for estimating of the risk for onset of a particular brain disease may be used for diagnosing of a particular brain disease or for follow up of the existing brain disease by using said neurons. Said method provides a precise numerical value indicating what is the current risk of Mitering a symptomatic phase of brain disease within the next 5 years frame.
  • the method comprises the steps of: plucking at least two hair samples and sending each hair sample to a laboratory in a vial containing a transporting medium, wherein sending of each hair sample is performed in a predetermined time interval; measuring of a level of at least one biomarker of interest from neurons obtained from each hair sample; and applying a scale, the scale is based on two subsequent measurements of the level of at least one biomarker of interest, wherein the scale defines a risk ranges for the onset of a particular brain disease, the risk ranges are based on a numerical difference between two subsequent measurements of the level of at least one biomarker of interest, wherein if more than one biomarker is measured, the numerical difference relates to the same biomarker for which a separate scale is established.
  • the numerical difference of the measured levels of biomarkers is expressed as a percentage change, wherein to each risk range is assigned a corresponding range of the percentage change.
  • - the numerical difference - of the measured levels of biomarkers obtained in two subsequent measurements is said percentage change.
  • a corresponding range of the percentage change is assigned.
  • Said method can be applied for various brain diseases, including, but not limited to Alzheimer’s disease, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, dementia wife Lewy Bodies and other neurodegenerative or neuroinflammatoiy diseases. It can be applied for any disease in which the protein-biomarker is known and in which one can have a reliable scale for estimating the level of risk for developing the first symptoms.
  • the scale comprises 5 risk ranges based on the corresponding range of the percentage change of measured levels of biomarkers between two subsequent measurements.
  • the risk range of up to 20% indicates a very low risk
  • the risk range of 20-40% indicates a low risk
  • the risk range of 40-60% indicates a moderate risk
  • the risk range of 60-80% indicates a high risk
  • Each corresponding range of the percentage change is categorized according to the decades of life of a human subject.
  • a predetermined time interval between two subsequent measurements is in a range between 1 and 5 years, preferably in the range between 1 and 2 years. The predetermined time interval depends on the measured level of biomarkers measured for the very first time.
  • the scale of the present invention is relative with time perspective.
  • the method is based on detection of the time point in which a significant increase of measured levels of biomarker of interest occurs, thus giving a numerical value of the risk expressed in the scale.
  • said predetermined time interval depends on the measured level of biomarkers of interest at the first measurement.
  • Biomarkers are selected firm the group consisting of a protein, peptide, lipid, metabolite, nucleic-acid based molecule, cellular action potential, influx and efflux of certain molecules, status of mitochondria, status of microtubules, status of ribosomes or combinations thereof.
  • biomarker is beta amyloid 42, but it may be used for other brain diseases characterized by other biomarkers.
  • Example 5 Stratification of subjects according to the risk of being affected by Alzheimer's disease. based on levels of beta 42 amyloid obtained from the hair follicle and on developed risk scale
  • the first neurological exam revealed no abnormalities and the subject did not report any problems with memory, mood or any element of general functioning.
  • Neurologic examination did not reveal any focal neurologic deficit
  • Routine laboratory tests including blood chemistry, electrolyte, and urine analyses, were normal.
  • Thyroid function tests were normal.
  • Subject two S2
  • age 56 Asian
  • MRI imaging of the brain showed no peculiarities.
  • Subject two S2
  • age 56 Asian
  • Subject two S2
  • age 56 Asian
  • the first neurological exam revealed no neurological abnormalities.
  • Neurologic examination did not reveal any focal neurologic deficit. Routine laboratory tests, including blood chemistry, electrolyte, and urine analyses, were normal.
  • Thyroid function tests were normal.
  • Subject three S3
  • age 69 mated Hispanic Asian race
  • S3 Subject three
  • age 69 mated Hispanic Asian race
  • Another statement which could not be confirmed was that two close relatives were affected by dementia in their 60s.
  • Genetic testing which was initiated by the subject revealed mutation in SORL1, which was known for both sporadic and familial cases of Alzheimer's disease.
  • the first neurological exam (point 0, age 69) revealed weaker reflexes and a slight lateralization, which was later checked on CT scans. No morphological signs for any brain disease have been found.
  • Routine laboratory tests revealed a borderline anemia and borderline decreased levels of potassium.
  • Urine analyses revealed a serious infection, which was then treated.
  • Thyroid function tests revealed signs of autoimmune hypothiroidism. On Mini mental state exam, he scored 26 and he did not express any problems suggesting Alzheimer's disease.
  • Mini mental state exam revealed a change in S2 - the number of points decreased from 25 to 24.
  • Mini mental state exam revealed a dramatic change in S2 - the number of points decreased from 24 to 20, so he entered a state of moderate dementia.
  • Graphs in Figs. 14-16 show comparison of history of follow up of 3 subjects with confirmed mutations and the history of familial Alzheimer's disease.
  • Subject 1 has shown a moderate increase in levels ofbeta42 amyloid in the first and significant increase in the 2nd and 3rd year. He developed dementia in the 5th year, which means 4 years from the moment of detected increase of the used biomarker. In the following years after disease onset, levels of beta 42 amyloid woe changing, but not significantly and the mental state was constantly deteriorating.
  • Subject 3 exhibited a constant low level of the biomarker in 4 subsequent years. Then, in year 5 we observed the first more significant increase, which was followed by three more years of a significant increase of amount of measured beta 42 amyloid. Similar to the Subject 1, Subject 3 exhibited onset of clinically relevant disease 4 years after observed a significant increase of the level of beta 42 amyloid.
  • the present invention provides a scale based on detection and measurement of levels of biomarkers such as beta amyloid proteins in human population which was analyzed separately in their 5th, 6th, 7th and 8th decade of life. It was needed more than a decade of meticulous follow of up of levels of those proteins, based on following families with a high risk of onset of Alzheimer's disease (familial disease forms), but also based on genetically non-peculiar families and discovering sporadic spontaneous onset of disease to collect sufficient data for building the scale.
  • biomarkers such as beta amyloid proteins in human population which was analyzed separately in their 5th, 6th, 7th and 8th decade of life. It was needed more than a decade of meticulous follow of up of levels of those proteins, based on following families with a high risk of onset of Alzheimer's disease (familial disease forms), but also based on genetically non-peculiar families and discovering sporadic spontaneous onset of disease to collect sufficient data for building the scale.
  • the present invention provides the scale which is based on estimation of importance of incremental increase or decrease of levels of biomarkers of interest
  • the innovative element which is in the core of this invention and which allows precise numerical estimation is detection of levels of proteins in for example 1- or 2-years * time interval distance, which then, interpolated to the scale according to the present invention gives precise levels of risk of onset of a particular brain disease.
  • the present scale is numerical and gives a precise information of the risk in categories up to 20%, 20-40%, 40-60%, 60-80% and above 80%, within 3 years’ time interval. If, for example, someone enters in the first category, 20% or less risk, it is advised to repeat the test in 5 years.
  • the scale gives a precise insight in the current status of the biomarkers accumulation and it positions it within the time perspective.
  • the scale is relative with time perspective and may be personalized for each tested subject, and more generally the scale is categorized according to the decade of life of the tested subject This is the only way to detect a significant increase or decrease of measured level of at least one biomarker, thus giving a numerical value of the risk, a precise prediction for the onset of a particular brain disease.
  • the scale for estimating the risk for the onset of a particular brain disease, diagnosing of a particular brain disease or for following up on an existing brain disease is based on two subsequent measurements of a level of at least one biomaiker of interest.
  • the scale defines a risk ranges for the onset of a particular brain disease, the risk ranges are based on a numerical difference between two subsequent measurements of the level of at least one biomarker of interest. If more than one biomarker is measured, the numerical difference relates to the same biomarker for which a separate scale is established.
  • the numerical difference of the measured levels of biomarkers is expressed as a percentage change.
  • the percentage change is a mathematical concept that represents the degree of change over time. To each risk range a corresponding range of the percentage change is assigned. Further, the scale comprises 5 risk ranges based on the corresponding range of the percentage range.
  • Each risk range classifies the tested subject for the onset of a particular brain disease in one of the following risk ranges: a very low risk (up to 20%), a low risk (20-40%), a moderate risk (40-60%), a high risk (60-80%) and a very high risk (above 80%).
  • the scale is categorized according to the decades of life of the subject
  • a predetermined time interval between two subsequent measurements is in a range between 1 and 5 years, preferably in a range between 1 and 2 years, wherein the predetermined time interval depends on the measured level of biomaricers measured for the very first time.
  • the biomarkers are selected from the group consisting of a protein, peptide, lipid, metabolite, nucleic-acid based molecule, cellular action potential, influx and efflux of certain molecules, status of mitochondria, status of microtubules, status of ribosomes or combinations thereof.
  • the brain disease is selected from the group consisting of including but not limited to, Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, and dementia with Lewy bodies and other neurodegenerative or neuroinflammatory diseases.
  • Graph in Fig. 17 shows the scale for entering into a symptomatic stage of Alzheimer’s disease for 50s and 60 years of life. (MMSE scale score lower than 25).
  • the risk is defined by increase in detected concentration of beta 42 amyloid, obtained as a measured difference in percentage between two subsequent measurements. Preferred time difference between two measurements is 1-2 years.
  • This scale is valid for subjects in their 50s and 60s years of life and only in the case when detected concentration in the first measurement is less than 40 fM/mg of the total protein concentration. Higher concentrations than 40 fM/mg of the total protein concentration are in majority of cases detected in clinically already detectable disease. Detected increases in concentrations of beta 42 amyloid correspond to the numerical risk of entering a symptomatic stage within up to next 5 years of life.
  • Graph in Fig. 18 shows the scale for entering into a symptomatic stage of Alzheimer's disease (MMSE scale score lower than 25).
  • the risk is defined by increase in detected concentration of beta 42 amyloid, obtained as a measured difference in percentage between the first and the second measurement. Suggested time difference between two measurements is 2 years.
  • This scale is valid for subjects in their 70s and 80s years of life and only in the case when detected concentration in the first measurement is less than 48 fM/mg of the total protein concentration. Higher concentrations than 48 fM/mg of the total protein concentration are in majority of cases detected in clinically already detectable disease.
  • Detected increases in concentrations of beta 42 amyloid correspond to the numerical risk of entering a symptomatic stage within up to next 5 years of life.
  • Average time distance for a start of a clinically significant cognitive decline, which corresponds to MMSE score lower than 25, in the case when estimated risk is higher than 50% is 3.8 years from the second measurement
  • Increase of beta 42 amyloid concentration for up to 32% suggests a very low risk (less than 20%), increase for 32-57% suggests a low risk (20-40%), increase for 57-71% suggests a moderate risk (40-60%).
  • Increases between 71 and 91% suggest a probability up to 80% to enter a clinically active disease within next 5 years.
  • Increase for more than 80% of concentration of beta 42 amyloid has been found in only few sporadic cases which were already clinically active.
  • the present invention also provides a kit to be used to transport a hair sample from the subject to the laboratory where the tests will be performed.
  • the kit includes a vial with a screw cap, a transporting medium (see fig. 11), a written instruction for sampling and a form comprising fields for name, age and the date and hour of sending the hair sample.
  • the written instruction consists of a simple drawing in which it is illustrated that forceps is needed in order to wrap a hair (one by one) around forceps and then pull it out applying a sudden movement and how to immerse a tip of each hair pulled out of the subject's scalp in the vial's transporting medium, while the opposite end can be cut (depending on the length of the individual hair) or, which is also possible, can be let to protrude outside when the vial is closed by complete insertion of the screw cap.
  • Number of pulled hairs is preferably in a range between 10 and 20.
  • the transporting medium comprises DMEM (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA) supplemented with Penicillin/Streptomycin (Gibco by Life Technologies), anti-fungal nystatin (Gibco by Life Technologies) and anti-mycoplasma (Gibco by Life Technologies).

