A method of generating human ipscs-derived forebrain-specific astrocytes, and uses thereof

Abstract

The present disclosure pertains to developing a method for generating iPSCs-derived forebrain-specific astrocytes (FSAs) from human subjects carrying Fragile X Syndrome (FXS) mutations, and uses thereof. The present invention also relates to a method of modelling neurodevelopmental disorders using human iPSCs-derived FSAs from a human subject affected by a neurodevelopmental disorder such as FXS.

Description

A METHOD OF GENERATING HUMAN iPSCs-DERIVED FOREBRAIN-SPECIFIC ASTROCYTES, AND USES THEREOFFIEED OF THE INVENTION

[0001] The present disclosure pertains to developing a method for generating iPSCs- derived forebrain-specific astrocytes (FSAs) from human subjects carrying Fragile X Syndrome (FXS) mutations, and uses thereof. The present invention also relates to a method of modelling neurodevelopmental disorders using human iPSC (hiPSC)-derived FSA from a human subject affected by a neurodevelopmental disorder.BACKGROUND OF THE INVENTION

[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the present invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Fragile X Syndrome (FXS), a common inherited form of intellectual disability and autism spectrum disorder (ASD), is caused by the lack of fragile X messenger ribonucleoprotein (FMRP) produced by the Fragile X Messenger Ribonucleoprotein 1 (FMRI) gene (OMIM: #300624, https: / / www.omim.org / entry / 300624). FMRP plays a role in the regulation of mRNAs translation, mRNA granule formation and transport, and micro RNA mediated regulation of gene expression (Santoro et al., 2012). Thus, loss of FMRP impacts not just brain development but also adult brain function. Both mRNA transcript levels of FMRI and immunostaining for FMRP in the brain have shown high neuronal expression, alongside significant expression in glial cells as well (Wang et al., 2004). However, a vast majority of earlier studies in animal models of FXS focused primarily on neurons and aberrations in their function. Consequently, little is known about the role of glia in FXS (Pacey and Doering, 2007). Traditionally thought of as “passive support” cells (Zhang et al., 2016), there is accumulating evidence for astrocytes being critical in mediating a wide range of neuronal function (Allen and Barres, 2009; Khakh and McCarthy, 2015), including promoting synaptogenesis (Allen et al., 2012), refinement of developing neural circuits (Chung et al., 2013), and neurotransmitter recycling (Rothstein et al., 1996). In parallel, there is growing evidence for the role of astrocytes in disease pathogenesis and manyneurodevelopmental conditions have been associated with astrocytic dysfunction (Almad and Maragakis, 2018).

[0004] While much of the earlier work using animal models of FXS focused on identifying and validating various molecular targets in neurons for treating FXS, these pre- clinical findings have not always led to successful clinical outcomes. Further, setbacks in recent clinical trials also underscore the need for human-based model systems. Models of neurodevelopmental disorders based on human stem cell-derived brain cells offer a powerful strategy to bridge this gap between mechanistic insights from animal studies and limited success with clinical outcomes for patients. However, only a handful of these studies have focused on astrocytes, and that too mostly on astrocytes that were not of forebrain origin.

[0005] Studies show that structure and function of astrocytes vary between brain regions (Zhang and Barres, 2010; Clarke and Barres, 2013). Thus, a better understanding of disease- induced changes in human astrocytes also needs to take into account these brain regionspecific difference in astrocytes. However, models of neurodevelopmental disorders using human stem cell-derived astrocytes that are specific to the forebrain remain relatively less explored (Bradley et al., 2019).

[0006] Therefore, general purpose of the invention is to develop methods for generating induced pluripotent stem cells (iPSCs)-derived FSAs from human subjects carrying FXS mutations and to a method of modelling neurodevelopmental disorders such as FXS using hiPSCs-derived FSA from a human subject affected by a neurodevelopmental disorder.OBJECTS OF THE INVENTION

[0007] Objects of the present invention is to generate FSAs from induced pluripotent stem cells (iPSCs) derived from human subjects carrying FXS mutations.

[0008] An object of the present invention is to provide a method for generating iPSCs- derived FSAs from human subjects carrying FXS mutations.

[0009] Another object of the present invention is to provide a method of modelling neurodevelopmental disorders such as Fragile X syndrome using human iPSCs (hiPSCs)- derived FSAs.

[0010] Another object of the present invention is to provide a method of studying neurodevelopmental disorders such as Fragile X syndrome using hiPSCs-derived FSAs.

[0011] Another object of the present invention is to provide biological markers for identifying neurodevelopmental disorders such as Fragile X syndrome using hiPSCs-derived FSAs.

[0012] Yet another object of the present invention is to provide a method for identifying a candidate compound from a library of compounds using hiPSCs-derived FSA for the treatment or prevention of FXS.SUMMARY OF THE INVENTION

[0013] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in detailed description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0014] Aspects of the present disclosure pertains to a disclosure pertains to a method for generating iPSCs-derived FSAs from human subjects carrying FXS mutations and to a method of modelling neurodevelopmental disorders using hiPSCs-derived FSA from a subject affected by a neurodevelopmental disorder, and uses thereof.

