Patent Application
20220333070
The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. Astrocytes are known to carry out functions that are essential for normal brain physiology. However, much of this knowledge is derived from studies of mouse astrocytes, which differ in complexity from human astrocytes. Current protocols for generating human astrocytes are time-consuming—with durations on the order of many weeks or months—and technically challenging, and the resulting cells are incompletely characterized with respect to function.
Herein disclosed is a new transcription-factor-based reprogramming protocol to rapidly and efficiently generate functional induced astrocytes (iAs) from human pluripotent stem cells (hPSC). Overexpression of the transcription factor(s) nuclear factor 1 B-type (Nfib), or sex-determining region Y-box 9 (Sox9) and Nfib, in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In further embodiments, Nfia is used instead of, or in addition to, Nfib. The induced astrocytes exhibited molecular and functional properties resembling those of adult human astrocytes. Thus disclosed embodiments include methods of establishing in vitro and in vivo models for the study of astrocyte biology and neural and neurologic physiology and pathophysiology. In some embodiments, the hPSC is a human embryonic stem cell (hESC). In other embodiments, the hPSC is a human induced pluripotent stem cell (hiPSC).
Further disclosed are a population of isolated iAs generated through overexpression the transcription factor Nfib and/or Nfia each alone or together, or further in combination with Sox9 in human pluripotent stem cells. These iAs are useful in the study of astrocyte biology and the modeling of neural or neurologic diseases, both in vitro, and in vivo, for example, by intracerebral implant. In various embodiments at least 85%, 90%, or 95% of the cells in the population of iAs generated with selection are positive for one or more astrocyte biomarkers. In various aspects of these embodiments the positive biomarker is S100 calcium-binding protein B (S100B), Glial fibrillary acidic protein (GFAP), vimentin (VIM), Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1), glutamate aspartate transporter (GLAST), CD44, Kir4,1, or any combination thereof. In some embodiments the iAs are positive for only a subset of these biomarkers to the exclusion of the assessment of others of the above listed biomarkers. In some embodiments the iAs are positive for only a subset of these biomarkers to the exclusion of the assessment of any other biomarkers. In some embodiments the iAs are positive for one, some, or all of S100B, GFAP, VIM, ALDH1L1, GLAST, CD44, Kir4.1, and one or more additional astrocyte biomarker. In some embodiments assessment of one or more additional astrocyte biomarkers is excluded. In aspects of these embodiments the additional astrocyte biomarker is nuclear factor 1 A-type (Nfia), glutamate transporter 1 (GLT-1), aldolase C (ALDOC), connexin 43 (CX43) also known as Gap junction alpha-1 protein (GJA1), hepatocyte cell adhesion molecule (HEPACAM), glutamate-ammonia Ligase (GLUL), tubulin, alpha 1A (TUBA1A), aquaporin-4 (AQP4), fatty acid binding protein 7 (FABP7), Sox9, or any combination thereof. In some embodiments the astrocyte biomarkers are S100B, GFAP, VIM, or any combination thereof. In further embodiments the population of isolated iAs do not express (or express only at very low levels) pluripotency, neural stem cell, neuronal, or oligodendrocyte genes.
In further embodiments the population of isolated iAs display one or more functional characteristics of astrocytes. In aspects of these embodiments the functional characteristic is having glycogen granules, glutamate uptake, ability to increase intracellular calcium levels (spontaneous or stimulated, for example, by ATP), support of synapse formation (for example, in cultures of induced neurons), an ability to increase cytokine expression in response to interleukin-1β (IL-1β) (e.g., IL-6, C-X-C motif chemokine ligand 8 (CXCL8), CXCL10, and/or C-C Motif Chemokine Ligand 5 (CCL5)), and formation of gap junctions.
In some embodiments, the isolated iAs are capable or surviving intracerebral implant. In aspects of these embodiments, the implanted iAs continue to express astrocyte biomarkers. In further aspects of these embodiments, the implanted iAs produce astrocytic processes that co-localize with synaptic structures on host neurons, for example, Bassoon+ and/or postsynaptic density-95+ (PSD95)+ synaptic structures. In still further aspects, the implanted iAs form gap-junctions with host cells.