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Abstract

The present invention provides an in vitro method of transformation of hair follicle cells into neurons comprising the steps of: plucking and sending a hair sample to a laboratory in a transporting medium; isolating of cells from the hair sample suitable for obtaining an early neuronal precursor cells; transformation of an isolated early neuronal precursors cells into a late neuronal precursor cells, wherein transformation of the early neuronal precursor into the late neuronal precursor cells comprises steps of: cultivating the isolated early neuronal precursor cells in a transformation culturing medium for the first 24 hours in a low ambient oxygen condition followed by applying a transient cycles of hypoxia and acidosis; cultivating of the late neuronal precursor cells in a proliferation medium in an low ambient oxygen condition; and differentiation of the late neural precursor cells into neurons on Poly-D-lysine and laminin coated surface in a differentiation medium. Further, the present invention provides a method and a scale of estimating the risk for the onset of a particular brain disease, diagnosing of a particular brain disease or for following up on an existing brain disease by the use of neurons obtained according to the in vitro method.

Description

METHOD OF TRANSFORMATION OF HAIR FOLICLE CELLS INTO NEURONS, METHOD AND SCALE FOR ESTIMATING RISK FOR ONSET OF A PARTICULAR BRAIN DISEASE
TECHNICAL FIELD
This invention provides an in vitro method of transformation of hair follicle cells into neurons, a method and a scale for estimating risk fin· onset of a particular brain disease. The method according to the present invention comprises the steps of substantially non-invasive taking of a hair sample from a subject, measuring of a level of at least one biomarker of interest, analyzing of said biomaifcers by using the scale of the present invention and estimating with great precision of the possible risk of onset of brain diseases, including, but not limited to Alzheimer’ s disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, dementia with Lewy bodies and other. Thus, the originality of this invention arises from obtaining of cells from a hair follicle, transforming said cells into neurons using said method, quantification of selected biomarkers, and then getting the numerical answer of the level of risk for onset of a particular brain disease based on a numerical difference of said biomarkers in at least two subsequent measurements. The present invention also provides a kit to be used to transport the hair sample from the subject to the laboratory where the tests will be performed.
BACKGROUND ART
Brain diseases represent the largest burden of human society. Expenses of care for patients affected by brain diseases are three times larger than treatment for all patients with all malignant tumors together. Even more, while incidence and prevalence of many diseases decrease and efficiency of their treatment increases, ratio of people affected by incurable brain diseases is growing. Of many brain diseases, the most critical is dementia linked to age, because it is predicted that humans will continue to increase average years of life. This means that number of people affected by various types of dementia will continue to grow almost exponentially. All projections predict that if something will not change, already in 20 to 30 years from now, majority of Western countries will face enormous problem of lack of manpower to take care of large number of people affected by dementia.
Alzheimer's is the most common cause of dementia and it accounts for 75% of all dementia cases. It is very strongly connected to the age and it is much more present among old population. On the other hand, there is a visible increase in number of young people affected by this type of dementia. Approximately 200,000 Americans under the age of 65 have early onset Alzheimer’s disease. People affected by Alzheimer’s disease are faced by memory loss and decrease in majority of cognitive abilities which impairs their daily life. As Alzheimer's advances it leads to increasingly complex symptoms, including disorientation, mood and behavior changes. In many cases, there is no clear family risk for Alzheimer's, but every presence of family history increases risk of getting it in the old age. One of the most commonly mentioned gene is the apolipoprotein E (APOE). This gene has several forms. One of them, APOE e4, increases a person's risk of developing the disease and is also associated with an earlier age of disease onset.
The most typical and from pathology point of view, most stressed hallmark of disease is presence of abnormal clumps (called amyloid plaques) and tangled bundles of fibers (neurofibrillary, or tau, tangles). Those accumulations are made of beta amyloid and tau proteins, which are one of the biomarkers of this disease. Unfortunately, pathology of Alzheimer’s is much more complex: it is possible that someone has a large number of visible plaques and clinically early stage of disease and vice versa -there are many reported cases of people which have died in a very advanced stage of disease and post-mortally only few plaques have been found. This highly suggests that levels of those proteins need to be analyzed individually and from the perspective of their change over certain time interval. Based on all known facts, it is understandable to assume that every test which would enable to recognize that someone is approaching the moment when the symptoms of any brain disease might appear, could be used as an important signal to start with the early therapy. Early therapy postpones onset of symptoms and possibly, stops their appearance.
PCT publication WO2011144901A1 discloses directed differentiation of epidermal neural crest stem cells, and more specifically human epidermal neural crest stem cells (hEPI- NCSC). A method of WO2011144901 Alcomprises 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; and 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.
US publication US20180140637A1 describes compositions, kits, and methods for generating Schwann cells from epidermal neural crest stem cells (EPI-NCSC), Such EPI-NCSC can be obtained from the bulge of hair follicles. A method of generating Schwann cells of US20180140637A1 comprising: contacting one or more epidermal neural crest stem cells (EPI-NCSC) with a first differentiation culture medium comprising b-mercapto ethanol, SB431542, and a first amount of fetal bovine serum for 1-3 days to generate a first cell population; contacting the first cell population with a second differentiation culture medium comprising all-trans-retinoic acid, SB431542, and the first amount of serum for 1-4 days to generate a second cell population; and contacting the second cell population with a third differentiation culture medium comprising all-trans-retinoic acid, SB431542, fetal bovine serum, fibroblast growth factor-2, a platelet derived growth factor-BB, neuregulin-1, and Forskolin for about 4 to about 28 days to thereby generate EPI-NCSC-derived Schwann cells. While WO2011144901 A 1 declares that 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, KJLF4, SOX2, LJN28, OCT-4/POU5F1 and NANOG, cells Isolated from a hair follicle according to an in vitro method of the present invention express NES, SOX2, MSX2, LRIG1, VARS2, ENAH, MYOIO, SORB S3 and PFN1, but does not comprise MYC, KLF4 and POU5F1.
Clone-forming ability in WO2011144901 A1 was reported as 70.7±7.9%. Cells characterized by the genetic profile mentioned above exhibit 88.5±4.9% of the clone-forming ability.
In addition, our two-step in vitro method comprises early and late precursors and finally gives rise to unique homogenous neuronal cell population with 100% cells expressing DCX, TUBB3, MAP2 and SYP. Levels of produced beta amyloid, which is crucial for our scale are, in different parallel clones within 5% of difference.
Different from some other attempts in which information about status and levels of the biomarkers can be obtained using long, expensive and very invasive procedures, requiring to visit the hospital and wait a long time to get the information, the main object of this invention was to develop a simple, a non- invasive approach which allows anybody to provide between 10 and 20 plucked hairs, send them by a regular post in a kit suitable for transporting plucked hair from the subject to the laboratory where the tests will be performed and to get an answer about the current state of the risk of developing a clinically active brain disease. This includes Alzheimer's, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscle atrophy and other similar neurodegenerative or neurovascular diseases.
Technical solution of the present invention significantly improves previously known methods to obtain neurons. Known methods either use invasive approach (e.g. biopsies of the skin) which require clinical set up, combined with induced phiripotent stem cell technology, which is complex, long lasting and expensive. A method of the present invention is based on obtaining of homogenous population of neurons from a small population of cells between hair root bulge and surface of the skin. Different from some other known methods which use heterogenous phiripotent stem cells from hair, which are giving various neural crest derivatives, a method of the present invention is based on homogenous nestinpositive population with extraordinary efficiency in obtaining neurons. Our approach is simple, robust, non-invasive, faster and most importantly - much more precise (numerically supported) in comparison to other approaches. The present invention provides an in vitro method of transformation of hair follicle cells into DCX, TUBB3, MAP2 and SYP positive neurons which is performed by combination of decreased oxygen (hypoxia), decreased pH and with specific peptides. This altogether forms an innovative in vitro method of transforming hair follicle cells into neurons. One of the major advantages of our method is in bypassing the transformation over a pluripotent stem cell types (IPSC). In addition, in vitro method of this invention yields very specific cell sub-populations which allow obtaining homogenous neuronal populations without unwanted cell subpopulations (for example, cells with a strong expression of oncogene or markers for glia or any other unwanted cell populations). In this way we make the method more robust, reproducible and we eliminate the risk of uncontrolled growth of cells, which impairs their application in diagnostic and prognostic purposes.
US publication US2013034858A1 provides a method for detecting onset of or the risk of development of, a protein misfolding disease, and a method for predicting the age of onset of a protein misfolding disease using nerve cells derived from iPS cells. The method of US2013034858A1 comprising the steps of establishing iPS cells from somatic cells derived from the test subject; inducing differentiation of said iPS cells into nerve cells; measuring the amount of a causative protein, or the activity or the expression level of an enzyme involved in degradation of the causative protein in said nerve cells; and comparing said measured value with the amount of the causative protein in control cells, or with the activity or the expression level of the enzyme involved in degradation of the causative protein in control cells. Difference between the method disclosed in US2013034858A1 and the technical solution of the present invention is that present invention is completely avoiding application of iPS cells. In addition, according to the US2013034858A1 diagnosing whether a test subject has developed a protein misfolding disease or whether a test subject has a risk of developing it is based on comparison of a control subject who has not developed the protein misfolding disease at the same age with the test subject.
The present invention is based on a scale of the risk for certain decade of life which is based on a numerical difference in a level of at least one biomarker detected in two subsequent measurements, thus it is personalized for each subject.
Technical solution of the present invention provides the scale which is based on estimation of importance of an incremental increase or decrease of a level of at least one biomarker of interest over a time interval between two subsequent measurements. The innovative element which is in the core of this invention and which allows precise numerical estimation is detection of the level of at least one biomarker of interest in 1- or 2-years* time interval distance, which then, interpolated to the scale according to the present invention provides precise levels of risk of onset of a particular brain disease. Different from other methods which combine measurement of levels of different proteins and give information about increased or non-increased risk, our scale is numerical and gives a precise information of the risk in categories below 20%, 20-40%, 40-60%, 60-80% and above 80%, within 3 years* time interval. In addition, since it is possible to have rather high levels of proteins without symptoms and rather low levels with symptoms, the scale of the present invention is relative with time perspective and may be personalized for each tested subject, and more generally the scale is categorized according to the decade of life of the tested subject SUMMARY OF THE INVENTION
The present invention relates to an in vitro method of transformation of hair follicle cells into neurons, a method and a scale for estimating of the risk for onset of a particular brain disease. Said methods are not limited only for estimating of the risk for onset of a particular brain disease, but may be used for diagnosing of a particular brain disease or for follow up of the existing brain disease.
Accordingly, the present invention provides an in vitro method of transformation of hair follicle cells into neurons comprising the steps of: plucking and sending a hair sample to a laboratory in a transporting medium; isolating of cells from a hair follicle suitable for obtaining an early neural precursors cells; transformation of the early neuronal precursors cells into a late neuronal precursors cells; wherein transformation of the early into the late neuronal precursors cells comprises steps of: cultivating the isolated early neuronal precursor cells in a transformation culturing medium for the first 24 hours in a low ambient oxygen condition followed by applying a transient cycles of hypoxia and acidosis; cultivating the late neural precursors in a proliferation medium in a low ambient oxygen condition; and differentiation of the late neural precursors on Poly-D-lysine and laminin coated surface in a differentiation medium. Neurons generated by the in vitro method are positive for DCX, TUBB3, MAP2 and SYP.
In addition, the present invention provides a method of estimating of the risk for onset of a particular brain disease, diagnosing of particular brain disease or for follow up of the existing brain disease by using neurons obtained according to the in vitro method comprising the steps of: plucking and sending a hair sample to a laboratory in a vial containing a transporting medium; measuring of a level of at least one biomarker of interest, wherein said measuring is performed at least two times in a given time interval; and applying a scale, the scale is based on two subsequent measurements of the level of at least one biomarker, wherein the scale is function of a numerical difference between measured levels of at least one biomarker in said two subsequent measurements, wherein if more than one biomarker is measured, the numerical difference relates to the same biomarker for which a separate scale is established.