[0015] In an aspect, the present invention provides a method of generating FSA from hiPSCs, said method comprising the steps of: a. enzymatically lifting hiPSCs, followed by suspension culture in a chemically defined medium to obtain a cell suspension; b. developing corticospheres by culturing the cell suspension under constant shaking and normoxic conditions; c. transferring corticospheres into a cell proliferation medium for 5-10 days, followed by generating early gliospheres by subjecting to glial enrichment medium for 10-20 days; d. generating “late gliospheres” by subjecting early gliospheres to glial enrichment medium (EGF and bFGF) including leukemia inhibitory factor (LIF) with constant shaking for 3-6 weeks; e. dissociating the “late gliospheres” into monolayers of astrocyte progenitor cells (APCs) by enzymatic dissociation every fortnight; and f. differentiating APCs into FSAs by plating onto Matrigel® coated plates for 10-20 days with astrocyte differentiation medium comprising Neurobasal medium supplemented with ciliary neurotrophic factor (CNTF).

[0016] In an aspect, the present invention provides a terminally differentiated population of FSA derived from hiPSC of a human subject having FXS, wherein the terminally differentiated population of FSA is functional and expresses lower level of glial fibrillary acidic protein (GFAP) positive astrocyte percentage; lower level de novo protein synthesis; lower level of ATP -induced intracellular calcium transients; bioenergetic deficits; and lower mitochondrial oxygen consumption, compared to control levels as found in normal FSA. In an embodiment, the altered levels are identifying biomarkers for FXS .

[0017] In one aspect of the present invention, the FSA, related systems, and methods of this invention can also be useful for modelling neurological diseases. In some embodiments, the present invention provides a novel human-based platform to model FSA in vitro, in which hiPSCs-derived FSA can be used to study reactive states and interrogate their role in neurodevelopmental and neurodegenerative diseases, preferably FXS.

[0018] In another aspect, the present invention provides a method of modelling FXS in a human subject, said method comprises the steps of: a) programming terminally differentiated population of FSAs from the iPSCs as claimed in anyone of claims 1-9; and b) analyzing the terminally differentiated population of FSA for at least one of: GFAP positive astrocyte percentage, de novo protein synthesis, ATP- induced intracellular calcium transient levels, and mitochondrial oxygen consumption and comparing the measured expression to control levels.

[0019] In yet another aspect, the present invention provides a method for identifying a candidate compound from a library of compounds for the treatment or prevention of FXS, said method comprising the steps of: a) obtaining terminally differentiated population of FSA from the hiPSCs by the methods as disclosed herein; b) exposing the terminally differentiated population of FSA to a library of compounds; c) measuring at least one of: GFAP positive astrocyte percentage, de novo protein synthesis, ATP -induced intracellular calcium transient levels, and mitochondrial oxygen consumption upon exposure to each of the compound from the library of compounds; d) comparing the measured levels from step c) to control levels; ande) identifying the candidate compound from the library of compounds based on the comparative results as per step d).

[0020] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.BRIEF DESCRIPTION OF DRAWINGS

[0021] Characteristics and advantages of the subject matter as disclosed in the present disclosure will become clearer from the detailed description of an embodiment thereof, with reference to the attached drawing, given purely by way of an example, in which:

[0022] FIG. 1 (A-F): relates to derivation of forebrain specific astrocytic progenitor cells (APCs) from hiPSCs. (A) Illustrative workflow for generation of astrocytes from hiPSCs. (B) Representative images of hiPSCs display comparable expression of Oct4 / Nanog in control and FXS lines. (C) Homogenous population of hiPSC derived APCs expressed a similar number of Vimentin and NF1A positive cells. (D) Quantification of Vimentin and NF1A positive APCs showed no significant difference across lines (Statistical analysis by two factor ANOVA followed by Tukey’s pairwise comparison). (E) Schematic illustrating region specific expression of FOXG1 and HOXB4 during development in vivo. (F) Graphical representation of FOXG1 expression compared with HOXB4 (using qRT-PCR) across genotypes suggesting propensity towards forebrain lineage. Statistical analysis by two factor ANOVA followed by Sidak’s multiple comparison test. For all experiments N=3 biological replicates. Scale bar = 50pm. ***p<0.001. Error bars represented as SEM

[0023] Figure 2 (A-D): relates to hiPSC derived FXS astrocytes reveal reduced number of glial fibrillary acidic protein (GFAP) cells. (A) Representative images of hiPSC derived astrocytes exhibiting S 100(3 and GFAP expression. (B) Percentage of GFAP positive astrocytes was significantly lower in FXS derived astrocytes compared to controls although both exhibited similar S 100(3 expression. Statistical significance by two factor ANOVA followed by Tukey’s pairwise comparison. (C) Immunoblot representing expression of FMRP in astrocytes from control and FXS. (D) Graphs showing absence of FMRP in hiPSC derived FXS astrocytes. Statistical analysis by single factor ANOVA with Tukey’s pairwise comparison. For all the above experiments N=3 biological replicates. Scale bar = 50pm. ***p<0.001. Error bars represent SEM.

[0024] Figure 3 (A-B): relates to Differentiation from APCs to astrocyte causes reduction in protein synthesis. (A) Representative images of (top) Vimentin positive hiPSC derived APCs and (bottom) GFAP positive astrocytes with FUNCAT / hNA label in control and FXS derived cells. (B) Graphical representation of FUNCAT / volume of hiPSC derived APCs was significantly higher than corresponding derived astrocytes. For all experiments N=3 biological replicates. Scale bar = 50pm. **p<0.01, ***p<0.001. Whiskers represent 1.5*IQR.