In some embodiments, the isolated iAs are genetically modified to have a genetic lesion associated with a neural or neurologic disease or disorder. In some embodiments human pluripotent stem cells (e.g., hESC or iPSCs) are genetically modified to have a genetic lesion associated with a neurologic disease or disorder, and then treated to produce iAs bearing the genetic modification. In some embodiments, these genetically modified iAs are capable of being implanted in the central nervous system (for example, intracerebrally, or into spinal cord) or peripheral nervous system, and reproducing structural and/or functional characteristics of the disease or disorder.
Herein disclosed is a controlled and simple protocol to rapidly and efficiently induce highly enriched, mature, and bona fide functional human astrocytes. By “functional” herein is meant the ability to display previously described astrocytic functional properties at similar efficiency as compared to normal human or mouse astrocytes in vitro. Examples of functional astrocyte include, but are not limited to, (1) the expression of glutamate transporters such as GLT1 and GLAST and the ability to take up glutamate added to the culture medium; (2) the expression of gap junction proteins such as Cx43, establishment of gap junctions between cells and the ability to transfer biocytin between cells trough gap junctions; (3) the ability to show spontaneous and induced elevations of intracellular calcium levels; and (4) the ability to increase expression of cytokines upon an inflammatory stimulus such as exposure to IL-1beta; and (5) the ability to support formation of synapses in co-cultured neurons, or combination thereof
By overexpressing Nfib and/or Nfia alone or in combination with Sox9 in hESCs or hiPSCs, it has been possible to generate astrocytes that closely resemble adult human primary astrocytes at the phenotypic, molecular, and functional levels. These iAs are useful for studies on astrocyte biology and for neurological disease modeling.
Overexpression of the transcriptions factor(s), Nfib, Nfia, or Nfib and Sox9, or Nfia and Sox9 is all that is needed to induce astrocyte differentiation, in a period of 7-14 days. By “overexpression” herein is meant a higher level of expression compared to that in human pluripotent stem cells not bearing an expression vector for the transcription factor, which would be the baseline level of expression. Thus exogenous expression of these transcription factors should be under the control of a switchable promoter system, especially one that is inactive under baseline conditions and active in a manipulated condition, typically the presence of an added reagent. Flexibility of experimental design for use of the iAs is enhanced by use of a manipulated condition that can be easily avoided. In the working examples below the switchable promoter system is the reverse tetracycline-controlled transactivator and the manipulated condition is the inclusion of doxycycline (Dox) in the culture medium, however, while this is a robust and well-understood expression control system, others are known in the art, and any other expression control system with similar attributes would be similarly appropriate. Use of the reverse tetracycline-controlled transactivator system in particular is not essential, though it can be preferred in some embodiments.
As exogenous overexpression of the transcription factors is a temporary requirement, which need not be maintained (and in some embodiments, is preferably not maintained) after the pluripotent stem cells have differentiated into astrocytes, either integrating or episomal expression vectors are used . Alternatively, RNA (e.g. self-replicating RNA), or transgene expression from a safe harbor locus is used. The expression vector will typically encode a selectable marker, for example, an antibiotic resistance, in addition to the transcription factor. In the working examples below a lentiviral expression vector system is used. While this is a robust and well-understood expression vector system, many other expression vectors and their properties are known in the art, and any other expression vector system operable in the cells in the astrocyte differentiation pathway would be similarly appropriate. Use of a lentiviral expression vector in particular is not essential, though it can be preferred in some embodiments.