The scale is available to a very wide human population, and more importantly, the idea is to perform testing of a subject, in at least two subsequent measurements in a time interval ranging from 1 to 2 years, which results will detect levels or features of biomarkers and then provide in a form of the scale an information with great precision about the current risk of developing active brain disease. In addition, methods and the scale of the present invention may be used for diagnosing of a particular brain disease or for follow up of the existing brain disease by using neurons obtained according to the in vitro method of the present invention. DETAILED DESCRIPTION OF THE INVENTION
The present disclosure provides an in vitro method of transformation of hair follicle cells into neurons. The present disclosure also provides an early neuronal precursors cells obtained by the in vitro method which are positive for NES, SOX2, MSX2, LRIG1, VARS2, ENAH, MYOIO, SORBS3 and FFN1, but does not comprise MYC, KLF4 and POU5F1. Further, the present invention provides a late neuronal precursors cells which are positive fin· NES and TBR1. The present disclosure also provides a method and a scale for estimating of the risk for onset of a particular brain disease, diagnosing of particular brain disease or for follow up of the existing brain disease by using neurons obtained according to the in vitro method.
The main problem with brain diseases is that train is not accessible using usual diagnostic procedures.
Different from other organs which mirror their status in blood or they are easily approachable by biopsy, ultrasound or similar methods, the brain tissue is hidden behind the blood brain barrier. Thus, the major problem which present invention solves is to provide the in vitro method of transformation of hair follicle cells into neurons which mirror neurons in the brain of a tested subject. Different from other methods which use induced pluripotent stem cells and different from other methods which use multrpotent neural crest stem cells, our method uses cell population obtained from a small region in the hair, which differs from other reported cell populations. Although we still don't have a very efficient drug to treat Alzheimer's disease, it is well known that starting with pharmacological therapy and other approaches, which include changing of life style, cognitive brain exercise, changing of diet, etc. - they all slow down disease progression. No one yet knows what would happen if we would recognize that the tested person/subject is very close to start of the Alzheimer's disease symptoms or amyotrophic lateral sclerosis or multiple sclerosis. According to the method and the scale of the present invention it may be recognized that the tested subject is very close to start of tested brain disease symptoms. If this person would start with intensive anti-disease approach, it is highly possible that onset of disease would be postponed or disease would even never enter into a visible clinical stage.
Apart from significantly improved protocol to obtain neurons from the hair, which allows everybody to provide samples of plucked hair sample without need to go to the hospital, the most innovative element of this invention is the scale based on detection and measurement of levels of beta amyloid proteins in human population which was analyzed separately in their 5th, 6th, 7th and 8th decade of life. More than a decade of meticulous follow of up of levels of those proteins, based on following families with high risk of onset of Alzheimer’s disease (familial disease forms), but also based on genetically non-peculiar families and discovering sporadic spontaneous onset of disease, allowed us to create the scale. The scale is based on an incremental increase or decrease of a level of at least one biomarker of interest, wherein the incremental increase or decrease is a numerical difference of the measured level of at least one biomarker between two subsequent measurements. The numerical difference is expressed in the scale as a percentage change in relation to the preceding measured level of biomarker, wherein the increase or decrease of the subsequent measured level of biomarkers expressed in percentages is said percentage change. The innovative element which is in the core of this invention and which allows precise numerical estimation is detection of levels of proteins in for example 1- or 2-years* time interval distance, which then, interpolated to the scale according to the present invention gives precise levels of risk of onset of a particular brain disease. Different from other methods which combine measurement of levels of different proteins and give information about increased or nan-increased risk, our scale is numerical and gives a precise information of the risk in categories below 20%, 20-40%, 40-60%, 60- 80% and above 80, within 3 years* time interval. If, for example, someone enters in the first category,
20% or less risk, it is advised to repeat the test in 3 years. If someone, for example, enters the category of 80% risk and above, we highly advise additional neurological tests and start of the therapy with the goal to postpone disease onset Thus, the scale gives a precise insight in the current status in the terms of a change of at least one biomarker between two subsequent measurements. Subsequent measurements may be measurements between a first and a second measurement, the second and a third measurement and so on. The first measurement refers to the measurement of the level of at least one biomarker that occurred for the first time. Different from others, which by increased or non-increased levels of some markers suggest presence of increased risk, which might be misleading, since it is possible to have rather high levels of proteins without symptoms and rather low levels with symptoms, the scale is relative with a time perspective and is categorized according to the decade of life of the tested subject The invention described here is based on detection of a time interval in which a significant increase or decrease is occurring, thus giving a numerical value of the risk for onset of a particular brain disease.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates a tip of the plucked human hair - hair follicle;
Fig. 2 illustrates a part of a hair root from which the cells from a hair follicle are selected, namely an upper portion of a hair follicle, above a main bulb, near the connection with a sebaceous gland;
Fig. 3 is a higher magnification of a portion of a hair follicle which demarcates the upper border of the region from where we isolate cells;
Fig. 4 illustrates a hair follicle after treatment with enzymes Collagenase and Dispase for separation of an epithelial layer (root sheath) from the major portion of the hair;
Fig. 5 illustrates an elongated cell which migrate out the isolated (upper) portion of the hair follicle; Fig. 6 illustrates typical thick colonies formed 5-7 days after migration of a hair follicle cells; Fig. 7 illustrates cells isolated from the upper portion of the hair follicle where (A)-; are native cells, (B)-nuclei; and (C)- nestin positive signal of those cells;
Fig. 8 is a graph illustrating comparison of efficiency in obtaining neuronal precursors from 6 subjects, 3 males (age 23, 36 and 59) and 3 females (age 23, 35, 61);
Fig. 9 illustrates cells three days after they have been exposed to the transformation culturing medium;
Fig. 10 illustrates Map2 expression in the protocol which includes two steps of neural precursors (A) and in the one which includes three steps of neural precursors (B);
Fig. 11 illustrates a vial filled with a transporting medium for sending hair samples;
Fig. 12 is a graph illustrating comparison in efficiency of obtaining hair follicle cells after sending different hair samples in a vial, and processing some hair samples after three days when compared to samples which were processed after five days;
Fig. 13 is a graph illustrating a correlation of concentration of beta amyloid 42 and symptomatic stage of Alzheimer's disease;
Figs. 14 to 16 are graphs illustrating comparison of history of follow up of 3 subjects with confirmed mutations and the history of familial Alzheimer's disease; and
Fig. 17 is a graph illustrating an increasing risk scale in the 50s and 60 years of life for entering into a symptomatic stage of Alzheimer's disease; and
Fig. 18 is a graph illustrating an increasing risk scale in the 70s and 80 years of life for entering into a symptomatic stage of Alzheimer's disease.
DEFINITIONS
As used herein, the term "neural stem cell" (NSC) refers to an undifferentiated cell that can proliferate, self-renew, and differentiate into the adult neural cells of the brain. NSCs are capable of selfmaintenance (self-renewal), meaning that with each cell division, one daughter cell will also be a stem cell. The non-stem cell progeny of NSCs are termed neural progenitor cells. Neural progenitors cells generated from a single multi potent NSC are capable of differentiating into neurons, astrocytes (type I and type P), and oligodendrocytes. Hence, NSCs are "multipotent" because their progeny has multiple neural cell fetes. Thus, NSCs can be functionally defined as a cell with the ability to: 1) proliferate, 2) self-renew, and 3) produce functional progeny that can differentiate into the three main cell types found in the central nervous system: neurons, astrocytes and oligodendrocytes.
As used herein, the terms "neural progenitor cell" or "neural precursor cell" refer to a cell that can generate progeny such as neuronal cells (c.g., neuronal precursors or mature neurons), glial precursors, mature astrocytes, or mature oligodendrocytes. Typically, the cells express some of the phenotypic markers that are characteristic of the neural lineage. A "neuronal progenitor cell" or "neuronal precursor cell" is a cell that can generate progeny that are mature neurons.
As used herein, the term "isolated" with reference to a cell, refers to a cell that is in an environment different from that in which the cell naturally occurs, e.g., where the cell naturally occurs in a multicellular organism, and the cell is removed from the multicellular organism, the cell is "isolated." This usually means that the cell is“isolated" from its original natural environment (organism) and placed into an artificial environment. The artificial environment usually means a medium for cell growth and/or differentiation. This is usually referred as“in vitro cell cultivation”.
The terms "individual," "subject,",“test subject” "host," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, murines (rats, mice) non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.
As used herein, the term“level of biomarket" refers to a, quantity or status of molecule which can be protein, peptide, lipid, metabolite, nucleic-acid based molecule (for example, RNA or DNA), then marker based on physiological functions like cellular action potential, influx and efflux of certain molecules, status of mitochondria, status of microtubules, status of ribosomes or combinations thereof. Further as used herein“the biomarker of interest” refers to a molecule such as a protein, a peptide, derivative of tyrosine, derivative of cholesterol that is deposited outside the cells or a molecule that accumulates inside the cells. In cases where we analyze the risk of Alzheimer's disease, examples of the causative protein include, but are not limited to, amyloid beta and tau.
Before the present invention is ftirther described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a neuron" includes a plurality of such cells and reference to "Sox2 " includes reference to one or more Sox2 polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely,” "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. IN VITRO METHOD OF TRANSFORMATION OF HAIR FOLICLE CELLS INTO NEURONS
The present disclosure provides an in vitro method of transformation of hair follicle cells into neurons from human subjects in a very simple, fast, efficient and non-invasive way. Neurons obtained by said method are used to get a quantified, precise estimation of the risk to enter a symptomatic phase of brain disease, for example Alzheimer’s disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy and other neurodegenerative or neuroinflammatory disease within the 3 years* time interval from the moment of taking a very first hair sample. The in vitro method generally comprises steps of; plucking and sending a hair sample to a laboratory in a transporting medium; isolating of cells from the hair sample suitable for obtaining of an early neural precursors cells; transformation of the early neural precursors cells into a late neuronal precursors cells, wherein transformation of an isolated early neuronal precursors cells into a late neuronal precursor cells comprises steps of: cultivating the isolated early neuronal precursor cells in a transformation culturing medium for the first 24 hours in a low ambient oxygen condition followed by applying a transient cycles of hypoxia and acidosis; cultivating of the late neural precursors cells in a proliferation medium in an low ambient oxygen condition; and differentiation of neural precursors cells into neurons on Poly-D-lysine and laminin coated surface in a differentiation medium. Isolating of cells from the hair sample comprises steps of immersing the hair sample in a culture dish comprising Collagenase and Dispase enzymes for 12 hours at 4 °C, followed by rinsing in Phosphate buffered saline and isolating of upper portion of the hair root, above the main bulb, near the connection with sebaceous gland. The transporting medium comprises DMEM supplemented with Penicillin/Streptomycin, anti-fungal nystatin and anti-mycoplasma. An isolated cells are immersed in DMEM supplemented with Penicillin/Streptomycin and anti-fungal nystatin and adhering the isolated cells to a culture dish treated by fibronectin. The transformation culturing medium comprises Opti-MEM, B27, N2, BDNF (all Gibco), Penicillin/Streptomycin, and peptides Forskolin, DNP-derived active peptide, NAP, CHIR99021, LIF, and FGF2. It has been found that applying of low ambient oxygen condition for cultivating the isolated cells provides better efficiency m their proliferation and survival. In addition, applying of transient cycles of hypoxia and acidosis for cultivating the isolated cells resulted in higher rates of transformation from early to late neuronal precursors. Therefore, the in vitro method comprises cultivating the isolated cells in the transformation culturing medium for the first 24 hours in an ambient oxygen condition of 10% oxygen followed by applying a transient cycles of hypoxia and acidosis. After cultivating the isolated cells in the transformation culturing medium is followed by cultivating of the neural precursors cells in the proliferation medium in a low ambient oxygen condition of 5% oxygen. The proliferation medium comprises DMEM/F-12, B-27 Supplement, N-2 Supplement, Penicillin/Streptomycin, FGFb and EGF. Finally, the neural precursors cells are differentiated into neurons on Poly-D-lysine and laminin coated surface in the differentiation medium, the differentiation medium comprises Neurobasal and Penicillin/Streptomycin with glutamine. A time frame within which occurs transformation of hair follicle cells into neurons according to said in vitro method is 20 days.