[0025] Figure 4 (A-F): relates to deficient Ca2+signaling evoked by ATP in hiPSCs- derived FXS astrocytes. (A) Representative traces of Ca2+transients recorded from individual astrocytes upon external application of ATP at 25thsecond. (B) Averaged F340 / F380 ratios depicting Ca2+transients over 250 seconds after ATP application. (C) Grouped data showing higher percentage of non-responders to ATP in hiPSCs-derived FXS astrocytes. Statistical significance by two factor ANOVA with Tukey’s pairwise comparison. (D-F) Quantification of first peak response (amplitude and duration) and frequency of events. (D) Shows a significant reduction in amplitude. Statistical significance by single factor ANOVA with Tukey’s pairwise comparison and (E) duration in hiPSC derived FXS astrocytes, Statistical significance by Kruskal-Wallis test with Dunn’s multiple comparison test, data set represent as mean ranks with SEM. (F) Graphical representation of Ca2+transient frequency in hiPSCs- derived FXS astrocytes. Statistical analysis by single factor ANOVA with Tukey’s pairwise comparison. For all experiments N=3 biological replicates; n=19 cells for each cell line. *p<0.05, **p<0.01, ***p<0.001. Error bars represent SEM.

[0026] Figure 5 (A-D): relates to the bioenergetic deficits in hiPSC derived FXS astrocytes. (A) Line graph representing extracellular acidification rate (ECAR) of hiPSC derived astrocytes (Control and FXS) after addition of 10 mM Glucose, 1 pM Oligomycin and 50 mM 2-DG sequentially and plotted on a line graph. (B) Glycolysis, glycolytic capacity and glycolytic reserve quantification showed increase in hiPSC derived FXS astrocytes. Statistical analysis by two factor ANOVA with Tukey’s pairwise comparison. (C) Line graph depicting oxygen consumption rate (OCR) measurement by sequential addition of 1.5 pM Oligomycin, 1 pM FCCP and 0.5 pM antimycin A and Rotenone. (D) Basal respiration, maximal respiration and ATP production was quantified from the line graph and hiPSC derived FXS astrocytes showed significant decrease in maximal respiration in comparison to control astrocytes. Statistical analysis by two factor ANOVA with Tukey’s pairwisecomparison. For all experiments N=2 biological replicates. *p<0.05, **p<0.01, ***p<0.001. Error bars represent SEM.DETAILED DESCRIPTION OF THE INVENTION

[0027] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0028] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0029] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0030] In some embodiments, numbers have been used for quantifying weight percentages, angles, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0031] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0032] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0033] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0034] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0035] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0036] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.

[0037] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of theprinciples and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0038] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0039] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0040] Various terms are used herein. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0041] While a particular form of the invention has been illustrated and described, it will be apparent that various modifications can be made without departing from the spirit and scope of the invention.

[0042] Embodiments of the present disclosure pertains to developing a method for generating iPSCs-derived forebrain-specific astrocytes (FSAs) from human subjects carrying Fragile X Syndrome (FXS) mutations, and uses thereof. The present invention also relates to a method of modelling neurodevelopmental disorders using human iPSC (hiPSC)-derived FSA from a human subject affected by a neurodevelopmental disorder

[0043] Astrocytes are critical in all types of brain pathologies from acute lesions to chronic neurodegenerative processes (For e.g. Alexander's disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis and the like) and psychiatric diseases. FSA are the most numerous and diverse neuroglial cells in the CNS. An archetypal morphological feature of FSA is their expression of intermediate filaments, which form the cytoskeleton. The main types of astroglial intermediate filament proteins are glial fibrillary acidic protein (GFAP) and vimentin.

[0044] In an embodiment of the present invention, the neurodevelopmental disorders or syndrome is selected from the group consisting of Fragile X syndrome, autism, RETT syndrome, schizophrenia, Angelman syndrome and Timothy syndrome. Fragile X syndrome (FXS), the most common cause of congenital neurodevelopmental conditions, is resulted byFMRI mutation. FXS patients show cognitive impairment, autistic features, attention deficits, increased rates of epilepsy, and motor abnormalities. In humans, polyglutamine repeats in the FMRI gene lead to loss of FMRP protein expression.

[0045] In an embodiment of the present invention, the FSA means a cell derived from hiPSC upon differentiation into the Astrocytic Progenitor Cells (APCs).

[0046] In an embodiment, the present invention provides a method of generating FSAs from hiPSCs, said method comprising the steps of: a. enzymatically lifting hiPSCs, followed by suspension culture in a chemically defined medium to obtain a cell suspension; b. developing corticospheres by culturing the cell suspension under constant shaking and normoxic conditions; c. transferring corticospheres into a cell proliferation medium for 5-10 days, followed by generating early gliospheres by subjecting to glial enrichment medium for 10-20 days; d. generating “late gliospheres” by subjecting early gliospheres to glial enrichment medium (EGF and bFGF) including leukemia inhibitory factor (LIF) with constant shaking for 3-6 weeks; e. dissociating the “late gliospheres” into monolayers of astrocyte progenitor cells (APCs)by enzymatic digestion (such as papain, accutase etc); and f. differentiating APCs into FSAs by plating in matrix coated plates for 10-20 days with astrocyte differentiation medium comprising of Neurobasal medium supplemented with ciliary neurotrophic factor (CNTF).