Some astrocytic phenotypes are stably established within seven days overexpression of Nfib or Nfia alone, Nfib plus Sox9, Nfia plus Sox9, or Nfib plus Nfia plus Sox9 (for example, expression of S100B or VIM), whereas the number of cells displaying the phenotype will diminish somewhat unless overexpression is maintained for a longer period of time, up to 14 days (for example, expression of GFAP). Thus, iAs become independent of the exogenous overexpression the transcription factors between 7 and 14 days of such treatment. In some embodiments the lower yield of fully differentiated iAs is acceptable and overexpression of the transcription factors is not maintained past 7 days. In other embodiments, overexpression of the transcription factors is maintained for 8, 9, 10, 11, 12, 13, or 14 days, for example, because a better yield of fully differentiated iAs is desired.
A detailed exemplary protocol is described in the examples below, but in general human pluripotent stem cells (either embryonic stem cells “ESCs”, or induced pluripotent stem cells “iPSCs”) are dissociated (for example, with a protease preparation) and plated on an appropriate surface (e.g., a MATRIGEL-coated surface) and cultured in an appropriate medium for a day. The cultured pluripotent stem cells are then exposed to the expression vector and cultured to allow incorporation of the vector. After sufficient time (for example, overnight culture) for the vector to be incorporated (integrated, in the case of an integrating vector) the culture conditions are manipulated to switch on expression from the vector so that the Nfib, Nfia, Nfib+ Nfia, Nfib+Sox9, Nfia+Sox9, or Nfib+Nfia+Sox9, and the selectable markers are expressed. After an interval of time for expression to become established (for example, overnight culture) the culture medium is changed to an Expansion Medium supplemented with the selection reagent(s) for the selectable marker carried by the expression vector and the factor required to keep the expression system switched on (expression factor). This allows the cells to recover from infection with the viral vector and from selection, and also allows for a degree of proliferation. Over the course of several days, with daily medium changes, a mixed medium containing decreasing amounts of Expansion Medium and increasing amounts of an astrocyte-appropriate (alternatively termed astrocyte-permissive) medium (for example, FGF Medium) is provided to the culture, until the medium is 100% the astrocyte-appropriate medium at which point inclusion of the selection reagents is discontinued; the expression factor is included throughout. FGF medium promotes the expression of glutamate transporters and thus induces glutamate uptake capabilities. Over this period of time, cells that did not incorporate the expression vector, or from which it has been lost, have been eliminated by the selection reagent, and expression of the transcription factors promote differentiation. The day after reaching 100% astrocyte-appropriate medium, the cells are dissociated, pelleted, and replated on an appropriate surface and cultured for a few days in the astrocyte-appropriate medium supplemented with the expression factor. Subsequently, half of the culture medium is replaced with a Maturation Medium every 2-3 days. During this interval the cells take on a more mature morphology and develop a more mature phenotype. The Maturation Medium can be supplemented with the expression factor, which is preferably present through the 14th day of culture. The expression factor can be supplied indefinitely, to ensure expression of the transcription factors is not lost, but can also be withdrawn temporarily or permanently to avoid interference with experimental measurements, for example if analyzing global gene expression. In some embodiments, the exposure to the expression vector takes place on day −1 of the procedure. In some embodiments, the expression factor is first included on day 0 of the procedure. In some embodiments, the selection reagents are first included on day 1 of the procedures. In some embodiments, use of mixtures of Expansion Medium and astrocyte-appropriate medium commences on day 3 of the procedure and 100% astrocyte-appropriate medium is reached on day 6 of the procedure. In some embodiments, dissociation and re-plating takes place on day 7 of the procedure. In some embodiments, replacement of half of the medium commences on day 10 of the procedure.