A subject neurons generated by the in vitro method are positive for DCX, TUBB3, MAP2 and SYP. Said neurons transformed from a hair follicle cell of a subject are used in a method of estimating the risk for the onset of a particular brain disease, diagnosing of a particular brain disease and for following up on an existing brain disease. The subject is a human subject The brain disease is selected from the group consisting of, including but not limited to, Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, and dementia with Lewy bodies and other neurodegenerative or neuroinflammatory diseases.
An early neuronal precursors cells generated by the in vitro method are positive for NES, SOX2, MSX2,
LRJG1, VARS2, ENAH, MYOIO, SORBS3 and PFN1, but does not comprise MYC, KLF4 and POU5F1.
A late neuronal precursors cells generated by the in vitro method are positive for NES and TBR1.
Obtaining a hair samples from a human subject
With the goal to develop a test which will be based on a simple self-sampling available to every human subject, present invention provides a vial of 2 ml comprising a transporting medium (see fig. 11). hi addition to the vial, a simple drawing is provided in which it is shown that forceps is needed in order to wrap a hair (one by one) around forceps and then pull it out applying a sudden movement The tip of the hair which was pulled out of the subject’s scalp has to be immersed in the transporting medium, while the opposite end can be cut (depending on the length of the individual hair) or, which is also possible, can be let to protrude outside when the vial is closed by complete insertion of the screw cap. Number of pulled hairs is preferably in a range between 10 and 20. After hair samples are collected, immersed in the transporting medium and the vial is properly closed, the subject is instructed to put the vial into the middle size envelope with absorbing cushions. The subject is also instructed to fill in the form in which name, age and the date and hour of sending is clearly written. Then, the samples are to be sent to a laboratory using express postal service.
The transporting medium comprises DMEM (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA) supplemented with Penicillin/Streptomycin (Gibco by Life Technologies), anti-fungal nystatin (Gibco by Life Technologies) and anti-mycoplasma (Gibco by Life Technologies).
Isolating of cells from a Hair sample received in a vial suitable for transfnrrnatinn into early neural
After receipt of a vial containing a hair sample, under control of a microscope, upper portion of a hair root, above a main bulb, near the connecting with sebaceous gland is isolated. This protocol is efficient and fast to perform. An isolated tissue is immersed in Collagenase and Dispase (Roche, Fisher Scientific), using 35-mm cell culture dish (Thermo Fisher Scientific) on +4°C for 12 hours (overnight). After rinsing in Phosphate buffered saline (PBS) and mechanically removing an epithelial layer, small pieces of the isolated tissue are immersed in DMEM (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA) supplemented with Penicillin/Streptomycin (Gibco by Life Technologies) and anti-fungal nystatin (Gibco by Life Technologies) and again plated on 35-mm culture dish treated by fibronectin. Small pieces of the isolated tissue adhere to the surface treated by fibronectin within 1 h. Preferred incubation parameters are 37 °C, 5 % CQi and 10 % (O2. Lower oxygen concentrations, mentioned in protocols for isolation of fast migrating cells do support neural crest with fast migratory activity. The first cells migrating out of the bulk of the isolated tissue are fast migrating neural crest cells. However, our FBS-fiee protocol does not yield a lot of those cells and we let cells which are protruding out, but not far from the bulk to multiply. After 5 days of the start of cultivation we start to add human recombinant epidermal growth factor (Merck, catalog number: GF144) and wait 5 more days for cells to multiply. Preferred concentration of cells can reach 104 -105 cells per 35 mm culture plate. Colonies with not migratory features, made by round or slightly elongated cells are selected and transferred in medium for transformation into neural stem cells. One human hair follicle yields on average 0.5 million of cells per bulge within 10 days which are suitable for transformation into early neuronal precursors cells.
The early neuronal precursors cells are positive for NES, SOX2, MSX2, LRIG1, VARS2, ENAH, MYO10, SORBS3 and PFN1, but does not comprise MYC, KLF4 and POU5F1.
Table 1 bellow shows expression of genes which define a population of early neuronal precursors cells isolated from the upper portion of the hair bulb.
Figure imgf000014_0001
Figure imgf000015_0001
Table 1
Figure imgf000015_0002
Transformation of isolated early neuronal precursors cells into late neuronal precursors cells
Transformation of an isolated early neuronal precursors cells into a late neuronal precursor cells, comprises steps of cultivating the isolated early neuronal precursors cells in a transformation culturing medium for the first 24 hours in a low ambient oxygen condition followed by applying a transient cycles of hypoxia and acidosis, and afterwards culturing in the transformation culturing medium in an ambient oxygen condition of 5% oxygen. The transformation culturing medium comprises following peptides for promoting differentiation of the early neuronal precursors into the late neuronal precursor cells: fbrskolin, 4-24 mM; NAP, 1-2 mM; CHIR99021, 1-5mM; LIF, 10-30 ng/ml; and FGF2, 10-40 ng/ml. Cultivating of the isolated early neuronal precursors cells in the transformation culturing medium for the first 24 hours comprises an ambient oxygen condition of 10% oxygen. Next step is applying the following 3 transient cycles of hypoxia and acidosis:
a. 5 hours in an ambient oxygen condition of 3.5% oxygen followed by 2 hours in an ambient oxygen condition of 10% oxygen,
b. 20 min of 6.4 pH in an ambient oxygen condition of 10% oxygen followed by increasing pH to 7.4,
c. 5 hours in an ambient oxygen condition of 3.5% oxygen followed by 12 hours in an ambient oxygen condition of 10% oxygen, d. 20 min of 6.4 pH in an ambient oxygen condition of 10% oxygen followed by increasing pH to 7.4, and
e. 5 hours an ambient oxygen condition of 3.5% oxygen followed by 2h an ambient oxygen condition of 10% oxygen.
After said cycles of hypoxia and acidosis, the late neural precursors cells are cultured in a proliferation medium in an ambient oxygen condition of 5% oxygen. A time frame for cultivation said cells depends on required number of cells, preferably between 3-7 days.
In total, the procedure from the isolation of the hair sample to obtained late neuronal precursor cells lasts 14 days.
The late neuronal precursor cells are positive for NES and TBR1.
Table 2 below shows expression of genes which define a population late neuronal precursors cells.
Figure imgf000016_0001
Figure imgf000017_0001
Table 2
Proliferation neuronal precursor cells
Isolated late neuronal precursor cells are cultivated in a proliferation medium comprising DMEM/F- 12 (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA), B-27 Supplement (Gibco by Life Technologies), N-2 Supplement (Gibco by Life Technologies), Penicillin/Streptomycin (Gibco by Life Technologies), 20 ng/mL Recombinant Mouse Fibroblast Growth Factor-basic (FGFb, ThermoFisher Scientific, Pittsburgh, PA, USA) and 20 ng/mL Recombinant Mouse Epidermal Growth Factor (EGF, ThermoFisher Scientific). The late neuronal precursor cells are cultivated in suspension, and after two days neurospheres are formed.
Differentiation of late neuronal precursors cells into neurons
For the purpose of differentiation analyses neurospheres are dissociated and then obtained single late neuronal precursors cells are plated on 12-mm coverslips (200 to 250 000 cells/coverslip) as well as on 6-well plates (1 000 000 cells/well). Coverslips and wells were previously coated with Poly-D-lysine (500 mg/mL, 24h at 37°C, Sigma) and laminin (10 mg/mL, 24h at 37°C, Sigma). 24 hours after plating, the medium is changed into a differentiation medium comprising Neurobasal (Gibco by Life Technologies) which supports neurons in vitro and antibiotic with glutamine (Pen Strep Glutamine, Gibco by Life Technologies).
Table 3 below shows expression of genes which define differentiated neurons obtained by the in vitro method of fee present invention.
Figure imgf000017_0002
Figure imgf000018_0001
Table 3
Components/equipment is used in following examples:
6-well pistes (Coming, Falcon®, catalog number 353934)
NIH-3T3 cells (ATCC, catalog number: CRL-1658)
Collagenase A (Sigma-Aldrich, Roche Diagnostics, catalog number 10103578001) Dispase P (Sigma-Aldrich, catalog number 4942078001)
Manufacturer Roche Diagnostics, catalog number 04942078001.
Trypsin (2.5%) (Thermo Fisher Scientific, Gibco™, catalog number: 15090046) Dulbecco’s modified Eagle medium (DMEM) without calcium and magnesium (Thermo Fisher Scientific, Gibco™, catalog number: 21068028)
Ham’s FI2 Nutrient Mix (Thermo Fisher Scientific, Gibco™, catalog number: 11765047) L-Glutamine (Thermo Fisher Scientific, Gibco™, catalog number 25030081) Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco™, catalog number: 15140148)
Amphotericin B (Thermo Fisher Scientific, catalog number: 15290026)
Dulbecco’s modified Eagle medium (DMEM) high glucose (Thermo Fisher Scientific, Gibco™, catalog number: 11960044)
Example 1: Isolation of early neuronal precursors cells from hair follicle from six subjects of different sent and age
In this example is demonstrated that the claimed method can be used to obtain early neuronal precursors cells from a human hair follicle. In order to compare efficiency of obtaining multipotent stem cells from the hair root, we tested the protocol in 6 subjects: 3 males (age 23, 36 and 59) and 3 females (age 23, 35 and 61). All 6 people were Caucasians without any history of skin or any other serious disease. None of 6 subjects had a history of application of any treatment for their hair. All 6 subjects signed informed consent in which they were informed about possible risks of the procedure. Five hairs were taken from each of the volunteer by the same person (PhD student). A simple metal forceps and the same procedure were used. One by one hair was twice wrapped around the tip of the forceps and then pulled out by a fast movement. Fig. 1 illustrates a tip of the plucked human hair - hair follicle. If pulled out by the fast movement, it regularly contains a whole follicle which ends with the hair bulb. In this figure, the hair root sheath is also well visible. The length of here-shown follicle is 2 mm. Fig. 2 illustrates an upper portion of the hair follicle which we isolate during the claimed protocol. Tip of the arrow is showing the region from which the majority of our cells are originating. The length of the here-shown part of the follicle is 350 mm. Fig. 3 is a higher magnification of a portion of a hair follicle which demarcates the upper border of the region from where we isolate cells claimed in this protocol. Tip of the arrow is marking a junction point of the smooth muscle. A region rich with cells we are isolating is just beneath this segment Under control of a microscope, an upper portion of the hair root, above the main bulb, near the connection with sebaceous gland was cut and then immersed in Collagenase and Dispase (Roche, Fisher Scientific), using 35 -mm cell culture dish (Thermo Fisher Scientific) and left at 4°C for 12 hours (overnights). Fig. 4 illustrates a hair follicle after treatment with enzymes Collagenase and Dispase for separation of an epithelial layer (root sheath) from a major portion of a hair sample. In Fig. 4 the region rich with cells used in subject method is marked by circle. After rinsing in Phosphate buffered saline (PBS) and mechanical removing of epithelial layer, small pieces of tissue were immersed in a medium comprising DMEM (Gibco by Life Technologies, Thermo Fisher Scientific, Pittsburgh, PA) supplemented with Penicillin/Streptomycin (Gibco by Life Technologies), anti-fungal nystatin (Gibco by Life Technologies) and plated on 35-mm culture dish treated by fibronoctin. Small pieces of tissue adhered to the surface treated by fibronectin within 1 h. Preferred incubation parameters were 37 °C, 5% CO2 and 10% O2. Fig. 5 illustrates an elongated cell which migrate out the isolated (upper) portion of the hair follicle. They very strongly attach to the fibronectin-coated surface and migrate around the pieces of follicle. After 5 days of the start of cultivation, human recombinant epidermal growth factor (Merck, catalog number GF144) was added and then 5 more days were given to cells to multiply. Fig. 6 shows typical thick colonies formed 5-7 days after migration of the hair follicle cells. Cells are not anymore so elongated and spindle shaped, but slowly became more rounded. They exhibit high stability in the cultivation medium and very low cell death, less than 10%. Pieces of hair tissue were removed, cells were detached from the surface using trypsin and then counted using counting chambers. After 5 days, we obtained approximately 10.000 of early neuronal precursors cells from each hair - they were seeded on fibronectin treated glass in DMEM. After letting them 4 hours to attach, they were fixed by 4% Paraformaldehyde solution. Immunocytochemistry with DAPI by primary antibody nestin and secondary antibody Alexa-488 allowed to recognize that more than 95% of early neuronal precursors cells were strongly nestin positive. Fig.7-(A) illustrates cells isolated from the upper portion of the hair follicle. Fig. 7- (B) illustrates cells obtained five days after start of the cultivation. Fig. 7 (C) illustrates stem cells which are nestin positive. It revealed that isolated early neuronal precursors cells express nestin - early marker of neuronal precursors. Fig. 8 is a graph illustrating comparison of efficiency in obtaining neuronal precursors from 6 volunteers - 3 males (age 23, 36 and 59) and 3 females (age 23, 35, 61) obtained from 5 hairs after 5 days. Although no statistically significant difference was observed in regard to age or sex, there was a slight tendency to obtain more cells in volunteers of younger age. Average number of obtained early neuronal precursors cells from total of 5 hairs after 5 days was 51.000ti.200.