[0047] In an embodiment, the corticospheres are maintained in the cell proliferation medium for 7 days with constant shaking

[0048] In an embodiment, the generation of early gliospheres by subjecting the corticospheres to glial enrichment medium for 14 days with constant shaking

[0049] In an embodiment, the generation of “late gliospheres” by subjecting early gliospheres to glial enrichment medium (EGF and bFGF) including leukemia inhibitory factor (LIF) with constant shaking for 4 weeks.

[0050] In an embodiment of the present invention, the differentiation of APCs into FSAs is affected by placing in a matrix coated plate for 14 days with astrocyte differentiation medium comprising Neurobasal medium supplemented with ciliary neurotrophic factor (CNTF). In some embodiments, the preferable matrix is a protein mixture secreted byEngelbreth-Holm- Swarm (EHS) mouse sarcoma cells, sold under trade names including but not limited to Cultrex™, Matrigel™, and Geltrex™, more preferably Matrigel™. Other suitable matrices include, without limitation, collagen, fibronectin, gelatin, laminin, polylysine, vitronectin, and combinations thereof.

[0051] In an embodiment of the present invention, the chemically defined medium is supplemented with forebrain patterning mitogens such as N-Acetyl Cysteine, LDN 193189 and SB431542.

[0052] In an embodiment of the present invention, the shaking is effective between 10- 100 rpm. Preferably 40 rpm.

[0053] In an embodiment of the present invention, the cell proliferation medium containing Advanced DMEM / F12 is supplemented with N2, Glutamax, B27, anti -anti and basic fibroblast growth factor (bFGF).

[0054] In an embodiment of the present invention, the cell proliferation medium containing Advanced DMEM / F12 is supplemented with, 0.1-10% N2, 0.1-10% Glutamax, 0.01-1% B27, 0.1-10% Anti-anti and 1-10 ng / ml basic fibroblast growth factor (bFGF).

[0055] In a preferable embodiment of the present invention, the cell proliferation medium containing Advanced DMEM / F12 is supplemented with, 1% N2, 1% Glutamax, 0.1% B27, 1% Anti-anti and 2.5 ng / ml basic fibroblast growth factor (bFGF).

[0056] In an embodiment of the present invention, the glial enrichment medium containing Advanced DMEM / F12 is supplemented with, N2, Glutamax, B27, Anti-anti, Epidermal Growth Factor (EGF), bFGF, and heparin.

[0057] In an embodiment of the present invention, the glial enrichment medium containing Advanced DMEM / F12 of step d) is supplemented with 0.1-10% Anti -anti, 0.1- 10% N2, 0.1-10% Glutamax, 0.01-1% B27, 10-30 ng / ml epidermal growth factor (EGF), 10- 30 ng / ml bFGF, and 1-10 mg / ml heparin.

[0058] In a preferable embodiment of the present invention, the glial enrichment medium containing Advanced DMEM / F12 of step d) is supplemented with 1% Anti -anti, 1% N2, 1% Glutamax, 0.1% B27, 20 ng / ml epidermal growth factor (EGF), 20 ng / ml bFGF, and 5mg / ml heparin.

[0059] In an embodiment of the present invention, the glial enrichment medium (EF20) of step e) further comprises EGF. In an embodiment, EF20 comprises 10-30 ng / ml EGF.

[0060] In a preferable embodiment of the present invention, the glial enrichment medium (EF20) of step e) further comprises 20 ng / ml EGF.

[0061] In an embodiment of the present invention, the astrocyte differentiation medium containing Neurobasal medium is supplemented with Anti-anti, Glutamax, N2, B27, non- essential amino acid (NEAA) medium and ciliary neurotrophic factor (CNTF).

[0062] In an embodiment of the present invention, the astrocyte differentiation medium containing Neurobasal medium is supplemented with 0.1-10% Anti-anti, 0.1-10% Glutamax, 0.1-10% N2, 0.01-1% B27, 0.1-10% NEAA medium and 1-20 ng / ml ciliary neurotrophic factor (CNTF).

[0063] In a preferable embodiment of the present invention, the astrocyte differentiation medium containing Neurobasal medium is supplemented with 1% Anti -anti, 1% Glutamax, 1% N2, 0.2% B27, 1% NEAA medium and 10 ng / ml ciliary neurotrophic factor (CNTF).

[0064] In an embodiment of the present invention, the iPSCs are obtained from a subject having a neurodevelopmental disorder or syndrome.

[0065] In an embodiment, the present invention provides a terminally differentiated population of FSA derived from hiPSC of a human subject having FXS, wherein the terminally differentiated population of FSA are functional and expresses lower percentages of glial fibrillary acidic protein (GFAP; lower level of de novo protein synthesis; reduction in intracellular calcium transients induced by ATP; bioenergetic deficits and lower mitochondrial oxygen consumption, compared to control levels as found in normal FSA. In an embodiment, the altered levels are biomarkers for identifying FXS in a human subject.