This method of producing iAs offers advantages in speed, simplicity (fewer steps, less complex and cheaper media) robustness, and characterization of the iAs produced, as compared to previous methods (see Table 1). A particular advantage is that there is no need to first perform a neural induction, nor a need to make embryoid bodies. Perhaps the most immediately recognizable advantage is the speed with which the iAs are generated. Important astrocyte biomarkers are observed by seven days for example, by flow cytometry or immunohistochemistry, and as early as by three days by QPCR, after exogenous expression of the transcription factor(s) commence. This compares with from 4 to 84 weeks for biomarker appearance in earlier protocols. Moreover the portion of the population of cells that are positive for the biomarker is at least similar to, if not greater than, that observed in the earlier protocols. Various functional characteristics of astrocytes are observed in the iAs produced by the herein disclosed protocol within 14-21 days. In comparison, the earlier protocols required from 4 to 71 weeks for these various functional characteristics to be observed, if the cells were even assessed for their presence. Table 1 includes seven functional characteristics of astrocytes for which the iAs produced by the herein disclosed protocol have been characterized; none of the iAs produced by the earlier protocol have been characterized for more than 4 of these functional characteristics. This more complete characterization means that iAs produced by the herein disclosed protocol can be used with greater confidence that they faithfully represent natural astrocytes, and provides a larger number of assayable features for whatever use may be made of them.
The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments now contemplated. These examples should not be construed to limit any of the embodiments described in the present specification. The inventor(s) published a version of these experiments as Canals et al., Nature Methods 15:693-696 (2018), which is incorporated by reference herein in its entirety.
Example 1
Glioqenic Transcription Factor Nfib Alone or Together with Sox9 Efficiently Induced Expression of Astrocyte Biomarkers
Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed by astrocytes amongst other cell types and is considered the hallmark intermediate filament protein of astrocytes. GFAP is widely used as an immunohistochemical marker of astrocytes, though it is not universally detectable in all astrocytes and can be found in other cell types in the central nervous system and in cells of the extended astroglial family elsewhere in the body.
S100B calcium-binding protein B (S100B) is a glial specific protein expressed primarily by astrocytes, particularly mature and NG2-expressing astrocytes. Thus, S100B is also useful as a biomarker of astrocytes.
To test the ability of the gliogenic transcription factors Nfia, Nfib, and Sox9 to induce astrocytic phenotypes, human embryonic stem cells (hESC) where infected with a combination of lentiviral vectors capable of expressing these factors under control of a Tet-On (rtTA) expression system. The cells were also infected with a lentiviral vector expressing enhanced green fluorescent protein (eGFP) under control of the GFAP promoter (GFAP::eGFP) (
After screening different combinations of transcription factors, we found that Nfib alone (B) or together with Sox9 (SB) showed the most efficient induction of a co-transduced GFAP::EGFP reporter and S100B expression, with cells displaying stellate morphology, which is characteristic of astrocytes, after 14 days. Using RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), we found higher expression of GFAP and ALDH1L1 in cells that were co-transduced with combinations that included Nfib, whereas S100B expression was higher in cells treated with combinations that included Sox9 (
The induced astrocytes (iAs) that were obtained after treatment with Nfib (iAs-B) or with both Sox9 and Nfib (iAs-SB) (
Astrocytes are the main storage sites of glycogen in the central nervous system. We detected glycogen granules in >80% of iAs, similar to what we observed in primary astrocytes (
To provide further evidence for the astrocytic identity of iAs, we analyzed the expression of several astrocytic markers by RT—qPCR. We found that S100B and HEPACAM levels were higher in iAs-SB than in iAs-B and similar to those in adult human astrocytes, indicating that iAs-SB were more mature than iAs-B. Expression of other canonical astrocyte markers (ALDH1L1, ALDOC, AQP4, GFAP, GLAST, GLT1, GLUL HepaCAM, AND S100B) in iAs-B and iAs-SB closely resembled that in adult human astrocytes (
Functional characterization of induced astrocytes
To address whether the iAs could perform astrocytic functions their ability to clear neurotransmitters was evaluated. Glutamate uptake assays 14 days after induction showed that iAs took up glutamate to an extent similar to that of human astrocytes (
A structural property of astrocytes is the formation of gap junctions that can be detected by the propagation of biocytin. We filled single cells with biocytin and observed diffusion into neighboring astrocytes, which confirmed the presence of functional gap junctions (
Another well-described property of astrocytes is a spontaneous or stimulated increase in the amount of intracellular calcium. Using calcium imaging, we found a population that displayed spontaneous calcium waves at 14 and 21 days after induction (iAs-B, 5-9%; iAs-SB, 17-31%), at levels similar to that of adult human astrocytes (24%) (
Astrocytes are important in neuroinflammation. Therefore, we exposed iAs to 1L-1β and assessed the expression of IL-6, CXCL8, CXCL10, and CCL5, all of which have been reported to be induced in human primary astrocytes. Notably, iAs showed increased expression of these cytokines (
The ability of human-derived iAs to support synapse formation in human induced neurons (iNs) similarly to primary mouse astrocytes (mAs) was assessed. We co-cultured iNs on iAs and analyzed the occurrence of spontaneous postsynaptic currents (sPSCs). On iAs-SB, 75% of iNs expressed sPSCs with frequencies similar to those on mAs (
Taking Examples 4 and 5 together, our findings in five different assays provide strong evidence that iAs exhibit all major functional properties of human astrocytes.