In this example we have clearly shown that the claimed method may be used to obtain early neuronal precursors cells from human hair root By using 6 volunteers, 3 males and 3 females, we have shown show that a sufficient number of early neuronal precursors cells is obtained regardless of the age and sex.
Example 2: Comparison of efficiency of transformation of early neuronal precursors cells obtained from hair follicle cells into late neuronal precursors cells bv using hvpoxia and normoxia
Isolation of cells from different body regions can yield different results. Also, different protocols can differ in efficiency to obtain a sufficient number of cells. Protocols usually include description of specific surfaces needed for cell attachment, specific media and specific components which help to obtain needed type of cells. In here claimed method we developed a specific protocol which allow to obtain a sufficient number of hair follicle cells and to transform them efficiently into neurons. Our method is based on transformation medium which contains specific components needed to transform selected hair follicle cells into neurons. In addition, cells are exposed to cycles of hypoxia and acidosis. We have shown that combination of hypoxia and acidosis support transformation of cells into neurons.
Hair follicle cells obtained according to our protocol for isolation of cells (described in Example 1) were cultivated in a low ambient oxygen conditions (10% oxygen) for 24 h in DMEM (Gibco by Life Technologies, Thermo Fisher Scientific, Pittsburgh, PA) and Penicillin/Streptomycin (Gibco by Life Technolopes). Then there were transferred into a transformation culturing medium. The transformation culturing medium is modified cultivating medium comprising Opti-MEM (Gibco by Life Technologies, Thermo Fisher Scientific, Pittsburgh, PA) with components B27, N2, BDNF (all Gibco), Peniciliin/Streptomycin (Gibco by Life Technologies) in which following transformation components have been added: Forskolin, 14 mM; DNP-derived active peptide, NAP, 1 mM, CHIR99021, 3mM, LIF,
20 ng/ml and FGF2, 20 ng/ml. After adding above mentioned transformation components, two approaches have been tested: cultivation of cells in normoxia (20% of oxygen) and approach in which cycles of transient hypoxia and acidosis have been applied. Cells not treated by cycles of hypoxia were cultivated 48 hours with added above mentioned transformation components.
For cells exposed to hypoxia (low ambient oxygen conditions), alternating cycles of hypoxia and acidosis have been applied as follows:
10% oxygen (first 24 hours);
3 cycles of hypoxia and acidosis as follows:
5h 3.5% oxygen condition followed by 2h of 10% oxygen condition,
20 min of pH change and then back to normal pH (pH reduced from 7.4 to 6.4) at 10% oxygen condition,
5h 3.5% oxygen condition followed by 12h of 10% oxygen condition,
20 min pH change then back to normal pH at 10% oxygen condition, and
5h 3.5% oxygen condition followed by 2h 10% oxygen condition.
In order to compare efficiency in transformation into neurons, the transformation medium was exchanged with neuronal medium in both cell groups (normoxia and hypoxia).
Cells were plated on 12-mm coverslips (75000 cells/coverslip). Coverslips and wells were previously coated with Poly-D-lysine (500 mg/mL, 24h at 37°C, Sigma) and laminin (10 mg/mL, 24h at 37°C, Sigma). In the first 24 hours, the proliferation medium was DMEMZF-12 (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA), 20 ng/mL Recombinant Mouse Fibroblast Growth Factor- basic (FGFb, ThermoFisher Scientific, Pittsburgh, PA, USA) and 20 ng/mL Recombinant Mouse Epidermal Growth Factor (EOF, ThermoFisher Scientific) with B-27 Supplement (Gibco by Life Technologies), N-2 Supplement (Gibco by Life Technologies) and Penicillin/Streptomycin (Gibco by Life Technologies). 24 hours after plating, the medium was changed into a differentiation medium comprising Neurobasal (Gibco by Life Technologies) which supports neurons in vitro and antibiotic with glutamine (Pen Strep Glutamine, Gibco by Life Technologies).
In both groups, cells were let to differentiate for 5 days and then immunohistochemistry with marker of neurons - Map2 antibody (plus DAPI) was performed.
In the first group, cells cultivated in 20% oxygen, exposed to transformation components and then transferred to the differentiation medium, efficiency of transformation was reaching average of 65±7%. In 15±5% of cells, signs of cell death were observed. Presence of Map2 was rather heterogenous, with cells with a strong signal and cells which exhibited a very weak presence of Map2. In another group, which was exposed to cycles of hypoxia and acidosis, average presence of Map2 was higher, reaching up to 85%. Moreover, cell population was more homogenous with uniform expression of Map2. Fig. 9 illustrates cells three days after they have been exposed to the transformation culturing medium. It can be seen that cells continue to form a shape between elongated spindles and rounded cells. Despite cycles of hypoxia and acidosis, cell death is very rare and cells continue to multiply. Fig. 10 illustrates comparison of Map2 expression, marker of differentiated neurons in the protocol where nestin precursors were directly transferred into the differentiation medium (A) and when additional step, with transformation using the transformation medium, hypoxia and acidosis is used (B). For the specific purpose of collecting medium in which we need to detect levels of biomarkers it is important to get as much as possible homogenous cell population. This is present in the case when additional transformation step is added (B). Bar - 100 pm.
Example 3: Testing the potential to isolate hair follicle stem cells after sending them in a transporting medium
With the goal to develop a diagnostic test which will be based on a simple self-sampling available to every human subject, here we present results obtained by experiment in which 10 volunteers have been asked to send a sample of their hair in a vial filled with a transporting medium. It was hypothesized that the usual medium will be good enough for cells to survive in a sufficient amount, but we also wanted to test if 3 and 5 days of delay between plucking hairs and cell isolation will make a difference.
Ten volunteers, age 20 to age 88, median age 53, were given the vial of 2 ml, filled with the transporting medium consisting of DMEM (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA) supplemented with Penicillin/Streptomycin (Gibco by Life Technologies), anti-fungal nystatin (Gibco by Life Technologies) and anti-mycoplasma (Gibco by Life Technologies). Fig. 11 illustrates the vial filled with the transporting medium. Every subject received a simple drawing in which it was shown feat they need to take forceps, wrap a hair (one by one) around it and then pull it out applying a sudden movement. The tip of the hair which was pulled out of the scalp has to be immersed in the transport medium, while the opposite end can be cut (depending on the length of the individual hair) or, which is also possible, can be let to protrude outside when the vial is closed by complete insertion of the screw cap. Subjects have been asked to pull out 10 hairs from their scalp and immerse them one by one into the vial filled with the transporting medium. We instructed them that after they collect 10 hairs, they can decide either to cut all ten hairs by scissors above the opening of the vial or just to close the vial by careful inserting the screw cap. We also suggested if the person has problems with fine coordination that this procedure should be carried out by a more skillful member of the household, a neighbor or a friend. In this case, all procedures were carried out by original volunteers, without any help from family members, friends or neighbors. As instructed, upon the sampling procedure, vials were put into the middle size envelope with absorbing bubbles. The subjects were also instructed to fill in the form in which name, age and the date and hour of sending were clearly written. Then, the samples were sent to the laboratory using express postal service. All vials were received whhin 72 h of sending them. We separated them in two groups, one which were processed immediately (maximum 72 hours after sending) and the samples which were let to wait 2 more days, which means they were processed 5 days after sampling.
Of ten received samples, five samples had ten hairs, two samples had twelve hairs, one sample had nine hairs, one sample had eight hairs and one sample had four hairs. On average, we received 9.5 hairs per subject We did not observe any lack of sampling medium or hair roots which were dried out. Also, in all samples, it was the hair root which was correctly placed (facing the bottom of the vial). When we compared the age of the subjects sending the required (or even larger) number of hairs with tire samples in which we received smaller number of samples, there was some tendency that older subjects make more mistakes, but this was not found as relevant As shown in above and below presented tests, the final result can be obtained with only 3-5 hairs, so we concluded that if 10 hairs were required, in great majority of cases we will receive enough hairs to perform the measurement.
Upper portion of the hair root was recognized on the microscope, cut and then immersed in Collagenase and Dispase (Roche, Fisher Scientific), using 35-mm cell culture dish (Thermo Fisher Scientific) and left overnight - 12 hours at +4 °C. After rinsing in Phosphate buffered saline (PBS) and mechanical removing epithelial layer, small pieces of tissue were immersed in DMEM (Gibco by Life Technologies, Thermo Fisher Scientific, Pittsburgh, PA) supplemented with Penicillin/Streptomycin (Gibco by Life Technologies) and anti-fungal nystatin (Gibco by Life Technologies) and plated on 35- mm culture dish treated by fibronectin. Small pieces of tissue adhered to the surface treated by fibronectin within 1 h. Incubation parameters were 37º C, 5 % C02 and 10 % 02. After 5 days of the start of cultivation human recombinant epidermal growth factor (Merck, catalog number GF144) was added and then 5 more days were given to cells to multiply. Pieces of hair tissue were removed, cells were detached from the surface using trypsin and then counted using counting chambers. Five thousand of cells from each sample were seeded on fibronectin treated glass in DMEM. After letting them 4 hours to attach, they were fixed by 4% PFA. The main goal of the immunocytochemistry was to detect if stem cells from the hair root after 3 and 5 days of transport in the transporting medium can be obtained. For this purpose, we used primary antibody nestin, secondary antibody Alexa-488 and DAPL Fig. 12 is graph showing comparison in efficiency of obtaining hair follicle cells after sending them in the vial, with isolation occurring 3 and 5 days after sampling the hair. Graph is showing efficiency in 10 subjects of different age (20-88), number of hairs they sent to us (4-12) and number of cells obtained. One of the most important observations here is that the number of cells obtained 5 days after sampling is significantly lower than in the case when delay was 3 days. As shown in fig. 12, a significantly larger number of cells was obtained in samples which were processed after three, when compared to samples which were processed after five days. On the other hand, no significant differences were observed in expression of nestin, which was in both groups of samples rather homogenously expressed. Like in some previous experiments, samples obtained from younger subjects had tendency to give more cells, but this was not found as relevant However, number of cells received after five days would be in, most of the cases sufficient for needed analyses. Moreover, in the case when we would require subjects to send 15-20 hairs, our estimation is that in more than 98% of cases, we would be able to perform the test without a need to repeat hair sampling.
Measurement of levels of biomarkers
This invention comprises elements of measurement of levels of molecules for which we or others have shown that can be used as biomarkers. More precisely, in this invention we show in which way one needs to analyze levels of molecules (e.g. proteins) to get a reliable result
In the application where the molecule of interest is an intracellular protein, the quantity of tiie protein is measured using a lysate of cells cultured in above defined conditions.