[0066] In an embodiment of the present invention, the terminally differentiated FSAs derived from hiPSC of a human subject having FXS, have lower GFAP levels, which is suggestive of impaired maturity. Although the inventors did not find any difference in de novo protein synthesis in FXS astrocytes and APCs compared to controls, they found a reduction in de novo protein synthesis in the transition from APCs to astrocytes in both FXS and control lines.

[0067] In an embodiment, the present invention uses two metrics of astrocyte function, namely intracellular calcium dynamics and bioenergetics. Compared to control human astrocytes, FXS astrocytes exhibit a reduction in the peak amplitude and total duration of ATP-induced intracellular calcium transients. Further, a lower proportion of FXS astrocytes respond to ATP application relative to controls. Together, these alterations suggest that disruption in calcium homeostasis in human FXS astrocytes, in turn, could have important implications for neuronal function. Further, an increased rate of glycolysis, higher glycolyticcapacity and glycolytic reserve are found in FXS astrocytes, alongside a reduction in the rate of mitochondrial oxygen consumption.

[0068] In an embodiment, the control level may be the level of at least one of the following: percentage of GFAP positive astrocyte, de novo protein synthesis, ATP -induced intracellular calcium transient, and mitochondrial oxygen consumption of FSA derived from iPSC reprogrammed from a healthy individual.

[0069] In an embodiment, one feature of the of the present invention is the ability to generate FSAs from patient samples, allowing disease-relevant generation and screening of the FSAs for therapeutic drugs and treatment regimens, where the methods utilize in vitro cell cultures or animal models derived thereof for such purposes. The methods utilize iPSCs, which may be obtained from patient or carrier cell samples, e.g. adipocytes, fibroblasts, and the like. The iPSCs are induced to develop a glial fate in vitro, and then differentiated into cortical spheroids (CS), which contain FSAs, as well as other cortical progenitors. . The FSA populations can be derived from the CS, or the intact CS can be used as a model for interacting neuralcell populations.

[0070] In an embodiment of the present invention, the FSA, systems, and methods of the invention can also be useful for studying neurological diseases. In some embodiments, the present invention provides a novel human-based platform to model FSA in vitro, in which hiPSCs-derived astrocytes can be used to study reactive states and interrogate their role in neurodegenerative and neurodevelopmental diseases, preferably FXS.

[0071] In an embodiment, the present invention provides a method of modelling FXS in a human subject, said method comprises the steps of: c) terminally differentiating a population of FSA from iPSCs by the methods as disclosed herein; and d) analyzing the terminally differentiated population of FSA for at least one of: percentage of GFAP positive astrocyte, de novo protein synthesis, ATP-induced intracellular calcium transient levels, mitochondrial oxygen consumption and comparing the measured expression to control levels.

[0072] In an embodiment of the present invention, the terminally differentiated population of FSA from human subject having FXS are functional and expresses lower percentage GFAP positive astrocytes; lower level de novo protein synthesis; lesser number ATP-induced intracellular calcium transients; bioenergetic deficits; and lower mitochondrial oxygen consumption, compared to control levels as found in normal FSA.

[0073] In an embodiment, the present invention provides a method for identifying a candidate compound from a library of compounds for the treatment or prevention of FXS, said method comprising the steps of: a) obtaining terminally differentiated population of FSA from the iPSCs by the methods as disclosed herein; b) exposing the terminally differentiated population of FSA to a library of compounds; c) measuring at least one of: percentage of GFAP positive astrocytes, de novo protein synthesis, number of ATP -induced intracellular calcium transients , and mitochondrial oxygen consumption upon exposure to each of the compound from the library of compounds; d) comparing the measured levels from step c) to control levels; and e) identifying the candidate compound from the library of compounds based on the comparative results as per step d).

[0074] In an embodiment of the present invention, the FSAs can be subjected to a library of candidate compounds, or a plurality of doses of a candidate compound. Library of compounds include small molecules, large molecules, nucleic acid sequence, and biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. An important aspect of the invention is to evaluate candidate compounds with preferred biological response functions.

[0075] In certain embodiments, the FSAs obtained from hiPSCs according to the methods described herein may be used for therapeutic purposes, more particularly in regenerative medicine.