Whether iAs survived intracerebral implantation was assessed. It was found that grafted iAs-SB localized in cores or as isolated cells (
Alexander disease (AxD) is a leukodystrophy caused by mutations in GFAP and is characterized by the presence of aggregated GFAP. To model AxD, we introduced an R239C mutation into H1 hESCs by CRISPR—Cas9 gene editing followed by iAs-SB induction (
H1 (WA01) cells, H9 (WA09) hESCs from WiCell Research Institute (Wicell, Wiss.), and iPSCs (clone RB9-CB1 (Rönn, R. E. et al. Stem Cell Rep. 4:269-281 (2015)); kindly provided by Niels-Bjarne Woods (Lund University)) were cultured in feeder-free conditions using mTeSR1 medium (StemCell Technologies) on MATRIGEL-coated six-well plates (Corning), with medium changed daily. Cells were dissociated with Accutase (Thermo Fisher Scientific) when the culture reached ˜80% confluency and replated in mTeSR1 supplemented with 500 nM thiazovivin (Sigma-Aldrich) during the first 24 h.
For human adult astrocytes, fresh cortical tissue was obtained during resection surgery from patients who had pharmacologically intractable epilepsy. The use of human brain tissue was approved by the local ethical committee in Lund (212/2007) and was carried out in accordance with the Declaration of Helsinki. Prior to each surgery, written informed consent was obtained from all of the subjects. The samples were submerged in Hibernation medium (Thermo Fisher Scientific) immediately after surgery. Meninges were removed under a dissection stereomicroscope (Leica), and the tissue was chopped into small chunks with a sterile surgical blade and processed with the Adult Brain Dissociation Kit (Miltenyi Biotec) according to the manufacturer's instructions. A single-cell suspension was obtained and plated onto poly-d-lysine (PDL) and human-fibronectin-coated culture vessels (Thermo Fisher Scientific). Cells were cultured in Neurobasal medium supplemented with B27, Glutamax (Thermo Fisher Scientific), brain derived neurotrophic factor (hBDNF; Peprotech), glial-cell-derived neurotrophic factor (GDNF; Peprotech), and ciliary neurotrophic factor (CNTF; Peprotech). After 3-4 weeks of cultivation, the medium was switched to DMEM:F12 1:1 supplemented with the N2 and G5 supplements (Thermo Fisher Scientific).
For human-fetus-derived astrocytes, cortical tissue from dead, aborted human fetuses, aged 7-9 weeks after conception, was obtained from Lund and Malmo University Hospitals according to guidelines approved by the Lund/Malmo Ethical Committee (2017/1). Prior to abortion, written informed consent was obtained from all of the subjects. Fetuses were kept in Hibernation medium, and dissection was conducted under a stereomicroscope (Leica). The central nervous system was isolated and cleaned from the surrounding tissue. The cortex was cut open along and close to the dorsal midline and dissected out. To generate primary astrocytic monolayer cultures, we incubated the cortex in Neurobasal supplemented with B27 and Glutamax, and mechanically dissociated the tissue until a single-cell suspension was obtained. Cells were plated onto PDL- and laminin-coated dishes in DMEM:F12 1:1 supplemented with N2 and 10% fetal bovine serum (all from Thermo Fisher Scientific).