If tiie molecule of interest is the protein which can be detected only or preferably outside of cells, the total quantity of the protein found in the total medium is measured. Quantity of the protein of interest is normalized to the total quantity of detected proteins. When differentiated neurons are obtained using tiie in vitro method of the present invention, different biomarkers can be detected, with the important advantage that they are detected directly on the mature neurons developed from the tested human subject In the case when disease of interest is Alzheimer's disease, the molecule of interest which is here declared as the biomarker is human beta amyloid 1-42 (Ab42). This is the fragment produced by cleavage of amyloid precursor protein (APP) by B- and y-secretase. This peptide is composed of 42 amino acid residues (AB42).
In the case when disease of interest is amyotrophic lateral sclerosis, the biomarker of interest may be level of cellular excitability. In the case when disease of interest is Parkinson disease, the biomarker of interest may be diffuse cytoplasmic accumulations of a-synuclein and Lewy body precursor inclusions.
In general, assessment of the concentration of any biomarker from the medium can be obtained by methods routinely used, like ELISA, Western blotting, immunoprecipitation, quantitative immunocytochemistry, radioimmunoassay or similar methods. Concentration of the protein needs to be assessed in comparison to some of the house keeping genes, used as reference (e.g. GAPDH, HSP90, beta tubulin).
Neurons obtained by the present in vitro method are left 2 weeks to reach a mature stage. For all this time period, supernatant is constantly collected (3 times per week), centrifuged at 4 °C at 2,500 rpm for 7 minutes to remove the precipitate before use. To prevent protein degradation, 4-(2-aminomethyl) benzene-sulfonyl fluoride hydrochloride is added to the medium, which is stored at -80 °C till the use.
For the purpose of detection of concentration of beta amyloid, our own antibody raised against amino acids 33-42 of Human beta Amyloid 42 is used. To measure levels of proteins, ELISA test can be applied. ..Sandwich ELISA“measures concentration of antigen between two layers of antibodies (usually called - capture and detection antibody). The antigen of interest typically contains two antigenic sites capable of binding two different antibodies. The first step is composed of coating the 96 well plate with the capture antibody (cone 5 -20 mg/mL), in carbonate buffer pH 9.6. 12 hours, 4 C. After washing in phosphate buffer, blocking step using 5% non-fat dry milk, 2 hours in PBS is applied. After another washing round, the plate is ready to accept the peptide. 100 mL of medium from the cell cultures is added, quadriplicates, 120 min, 37 C. Washing step with phosphate buffer, 50 mL of detection antibody is added and left for 2 h for incubation at room temperature. Wash the well with phosphate buffer. Add secondary antibody conjugated with alkaline phosphatase, 2 h room temperature. Wash the well with phosphate buffer. Perform detection using PNPP substrate. Measure the yellow color of nitrophenol at 405 nm after 15-30 min incubation at room temperature and stop the reaction by adding equal volume of 0.75 M NaOH. Final concentration of protein is measured by preparing a standard curve from the serial dilutions (Thermo Scientific Pierce BSA Protein Assay Standards) data with concentration on the x axis (log scale) vs absorbance on the Y axis (linear). Concentration of the protein of interest is normalized to the total concentration of proteins.
Examnle 4: Measuring levels of beta 42 amyloid in Alzheimer's disease affected patients using neurons obtained from the hair follicle
The goal of this experiment was to use method we developed and to test if it can be used to detect levels of beta amyloid in patients affected by Alzheimer’s disease. The molecule of interest, which is here declared as the biomarker is human beta amyloid 1-42 (Ab42). This is the fragment produced by -leavage of amyloid precursor protein (APP) by B- and y-secretase. This peptide is composed of 42 amino acid residues (AB42).
For this purpose, we selected two patients clinically diagnosed is Alzheimer's disease. Subject 1, a female, age 75, with history of Alzheimer's dementia lasting for 4 years, with mini mental state score of 15 (moderate dementia). No other peculiar health problems. Subject 2, a male, age 77, with history of Alzheimer's dementia lasting for 5 years, with mini mental state score of 14 (moderate dementia). History of two heart infarctions and diabetes type 2.
In the same experiment we had healthy controls of the same age, a female, age 75 and a male, age 77. Both with controlled hypertension. In the case of healthy controls, they signed informed consent for sampling 10 hairs and in the case of Alzheimer’s patient, the informed consent was signed by legal representatives - family members (in both cases, daughters). All 4 subjects were Caucasians without any history of hair/skin disease. Ten hairs were taken from each of the subject by the same person. A simple metal forceps and the same procedure were used. One by one hair was twice wrapped around the tip of the forceps and then pulled out by a fast movement. Under control of a microscope, upper portion of the hair root, above the main bulb, near the connection with sebaceous gland was cut and then immersed in Collagenase and Dispase (Roche, Fisher Scientific), using 35 -mm cell culture dish (Thermo Fisher Scientific) on +4 overnight. After rinsing in Phosphate buffered saline (PBS) and mechanical removing epithelial layer, small pieces of tissue were immersed in DMEM (Gibco by Life Technologies, Thermo Fisher Scientific, Pittsburgh, PA) supplemented with PenicillinZStreptomycin (Gibco by Life Technologies) and anti-fimgal nystatin (Gibco by Life Technologies) and plated on 35- mm culture dish treated by fibronectin. Small pieces of tissue adhered to the surface treated by fibronectin within 1 h. Preferred incubation parameters were 37° C, 5 % CO2 and 10 % O2. After 5 days of the start of cultivation human recombinant epidermal growth factor (Merck, catalog number: GF144) was added and then 5 more days were given to cells to multiply. Pieces of hair tissue were removed, cells were detached from the surface using trypsin and then counted using counting chambers. The transformation culturing medium is comprising modified cultivation medium (Opti-Mem based) in which following components have been added: Forskolin, 14 mM; DNP-derived active peptide, NAP, I mM, CHIR99021, 3mM, LIF, 20 ngZml and FGF2, 20 ng/ml. After adding above mentioned components, two approaches have been tested: cultivation of cells in normoxia (20% of oxygen) and approach in which cycles of transient hypoxia and acidosis have been applied.
Cells not treated by cycles of hypoxia were cultivated 48 hours with added components.
For cells exposed to hypoxia, cycles of hypoxia and acidosis have been applied as declared here:
10% oxygen (first 24 hours); 3 cycles of hypoxia and acidosis as follows:
5h 3.5% oxygen condition followed by 2h of 10% oxygen condition,
20 min of pH change and then back to normal pH (pH reduced from 7.4 to 6.4) at 10% oxygen condition,
5h 3.5% oxygen condition followed by 12h of 10% oxygen condition,
20 min pH change then back to normal pH at 10% oxygen condition, and
5h 3.5% oxygen condition followed by 2h 10% oxygen condition.
Neurons are left 2 weeks to reach a mature stage. For all this time period, supernatant is constantly collected (3 times per week), centrifuged at 4 °C at 2,500 rpm for 7 minutes to remove the precipitate before use. To prevent protein degradation, 4-(2-aminomethyl) benzene-sulfonyl fluoride hydrochloride is added to the medium, which is stored at -80°C till the use.
In our experimental setup we used fused cell populations coming from different hairs. This was acceptable because in experiments in which we checked if different hairs would give different results, we, as expected, did not obtain any difference, neither in efficiency of transformation, neither in number of obtained cells, neither in number of obtained neurons, neither in levels of detected amyloid.
For the purpose of detection of concentration of beta amyloid, our own antibody raised against amino acids 33-42 of human beta amyloid 42 was used. The first step is composed of coating the 96 well plate with the capture antibody (cone 5 -20 ng/mL), in carbonate buffer pH 9.6. 12 hours, 4 C. After washing in phosphate buffer, blocking step using 5% non-frit dry milk, 2 hours in PBS is applied. After another washing round, the plate is ready to accept the peptide. 100 mL of medium from the cell cultures is added, quadriplicates, 120 min, 37 °C. Washing step with phosphate buffer, 50 mL of detection antibody is added 2 h incubation at room temperature, Wash the well with phosphate buffer. Add secondary antibody conjugated with alkaline phosphatase, 2 h room temperature. Wash the well with phosphate buffer. Perform detection using PNPP substrate. Measure the yellow color of nitrophenol at 405 nm after 15-30 min incubation at room temperature and stop the reaction by adding equal volume of 0.75 M NaOH. Final concentration of protein is measured by preparing a standard curve from the serial dilutions (Thermo Scientific Pierce BSA Protein Assay Standards) data with concentration on the x axis (log scale) vs absorbance on the Y axis (linear). After performing Elisa test in quadruplicate, the average concentration of beta A42 in in patients affected by Alzheimer's was 68 and 79 fM (normalized to the total quantity of detected proteins). In the same time average concentration of the protein in control healthy subjects was 24 and 16 fM. Fig. 13 shows that applying clamed in vitro method of transformation of hair follicle cells into neurons we can clearly distinguish between subjects affected by Alzheimer’s disease and healthy subjects. Average concentration of beta amyloid 42 for two patients was 68 and 79 fM, while for healthy subjects of the same age was 24 and 16 fM.
METHOD OF ESTIMATING OF THE BISK FOR ONSET OF A PARTICULAR BRAIN DISEASE
The present invention further provides a method of estimating of the risk for onset of a particular brain disease by using neurons obtained according to the in vitro method previously described. In addition, the method for estimating of the risk for onset of a particular brain disease may be used for diagnosing of a particular brain disease or for follow up of the existing brain disease by using said neurons. Said method provides a precise numerical value indicating what is the current risk of Mitering a symptomatic phase of brain disease within the next 5 years frame. Generally, the method comprises the steps of: plucking at least two hair samples and sending each hair sample to a laboratory in a vial containing a transporting medium, wherein sending of each hair sample is performed in a predetermined time interval; measuring of a level of at least one biomarker of interest from neurons obtained from each hair sample; and applying a scale, the scale is based on two subsequent measurements of the level of at least one biomarker of interest, wherein the scale defines a risk ranges for the onset of a particular brain disease, the risk ranges are based on a numerical difference between two subsequent measurements of the level of at least one biomarker of interest, wherein if more than one biomarker is measured, the numerical difference relates to the same biomarker for which a separate scale is established. The numerical difference of the measured levels of biomarkers is expressed as a percentage change, wherein to each risk range is assigned a corresponding range of the percentage change. An increase or decrease
- the numerical difference - of the measured levels of biomarkers obtained in two subsequent measurements is said percentage change. To each risk range a corresponding range of the percentage change is assigned. Said method can be applied for various brain diseases, including, but not limited to Alzheimer’s disease, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, dementia wife Lewy Bodies and other neurodegenerative or neuroinflammatoiy diseases. It can be applied for any disease in which the protein-biomarker is known and in which one can have a reliable scale for estimating the level of risk for developing the first symptoms.
The scale comprises 5 risk ranges based on the corresponding range of the percentage change of measured levels of biomarkers between two subsequent measurements. The risk range of up to 20% indicates a very low risk, the risk range of 20-40% indicates a low risk, the risk range of 40-60% indicates a moderate risk, the risk range of 60-80% indicates a high risk, and the risk range above 80% a very high risk of entering a clinically active disease within next 3 years. Each corresponding range of the percentage change is categorized according to the decades of life of a human subject. A predetermined time interval between two subsequent measurements is in a range between 1 and 5 years, preferably in the range between 1 and 2 years. The predetermined time interval depends on the measured level of biomarkers measured for the very first time. Since it is possible to have rather high levels of proteins without symptoms and on the other hand rather low levels with symptoms, the scale of the present invention is relative with time perspective. The method is based on detection of the time point in which a significant increase of measured levels of biomarker of interest occurs, thus giving a numerical value of the risk expressed in the scale. Generally, said predetermined time interval depends on the measured level of biomarkers of interest at the first measurement. Biomarkers are selected firm the group consisting of a protein, peptide, lipid, metabolite, nucleic-acid based molecule, cellular action potential, influx and efflux of certain molecules, status of mitochondria, status of microtubules, status of ribosomes or combinations thereof.
The scale is demonstrated for Alzheimer's disease, wherein measured level of biomarker is beta amyloid 42, but it may be used for other brain diseases characterized by other biomarkers.