[0076] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.EXAMPLES

[0077] The present disclosure is further explained in the form of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.Example 1: Generation of Astrocytic Progenitor Cells (APCs): hiPSCs were enzymatically lifted with enzymes - collagenase:dispase (2: 1) and plated in a suspension culture containing chemically defined medium (Bilican et al., 2012) supplemented with forebrain patterning mitogens N-Acetyl Cysteine (#A9165, Sigma-Aldrich, USA), LDN 193189 (#S2618-SEL, Stratech, UK) and SB431542 (#1614, Tocris, UK) for 7 days. The cell suspension was placed on an orbital shaker at 40 rpm under normoxic conditions to aid development of corticospheres. On day 8, corticospheres were transferred to a cell proliferation medium containing Advanced DMEM / F12 (#12634-010, ThermoFisher Scientific, USA) with 1% Anti-anti (#15240-062, ThermoFisher Scientific, USA), 1% N2 (#17502-048, ThermoFisher Scientific, USA), 1% Glutamax (#35050061, ThermoFisher Scientific, USA), 0.1% B27 (#17504-044, Thermo) and 2.5 ng / ml basic fibroblast growth factor (bFGF) (#450-33, PeproTech, USA) for 7 days. The spheres were subjected to glial enrichment medium (EF20) containing Advanced DMEM / F12 with 1% Anti -anti, 1% N2, 1% Glutamax, 0.1% B27, 20 ng / ml epidermal growth factor (EGF) (#236-EG-01M, R&D systems, USA), 20 ng / ml 5mg / ml heparin (#H3149, Sigma-Aldrich, USA) for 2 weeks to get early gliospheres. For maturation of early gliospheres, the medium (EL20) was supplemented with 20 ng / ml leukemia inhibitory factor (LIF) (#L5283, Sigma-Aldrich, USA) and 20 ng / ml EGF for 4 weeks. After maturation, spheres were propagated in EF20 medium with mechanical dissociation every fortnight to prevent aggregation and loss of viability. The gliospheres were dissociated into monolayers of APCs using Papain dissociation kit (#LK003150, Worthington Biochemical Corporation, USA) and plated onto Matrigel® (1:80 dilution) coated plates. Further, APCs were propagated in EF20 medium until confluent and enzymatically passaged using Accutase® (#A6964, Sigma-Aldrich, USA), or cryopreserved.Example 2: Characterization of Astrocytic Progenitor Cells (APCs):As a first step towards the generation of forebrain specific astrocytes, we started with a healthy control line and two FXS lines (Table 1). These human induced pluripotent stem cell (hiPSC) colonies were maintained using commercially available defined medium and wereimmunostained for pluripotency markers, Oct4 and Nanog (Figure IB). We then used cell populations with greater than 90% pluripotency to generate the desired yield of astrocytes for this study. Cellular aggregates of hiPSCs, were sequentially patterned towards forebrain specificity using chemically defined medium for 14 days supplemented with the small molecule inhibitors SB431542, LDN-193189 (LDN) (inhibitors of BMP and TGF-P signaling pathways), N-Acetyl Cysteine and bFGF. These were then referred to as “Corticospheres” (Figure 1A). On day 14, epidermal growth factors (EGF and bFGF) were added to the growth medium for an additional 14 days, thereby inducing glial specification and forming “early gliospheres” (Figure 1A). Treatment with leukemia inhibitory factor (LIF) for another 30 days led to the formation of “late gliospheres” (Figure 1A). Notably, the best yield was obtained by keeping the spheres on an orbital shaker during the growth phase with periodic mechanical chopping into smaller aggregates (Svendsen, C. N. et al., 1998) (day 15-88, Figure 1A). Gliospheres were next dissociated enzymatically, plated and then immunostained for vimentin and NFIA, markers for APCs (Pekny et al., 1999; Roybon et al., 2013; Chandrasekaran et al., 2016). FXS and control APCs showed comparable highly enriched proportions of cells immunopositive for vimentin and NFIA (Figure 1C-D). APCs were generated using transient and low level of retinoic acid in contrast to standard methods of astrocyte generation. To confirm that this resulted in more rostral forebrain identity we next undertook qPCR for forebrain (FOXG1) and caudal (HOXB4) markers of regional identity (Figure IE). We found that APCs derived from healthy and FXS hiPSCs showed substantial upregulation of FOXG1 compared to HOXB4 (Figure IF) consistent with a predominant forebrain identity.Example 3: Differentiation of Astrocytes from APCsAstrocytes were generated by differentiating APCs for 14 days with astrocyte differentiation medium containing Neurobasal medium (#21103-049, ThermoFisher Scientific, USA), 1% Anti -anti, 1% Glutamax, 1% N2, 0.2% B27, 1% non-essential amino acid medium (NEAA) (#11140-050, ThermoFisher Scientific, USA) and 10 ng / ml ciliary neurotrophic factor (CNTF) (#257-NT-10, R&D systems, USA).Generation and characterization of a homogenous population of forebrain specific astrocytesIn order to generate astrocytes, APCs were treated with CNTF for 14 days. Glial fibrillary acidic protein (GFAP) and SlOOp, were used to confirm astrocytic identity of these cells (Figure 2A).A significant reduction in the number of GFAP expressing astrocytes was observed in FXS groups compared to healthy astrocytes without affecting the number of SlOOp expressing astrocytes (Figure 2B).The expression of FMRP remained unaltered through the differentiation process; faithfully representing the disease phenotype. For example, FXS derived FSA did not express FMRP (Figure 2C) as quantified in Figure 2D.Example 4: Reduction in de novo protein synthesis in astrocytes compared to astrocyte progenitor cellsThere are numerous reports on altered protein synthesis in FXS models (Pal and Bhattacharya, 2019). These include S35 autoradiography from various brain areas from the FXS mouse model (Qin et al., 2005), non-radioactive amino acid incorporation using puromycin (Gantois et al., 2019) or azidohomoalanine (AHA) (Bowling et al., 2019) in the Fmrl knockout (KO) mouse brain tissue. Multiple studies in patient-derived