Example 5: Stratification of subjects according to the risk of being affected by Alzheimer's disease. based on levels of beta 42 amyloid obtained from the hair follicle and on developed risk scale
The purpose of this experiment was to demonstrate that the scale for prediction of the risk of Alzheimer's can be used to predict disease onset. For this purpose, we have followed 3 families with diagnosed familiar Alzheimer's disease. In two families, mutation of PSEN2 was known, with history of being affected in late 40s years of life. In the third family, mutation of SORL1 was previously recognized with the history of onset of AD in late 50s years of life. Upon signing the informed consent, males aged
48 (SI), 56 (S2) and 69 (S3) were followed for up to 8 years.
Subject one (SI), age 48, Asian, his father died of Alzheimer's disease when he was 51, with disease onset of 45. In addition, one of his 5 brothers already died and one was in the last stage of disease. Genetic testing revealed that he is a carrier of the same mutation in PSEN2 gene.
The first neurological exam (point 0, age 48) revealed no abnormalities and the subject did not report any problems with memory, mood or any element of general functioning. Neurologic examination did not reveal any focal neurologic deficit Routine laboratory tests, including blood chemistry, electrolyte, and urine analyses, were normal. Thyroid function tests were normal. On Mini mental state exam, he scored 27/30, confirming no signs of disease. MRI imaging of the brain showed no peculiarities. Subject two (S2), age 56, Asian, reported two deaths of Alzheimer's disease of his older relatives. His sister died at the age of 47 with no clearly confirmed, but highly suspicious history of Alzheimer's. Genetic testing revealed R62C mutation, already well known for familial form of disease. The first neurological exam (point 0, age 56) revealed no neurological abnormalities. Neurologic examination did not reveal any focal neurologic deficit. Routine laboratory tests, including blood chemistry, electrolyte, and urine analyses, were normal. Thyroid function tests were normal.
On Mini mental state exam, he scored 25, which is borderline to mild dementia. After more detailed conversation, he revealed normal functioning, but with some elements of loss of interest for life and very mild personality changes.
Subject three (S3), age 69, mated Hispanic Asian race, reported that both of his parents died of disease which was highly suspicious for Alzheimer's, although without written proof. Another statement which could not be confirmed was that two close relatives were affected by dementia in their 60s. Genetic testing which was initiated by the subject revealed mutation in SORL1, which was known for both sporadic and familial cases of Alzheimer's disease. The first neurological exam (point 0, age 69) revealed weaker reflexes and a slight lateralization, which was later checked on CT scans. No morphological signs for any brain disease have been found. Routine laboratory tests revealed a borderline anemia and borderline decreased levels of potassium. Urine analyses revealed a serious infection, which was then treated. Thyroid function tests revealed signs of autoimmune hypothiroidism. On Mini mental state exam, he scored 26 and he did not express any problems suggesting Alzheimer's disease.
Since in the moment 0, all three subjects did not exhibit signs of Alzheimer's disease, but with the suspicion that they might develop it, based on family history and proven mutations, they were chosen to be followed during time.
Within one month of this initial test hairs from each subject have been collected.
In SI detected concentration of beta 42 amyloid was 10 fM
In S2 detected concentration of beta 42 amyloid was 27 fM.
In S3 detected concentration of beta 42 amyloid was 24 fM.
Taking the samples of the hair was repeated after 1 year (n 1) and the obtained results were as followed.
In SI detected concentration of beta 42 amyloid was 14 fM.
In S2 detected concentration of beta 42 amyloid was 50 fM.
In S3 detected concentration of beta 42 amyloid was 29 fM.
In all 3 subjects mini mental state exam was repeated.
Mini mental state exam revealed a change in S2 - the number of points decreased from 25 to 24.
Taking the samples of the hair was repeated after 1 year (n+2) and the obtained results were as followed. In SI detected concentration of beta 42 amyloid was 24 fM.
In S2 detected concentration of beta 42 amyloid was 49 fM.
In S3 detected concentration of beta 42 amyloid was 25 fM.
Mini mental state exam revealed a dramatic change in S2 - the number of points decreased from 24 to 20, so he entered a state of moderate dementia.
Taking the samples of the hair was repeated after 1 year (n+3) and the obtained results were as followed. In SI detected concentration of beta 42 amyloid was 41 fM.
In S2 detected concentration of beta 42 amyloid was 48 fM.
In S3 detected concentration of beta 42 amyloid was 23 fM.
Mini mental state exam revealed change only in S2, who additionally decreased his score from 20 to
18.
Taking the samples of the hair was repeated after 1 year (n+4) and the obtained results were as followed. In SI detected concentration of beta 42 amyloid was 37 fM.
In S2 detected concentration of beta 42 amyloid was 53 fM.
In S3 detected concentration of beta 42 amyloid was 31 fM.
Mini mental state exam revealed change in S2, who additionally decreased his score from 18 to 17.
S3 decreased from 26 to 25.
Taking the samples ofthe hair was repeated after 1 year (iri-5) and the obtained results were as followed. In SI detected concentration of beta 42 amyloid was 34 fM.
In S2 detected concentration of beta 42 amyloid was 51 fM
In S3 detected concentration of beta 42 amyloid was 42 fM.
Mini mental state exam revealed change in S2, who additionally decreased his score from 17 to 15, but the most significant change was SI who decreased from 27 to 22.
Taking the samples of the hair was repeated after 1 year (n+6) and the obtained results were as followed. In SI detected concentration of beta 42 amyloid was 39 fM.
In S2 detected concentration of beta 42 amyloid was 57 fM.
In S3 detected concentration of beta 42 amyloid was 68 fM. Mini mental state exam revealed change in S2, who additionally decreased his score from 15 to 14, but SI continued with fast decrease from 22 to 19.
Taking the samples of the hair was repeated after 1 year (n+7) and the obtained results were as followed. In SI detected concentration of beta 42 amyloid was 44 fM.
In S2 detected concentration of beta 42 amyloid was 56 fM.
In S3 detected concentration of beta 42 amyloid was 81 fM.
Mini mental state exam revealed change in SI, who decreased from 19 to 18.
Taking the samples of the hair was repeated after 1 year (n+8) and the obtained results were as followed.
S2 died of heart infarction.
In SI detected concentration of beta 42 amyloid was 42.
In S3 detected concentration of beta 42 amyloid was 80.
Mini mental state exam revealed change in SI, who decreased from 18 to 16 and dramatic decrease in S3 from 25 to 19.
Graphs in Figs. 14-16 show comparison of history of follow up of 3 subjects with confirmed mutations and the history of familial Alzheimer's disease.
Every year we were measuring levels of beta 42 amyloid and performing a detailed test of cognitive status, which is here shown as MMSE score. Cut off value, below which dementia starts was 24 points.
Subject 1 has shown a moderate increase in levels ofbeta42 amyloid in the first and significant increase in the 2nd and 3rd year. He developed dementia in the 5th year, which means 4 years from the moment of detected increase of the used biomarker. In the following years after disease onset, levels of beta 42 amyloid woe changing, but not significantly and the mental state was constantly deteriorating.
Subject 2 on the first measurement already exhibited an increased level of the biomarker. Indeed, in congruency with our estimation, already next year he exhibited a significant increase in the levels of beta 42 amyloid. Onset of his dementia is detected in the 3rd measurement This confirms that high levels of detected biomaiker require repeated measurement in the next year and they highly suggest that the increase in levels of beta 42 amyloid started before the first measurement. Same like in the Subject 1, when active disease has started, levels of beta 42 amyloid were not changing significantly, although the subject continued to worsen his mental status.
Subject 3 exhibited a constant low level of the biomarker in 4 subsequent years. Then, in year 5 we observed the first more significant increase, which was followed by three more years of a significant increase of amount of measured beta 42 amyloid. Similar to the Subject 1, Subject 3 exhibited onset of clinically relevant disease 4 years after observed a significant increase of the level of beta 42 amyloid.
SCALE FOR ESTIMATING TOE RISK FOR THE ONSET OF A PARTICULAR BRAIN DISEASE
Apart from significantly improved in vitro method to obtain neurons from the hair sample, which allows everybody to provide samples of plucked hair sample without need to go to the hospital, the present invention provides a scale based on detection and measurement of levels of biomarkers such as beta amyloid proteins in human population which was analyzed separately in their 5th, 6th, 7th and 8th decade of life. It was needed more than a decade of meticulous follow of up of levels of those proteins, based on following families with a high risk of onset of Alzheimer's disease (familial disease forms), but also based on genetically non-peculiar families and discovering sporadic spontaneous onset of disease to collect sufficient data for building the scale. The present invention provides the scale which is based on estimation of importance of incremental increase or decrease of levels of biomarkers of interest The innovative element which is in the core of this invention and which allows precise numerical estimation is detection of levels of proteins in for example 1- or 2-years* time interval distance, which then, interpolated to the scale according to the present invention gives precise levels of risk of onset of a particular brain disease. Different from other methods which combine measurement of levels of different proteins and give information about increased or non- increased risk, the present scale is numerical and gives a precise information of the risk in categories up to 20%, 20-40%, 40-60%, 60-80% and above 80%, within 3 years’ time interval. If, for example, someone enters in the first category, 20% or less risk, it is advised to repeat the test in 5 years. If someone, for example, enters the category of 80% risk and above, we highly advise additional neurological tests and start of the therapy with the goal to postpone disease onset Thus, the scale gives a precise insight in the current status of the biomarkers accumulation and it positions it within the time perspective. Different from others, which by increased or non-increased levels of some markers suggest presence of increased risk, which might be misleading, since it is possible to have rather high levels of proteins without symptoms and rather low levels with symptoms, the scale is relative with time perspective and may be personalized for each tested subject, and more generally the scale is categorized according to the decade of life of the tested subject This is the only way to detect a significant increase or decrease of measured level of at least one biomarker, thus giving a numerical value of the risk, a precise prediction for the onset of a particular brain disease. The scale for estimating the risk for the onset of a particular brain disease, diagnosing of a particular brain disease or for following up on an existing brain disease is based on two subsequent measurements of a level of at least one biomaiker of interest. The scale defines a risk ranges for the onset of a particular brain disease, the risk ranges are based on a numerical difference between two subsequent measurements of the level of at least one biomarker of interest. If more than one biomarker is measured, the numerical difference relates to the same biomarker for which a separate scale is established. The numerical difference of the measured levels of biomarkers is expressed as a percentage change. The percentage change is a mathematical concept that represents the degree of change over time. To each risk range a corresponding range of the percentage change is assigned. Further, the scale comprises 5 risk ranges based on the corresponding range of the percentage range. Each risk range classifies the tested subject for the onset of a particular brain disease in one of the following risk ranges: a very low risk (up to 20%), a low risk (20-40%), a moderate risk (40-60%), a high risk (60-80%) and a very high risk (above 80%). In addition, the scale is categorized according to the decades of life of the subject A predetermined time interval between two subsequent measurements is in a range between 1 and 5 years, preferably in a range between 1 and 2 years, wherein the predetermined time interval depends on the measured level of biomaricers measured for the very first time. The biomarkers are selected from the group consisting of a protein, peptide, lipid, metabolite, nucleic-acid based molecule, cellular action potential, influx and efflux of certain molecules, status of mitochondria, status of microtubules, status of ribosomes or combinations thereof. The brain disease is selected from the group consisting of including but not limited to, Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, and dementia with Lewy bodies and other neurodegenerative or neuroinflammatory diseases.
Graph in Fig. 17 shows the scale for entering into a symptomatic stage of Alzheimer’s disease for 50s and 60 years of life. (MMSE scale score lower than 25). The risk is defined by increase in detected concentration of beta 42 amyloid, obtained as a measured difference in percentage between two subsequent measurements. Preferred time difference between two measurements is 1-2 years. This scale is valid for subjects in their 50s and 60s years of life and only in the case when detected concentration in the first measurement is less than 40 fM/mg of the total protein concentration. Higher concentrations than 40 fM/mg of the total protein concentration are in majority of cases detected in clinically already detectable disease. Detected increases in concentrations of beta 42 amyloid correspond to the numerical risk of entering a symptomatic stage within up to next 5 years of life. Average time distance for a start of a clinically significant cognitive decline, which corresponds to MMSE score lower than 25, in the case when estimated risk is higher than 50% is 3.8 years from the second measurement Increase of beta 42 amyloid concentration for up to 22% suggests a very low risk (less than 20%), increase for 22-32% suggests a low risk (20-40%), increase for 32-63% suggests a moderate risk (40-60%). Increases between 63 and 80% suggest a probability up to 80% (high risk) to enter a clinically active disease within next 5 years. Increase for more than 80% of concentration of beta 42 amyloid has been found in only few sporadic cases which were already clinical active.
Graph in Fig. 18 shows the scale for entering into a symptomatic stage of Alzheimer's disease (MMSE scale score lower than 25). The risk is defined by increase in detected concentration of beta 42 amyloid, obtained as a measured difference in percentage between the first and the second measurement. Suggested time difference between two measurements is 2 years. This scale is valid for subjects in their 70s and 80s years of life and only in the case when detected concentration in the first measurement is less than 48 fM/mg of the total protein concentration. Higher concentrations than 48 fM/mg of the total protein concentration are in majority of cases detected in clinically already detectable disease.
Detected increases in concentrations of beta 42 amyloid correspond to the numerical risk of entering a symptomatic stage within up to next 5 years of life. Average time distance for a start of a clinically significant cognitive decline, which corresponds to MMSE score lower than 25, in the case when estimated risk is higher than 50% is 3.8 years from the second measurement Increase of beta 42 amyloid concentration for up to 32% suggests a very low risk (less than 20%), increase for 32-57% suggests a low risk (20-40%), increase for 57-71% suggests a moderate risk (40-60%). Increases between 71 and 91% suggest a probability up to 80% to enter a clinically active disease within next 5 years. Increase for more than 80% of concentration of beta 42 amyloid has been found in only few sporadic cases which were already clinically active.
The present invention also provides a kit to be used to transport a hair sample from the subject to the laboratory where the tests will be performed. The kit includes a vial with a screw cap, a transporting medium (see fig. 11), a written instruction for sampling and a form comprising fields for name, age and the date and hour of sending the hair sample. The written instruction consists of a simple drawing in which it is illustrated that forceps is needed in order to wrap a hair (one by one) around forceps and then pull it out applying a sudden movement and how to immerse a tip of each hair pulled out of the subject's scalp in the vial's transporting medium, while the opposite end can be cut (depending on the length of the individual hair) or, which is also possible, can be let to protrude outside when the vial is closed by complete insertion of the screw cap. Number of pulled hairs is preferably in a range between 10 and 20. After hair samples are collected, immersed in the transporting medium and the vial is properly closed, the subject is instructed to put the vial into the middle size envelope with absorbing cushions. The subject is also instructed to fill in the form in which name, age and the date and hour of sending is clearly written. Then, the samples are to be sent to a laboratory using express postal service.
The transporting medium comprises DMEM (Gibco by Life Technologies, ThermoFisher Scientific, Pittsburgh, PA) supplemented with Penicillin/Streptomycin (Gibco by Life Technologies), anti-fungal nystatin (Gibco by Life Technologies) and anti-mycoplasma (Gibco by Life Technologies).

Claims

1. An in vitro method of transformation of hair follicle cells into neurons comprises the steps of:
- plucking and sending a hair sample to a laboratory in a transporting medium;
- isolating of cells from the hair sample suitable for obtaining an early neuronal precursor cells;
- transformation of an isolated early neuronal precursors cells into a late neuronal
precursor cells, wherein transformation of the early neuronal precursor into the late neuronal precursor cells comprises steps of
- cultivating the isolated early neuronal precursor cells in a transformation culturing
medium for the first 24 hours in a low ambient oxygen condition followed by applying a transient cycles of hypoxia and acidosis;
- cultivating the late neuronal precursor cells in a proliferation medium in a low ambient oxygen condition; and
- differentiation of the late neural precursor cells into neurons on Poly-D-lysine and
laminin coated surface in a differentiation medium.
2. The method of claim 1, wherein isolating of cells from the hair sample further comprises steps of immersing the hair sample in a culture dish comprising Collagenase and Dispase enzymes for 12 hours at 4 °C, followed by rinsing in Phosphate buffered saline and isolating an upper portion of the hair root, above the main bulb, near the connection with a sebaceous gland.
3. The method of claim 2, further comprising immersing the isolated cells in DMEM
supplemented with Penicillin/Streptomycin and anti-fungal nystatin and adhering the isolated cells to a culture dish treated by fibranectin.
4. The method of claim 3, wherein isolating of the early neuronal precursors cells further
comprising adding a human recombinant epidermal growth factor.
5. The method of claim 1, wherein the transporting medium comprises DMEM supplemented with Penicillin/Streptomycin, anti-fungal nystatin and anti-mycoplasma.
6. The method of claim 1, wherein the transformation culturing medium comprises Opti-MEM, B27, N2, BDNF, Penicillin/Streptomycin, and peptides Forskolin, DNP-derived active peptide, NAP, CHIR99021, LIF, and FGF2.
7. The method of claim 1, wherein the low ambient oxygen condition for cultivating the isolated cells in the transformation culturing medium for the first 24 hours comprises an ambient oxygen condition of 10% oxygen.
8. The method of claim 1 , wherein the transient cycles of hypoxia and acidosis for cultivating the isolated cells in the transformation culturing medium, said cycles comprising steps of: a. Sh in an ambient oxygen condition of 3.5% oxygen followed by 2h in an ambient oxygen condition of 10% oxygen,
b. 20 min of 6.4 pH in an ambient oxygen condition of 10% oxygen followed by
increasing pH to 7.4,
c. 5h in an ambient oxygen condition of 3.5% oxygen followed by 12h in an ambient oxygen condition of 10% oxygen,
d. 20 min of 6.4 pH in an ambient oxygen condition of 10% oxygen followed by
increasing pH to 7.4, and
e. 5h an ambient oxygen condition of 3.5% oxygen followed by 2h an ambient oxygen condition of 10% oxygen.
9. The method of claim 1, further comprising cultivating the late neural precursors cells in the transformation culturing medium in an ambient oxygen condition of 5% oxygen.
10. The method of claim 1, wherein the proliferation medium comprises DMEM/F-12, B-27 Supplement, N-2 Supplement, Penicillin/Streptomycin, FGFb and EGF.
11. The method of claim 1, wherein the differentiation medium comprises Neurobasal and
Peniciilin/Streptamycin wife glutamine.
12. A neurons generated by the method of claim 1, wherein said neurons are positive for OCX, TUBB3, MAP2 and SYP.
13. An early neuronal precursors cells generated by the method of claim 1 , wherein said neuronal precursors are positive for NES, SOX2, MSX2, LRIG1, VARS2, ENAH, MYO10, SORBS3 and PFN1, but does not comprise MYC, KLF4 and POU5F1.
14. A late neuronal precursors cells generated by the method of claim 1, wherein said neuronal precursors are positive for NES and TBR1.
15. A method of estimating a risk for the onset of a particular brain disease, diagnosing of a particular brain disease or for following up on an existing brain disease by the use of neurons obtained according to the method of claim I, the method comprising the steps of:
- plucking at least two hair samples and sending each hair sample to a laboratory in a vial containing a transporting medium, wherein sending of each hair sample is performed in a predetermined time interval;
- measuring of a level of at least one biomarker of interest from neurons obtained from each hair sample; and
- applying a scale, the scale is based on two subsequent measurements of the level of at least one biomarker of interest, wherein the scale defines a risk ranges for the onset of a particular brain disease, the risk ranges are based on a numerical difference between two subsequent measurements of the level of at least one biomarker of interest, wherein if more than one biomarker is measured, the numerical difference relates to the same biomarker for which a separate scale is established.
16. The method of claim 15, wherein the numerical difference is expressed as a percentage change, wherein to each risk range a corresponding range of the percentage change is assigned.
17. The method of claim 16, wherein the scale comprises 5 risk ranges, wherein each risk range classifies the risk for the onset of a particular brain disease, the risk ranges are classified in the following ranges: up to 20% indicates a very low risk, 20-40% indicates a low risk, 40-60% indicates a moderate risk, 60-80% indicates a high risk, and above 80% indicates a very high risk.
18. The method of claim 16, wherein each corresponding range of the percentage change is classified according to the decades of life of the subject
19. The method of clam 15, wherein the predetermined time interval between two subsequent measurements is in a range between 1 and 5 years, preferably in the range between 1 and 2 years.
20. The method of claim 19, wherein the predetermined time interval depends on the measured level of said biomarkers measured for the first time.
21. The method of claims 15-20, wherein the biomarkers are selected from the group consisting of a protein, peptide, lipid, metabolite, nucleic-acid based molecule, cellular action potential, influx and efflux of certain molecules, status of mitochondria, status of microtubules, status of ribosomes or combinations thereof.
22. The method of claim 15, wherein the brain disease is selected from the group consisting of, including, but not limited to Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, dementia with Lewy bodies and other neuro degenerative or neuroinflammatory diseases.
23. The method of claim 22, wherein the brain disease is Alzheimer's disease.
24. The method of claim 23, wherein measured level of biomarker is beta amyloid 42.
25. A scale for estimating the risk for the onset of a particular brain disease, diagnosing of a particular brain disease or for following up on an existing brain disease, wherein the scale is based on two subsequent measurements of the level of at least one biomarker of interest, wherein the scale defines a risk ranges for the onset of a particular brain disease, the risk ranges are based on a numerical difference between two subsequent measurements of the level of at least one biomarker of interest, wherein if more than one biomarker is measured, the numerical difference relates to the same biomarker for which a separate scale is established.
26. The scale of claim 25, wherein the numerical difference is expressed as a percentage change, wherein to each risk range a corresponding range of the percentage change is assigned.
27. The scale of claim 26, wherein the scale comprises 5 risk ranges, wherein each risk range classifies the risk for the onset of a particular brain disease, the risk ranges are classified in the following ranges: up to 20% indicates a very low risk, 20-40% indicates a low risk, 40-60% indicates a moderate risk, 60-80% indicates a high risk, and above 80% indicates a very high risk.
28. The scale of claim 26, wherein each corresponding range of the percentage change is classified according to the decades of life of the subject
29. The scale of claim 25, wherein a predetermined time interval between two subsequent measurements is in a range between 1 and 5 years, preferably in the range between 1 and 2 years.
30. The scale of claim 29, wherein the predetermined time interval depends on the measured level of said biomarkers measured for the first time.
31. The scale of claims 25-30, wherein said biomaikers are selected from the group consisting of a protein, peptide, lipid, metabolite, nucleic-acid based molecule, cellular action potential, influx and efflux of certain molecules, status of mitochondria, status of microtubules, status of ribosomes or combinations thereof.
32. The scale of claim 25, wherein the brain disease is selected from the group consisting of, including but not limited to, Alzheimer’s disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, and dementia with Lewy bodies and other neurodegencrative or neuroinflammatory diseases.
33. The scale of claim 32, wherein the brain disease is Alzheimer’s disease.
34. The scale of claim 33, wherein measured level of biomarker is beta amyloid 42.
35. A neurons transformed from a hair follicle cell of a subject for use in a method of estimating the risk for the onset of a particular brain disease, diagnosing of a particular brain disease and for following up on an existing brain disease, and wherein said neurons are as defined in claim
12.
36. A neurons of claim 12 for use according to claim 35, wherein said subject is a human subject.
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WO2022155708A1 (en) * 2021-01-22 2022-07-28 Skin2Neuron Pty Ltd Methods for manufacture of neuronal precursors
KR20220140431A (en) * 2021-04-09 2022-10-18 가천대학교 산학협력단 A biomarker for neurodegenerative diseases containing a glycotoxin, a specific protein bound to glycotoxin, or glycotoxin-specific protein complex
KR102490838B1 (en) 2021-04-09 2023-01-20 가천대학교 산학협력단 A biomarker for neurodegenerative diseases containing a glycotoxin, a specific protein bound to glycotoxin, or glycotoxin-specific protein complex

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