lymphoblastoid cells and fibroblasts have shown increased protein synthesis (Gross and Bassell., 2012; Kumari and Bhattacharya et al., 2014 and Jacquemont et al., 2018). As there are few published reports measuring protein synthesis in patient-derived neurons or astrocytes, protein synthesis was characterized in forebrain specific APCs and mature astrocytes. Proteomic evaluation in vitro was performed by applying tethered fluorescent non-canonical amino acid tagging (FUNCAT) immunocytochemistry using relevant cytochemical markers for astrocytic progenitor cells and terminally differentiated astrocytes (Figure 3A). FUNCAT intensity was calculated per unit volume for GFAP-positive astrocytic soma and vimentin- positive soma for APCs. Quantification of newly synthesized proteins did not reveal any significant difference between control versus FXS lines in either APCs or astrocytes (Figure 3B). However, protein synthesis was found to be consistently higher in APCs compare to their respective astrocytes for each line (Figure 3B). Taken together, these results suggest a stage-specific reduction in de novo protein synthesis from APCs to astrocytes in both FXS and control lines.Example 5: Alterations in ATP-induced calcium waves in neurodevelopment astrocytes such as FXSAstrocytes respond with spontaneous and stimulus induced calcium transients that play an important role in regulating their function (Wang, Takano and Nedergaard, 2009). Hence, as a final measure of functional readouts of astrocytes generated with our method, we sought to evaluate if hiPSCs-derived forebrain astrocytes respond to ATP as reported previously inprimary human astrocytes (Zhang et al., 2016). Control, as well as FXS astrocytes, exhibited ATP-induced calcium transients (Figure 4A-B). However, a more detailed analysis revealed several key differences between the individual calcium transients recorded from healthy, FXS1 and FXS2 astrocytes. The astrocytes were classified as responders and non-responders to ATP based on the presence or absence of a calcium response respectively (Figure 4C). The number of responders in FXS1 and FXS2 was significantly lower compared to healthy. Specifically, a significant reduction was observed in the peak amplitude of the first calcium transient evoked after ATP application in both FXS astrocyte lines (Figure 4D). Further, the total duration of the first calcium transient was significantly shorter in FXS 1 and FXS2 lines compared to healthy (Figure 4E). However, the number of calcium events (quantified as the total number of events per 250 seconds; Figure 4F), was comparable between lines. Taken together, these findings reveal alterations in calcium responses elicited by ATP in hiPSCs- derived FXS astrocytes.Example 6: Increased glycolysis and altered mitochondrial oxidative phosphorylation in NDD astrocytes such as FXSEmerging evidence suggests potential metabolic deficits in glial cells in both neurodegenerative and neurodevelopmental disorders including FXS (D’Antoni et al., 2020). Therefore, seahorse analyzer was used to investigate basal and mitochondrial and glycolytic stress response of FXS astrocytes. Astrocytes stimulated with glucose (to measure glycolysis) showed a higher ECAR in FXS2 astrocytes compared to control astrocytes whereas FXS1 astrocytes showed no significant difference (Figure 5A-B). Moreover, to measure the glycolytic capacity of astrocytes, we blocked mitochondrial ATP production with the specific inhibitor oligomycin. Addition of oligomycin shifted the energy demand of astrocytes towards glycolysis, leading to an increase in ECAR. The difference between the maximum and basal values of ECAR was used to measure the glycolytic capacity. This analysis revealed an increase ECAR in both FXS 1 and FXS2 astrocytes compared to healthy (Figure 5A-B). Collectively, the glycolytic capacity was found to be significantly higher in FXS astrocytes. Furthermore, the total glycolytic pathway was blocked with 2-DG to measure total glycolytic reserve capacity by calculating differences between ECAR levels. The changes in ECAR levels were higher in FXS1 astrocytes, but not in FXS2 astrocytes, compared to healthy. These results suggest a higher rate of glycolysis, glycolytic capacity and glycolytic reserve in hiPSCs-derived FXS astrocytes.ATP production through the mitochondrial oxidative phosphorylation pathway is known to be compromised in vivo in Fmrl KO mice (D’Antoni et al., 2020). Hence, key parameters were measured related to mitochondrial function in FXS and control astrocytes using the Seahorse analyzer, after sequential addition of modulators (Oligomycin, FCCP, Rotenone and Antimycin A) known to perturb mitochondrial function. First, basal respiration was recorded by measuring baseline cellular oxygen consumption rate (OCR in pmol / min / pg of protein) and subtracting this from non-mitochondrial respiration. We found basal respiration in control and FXS astrocytes not to be significantly different (Figure 5C-D). Addition of oligomycin (ATP synthase inhibitor of ETC complex V) caused a decline in mitochondrial respiration as evidenced by a decrease in OCR. This reduction in OCR is linked to cellular ATP production (difference between oligomycin OCR and baseline OCR) and showed no significant difference between control and FXS astrocytes (Figure 5C-D). Followed by oligomycin, FCCP (Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone) was added to measure maximal respiratory capacity. FCCP stimulation elicited significantly lower maximal respiration in FXS astrocytes compared to control astrocytes (Figure 5C-D). A combination of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor, shuts down mitochondrial respiration, thereby allowing estimation of non-mitochondrial respiration. Together, these results point to a complex yet flexible role of both metabolic pathways to meet the energy requirements of astrocytes, and this was found to be impaired in FXS astrocytes.

[0078] Various modification and variation of the described assays, techniques and various means disclosed herein to implement the assays / methods in accordance with the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.REFERENCES1. Allen, N. J., and Barres, B. A. (2009). Glia — more than just brain glue. Nature 457, 675-677. doi: 10.1038 / 457675a.2. Allen, N. J., Bennett, M. L., Foo, L. C., Wang, G. X., Chakraborty, C., Smith, S. J., et al. (2012). 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Claims

AMENDED CLAIMS received by the International Bureau on 25 July 2025 (25.07.2025)We Claim:

1. A method for generating forebrain specific astrocytes (FSAs) from human induced pluripotent stem cells (hiPSCs), said method comprising the steps of: a. enzymatically lifting hiPSCs, followed by suspension culture in a chemically defined medium to obtain a cell suspension; b. developing corticospheres by culturing the cell suspension under constant shaking and normoxic conditions; c. transferring corticospheres into a cell proliferation medium containingAdvanced DMEM / F12 with growth factors for 5-10 days, followed by generating early gliospheres by subjecting to glial enrichment medium comprising Advanced DMEM / F12 with growth factor for 10-20 days; d. generating “late gliospheres” by subjecting early gliospheres to glial enrichment medium (EF20) comprising leukemia inhibitory factor (LIF) with constant shaking for 3-6 weeks; e. dissociating the “late gliospheres” into monolayers of astrocyte progenitor cells (APCs) by enzymatic methods ; and f. differentiating APCs into FSAs by plating in matrix-coated plates for 10-20 days with astrocyte differentiation medium comprising Neurobasal medium supplemented with ciliary neurotrophic factor (CNTF).

2. The method as claimed in claim 1, wherein the chemically defined medium is supplemented with forebrain patterning mitogens N-Acetyl Cysteine, LDN 193189 and SB431542.

3. The method as claimed in claim 1, wherein the shaking is effective at 10-100 rpm.

4. The method as claimed in claim 1, wherein the cell proliferation medium containing Advanced DMEM / F12 is supplemented with 0.1-10% Anti-anti, 0.1-10% N2, 0.1- 10% Glutamax, 0.01-1% B27 and 1-10 ng / ml basic fibroblast growth factor (bFGF).

5. The method as claimed in claim 1, wherein the glial enrichment medium containing Advanced DMEM / F12 of step d) is supplemented with 0.1-10% Anti-anti, 0.1-10% N2, 0.1-10% Glutamax, 0.01-1% B27, 10-30 ng / ml epidermal growth factor (EGF), 10-30 ng / ml bFGF, and 1-10 mg / ml heparin.

6. The method as claimed in claim 1, wherein the glial enrichment medium (EF20) of step e) further comprises 10-30 ng / ml EGF.

7. The method as claimed in claim 1, wherein the astrocyte differentiation medium containing Neurobasal medium is supplemented with 0.1-10% Anti-anti, 0.1-10% Glutamax, 0.1-10% N2, 0.01-1% B27, 0.1-10% NEAA medium and 1-20 ng / ml ciliary neurotrophic factor (CNTF).

8. The method as claimed in claim 1, wherein the hiPSCs can be derived from a subject having a neurodevelopmental disorder or syndrome.

9. The method as claimed in claim 8, wherein the neurodevelopmental disorder or syndrome is selected from the group consisting of Fragile X syndrome, autism, RETT syndrome, schizophrenia, Angelman syndrome and Timothy syndrome and the like.

10. A method of modelling Fragile X syndrome (FXS) in a human subject, said method comprises the steps of: a) terminally differentiating a population of forebrain specific astrocytes (FSA) from the iPSCs as claimed in anyone of claims 1-9; and b) analyzing the terminally differentiated population of FSA for at least one of: percent of GFAP positive astrocyte , de novo protein synthesis, ATP- induced intracellular calcium transient, and mitochondrial oxygen consumption and comparing the measured expression to control levels.

11. The method as claimed in claim 10, wherein the terminally differentiated population of FSA is functional and expresses lower percentages of GFAP positive astrocyte; lower levels of de novo protein synthesis; reduced ATP-induced intracellular calcium transients; bioenergetic deficits; and lower mitochondrial oxygen consumption, compared to control levels as found in normal FSA.

12. A method for identifying a candidate compound from a library of compounds for the treatment or prevention of FXS, said method comprising the steps of: a) obtaining terminally differentiated population of FSA from theiPSCs as claimed in claims 1-9; b) exposing the terminally differentiated population of FSA to a library of compounds; c) measuring at least one of: percentage of GFAP positive astrocyte, altered de novo protein synthesis, reduced ATP-induced intracellular calcium transient levels, and mitochondrial oxygen consumption upon exposure to each of the compound from the library of compounds;d) comparing the measured levels from step c) to control levels; and e) identifying the candidate compound from the library of compounds based on the comparative results as per step d).

13. The method as claimed in claim 12, wherein the library of compounds is selected from the group consisting of small molecules, large molecules, nucleic acid sequence, and biomolecules.STATEMENT UNDER ARTICLE 19AMENDMENTS TO THE CLAIMS IN VIEW OF OBJECTIONS RAISED IN THEISR / WOClaim Amendments:Claim 1 amended to revise Matrigel® as “matrix” for which the support is available in para [0050] of the specification. No technical amendments or addition of new matter has been done by way of these amendments.

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