Full-length cDNAs of mouse Nfia, Nfib, and Sox9 genes was amplified from plasmids available at Addgene (#64901, #64900, and #41080, respectively), and specific restriction sites were added to allow cloning in tetO-FUW (tetracycline operator FUW) lentiviral vectors carrying genes for resistance to blasticidin, hygromycin (Addgene #97330), and puromycin (Addgene #97329) with EcoRI/BamHI (Nfia and Nfib) or EcoR1/Xbal (Sox9) restriction enzyme combinations. The rtTA (reverse tetracycline-controlled transactivator) and tetO-FUW-GFP lentiviral vectors were obtained from Addgene (#20342 and #30130, respectively), and the GFAP::GFP vector was a kind gift from Chun-Li Zhang (University of Texas Southwestern Medical Center). The primers used in the cloning process are shown in Table 2. For the lentiviral production protocol, see Example 9.
Generation of iAs From hESCs and iPSCs.
On day −2, H1, H9 hESCs, and human iPSCs at ˜80% confluency were dissociated with Accutase, and 5×105 cells were replated in MATRIGEL-coated six-well plates using mTeSR1 (StemCell technologies) medium with 10 μM ROCK (p160-Rho-associated coiled kinase) inhibitor (Y-27632; StemCell Technologies). One day later (day −1), medium was replaced by fresh mTeSR1 medium with 8 μg/ml polybrene (Sigma-Aldrich), and 1 μl of each virus was added per well. One day after infection (day 0), medium was replaced with fresh mTeSR1 medium containing 2.5 μg/ml doxycycline, which was kept in the medium throughout the experiments. On days 1 and 2, cells were cultured in Expansion medium (DMEM/F-12, 10% FBS, 1% N2 supplement, and 1% Glutamax, from Thermo Fisher Scientific). From day 3 to day 5, Expansion medium was gradually switched to FGF medium (Neurobasal, 2% B27 supplement, 1% NEAA, 1% Glutamax, and 1% FBS, from Thermo Fisher Scientific; 8 ng/ml FGF, 5 ng/ml CNTF, and 10 ng/ml BMP4, from Peprotech), with the exception of the case of AxD modeling, for which serum was kept in the medium to enhance GFAP expression. On day 6, the mixed medium was replaced by FGF medium. Selection was carried out on days 1-5 for vectors that rendered cells resistant to blasticidin (1.25 μg/ml) or hygromycin (200 μg/ml) and on days 1-2 for vectors that made cells resistant to puromycin (1.25 μg/ml). On day 7, cells were dissociated with Accutase and replated in MATRIGEL-coated wells or coverslips. The day after, FGF medium was replaced, and afterward 50% of the medium was replaced by Maturation medium (1:1 DMEM/F-12 and Neurobasal, 1% N2, 1% sodium pyruvate, and 1% Glutamax, from Thermo Fisher Scientific; 5 mg/ml N-acetyl-cysteine, 500 mg/ml dbcAMP, from Sigma-Aldrich; 5 ng/ml heparin-binding EGF-like growth factor, 10 ng/ml CNTF, 10 ng/ml BMP4, from Peprotech) every 2-3 d, and cells were kept for 14, 21, or 28 d. The detailed composition of each medium can be found in Example 9.
Coculture of iAs with Induced Neurons.
hESC iNs were produced via a previously described protocoll4. On day 7 of both protocols, iNs and iAs were dissociated with Accutase and replated together on poly-d-lysine-(Sigma) and laminin-coated (Sigma) coverslips (50,000 iAs with 100,000 iNs). Cells were kept for 32-35 additional days in culture medium consisting of 1:1 Maturation medium and Neuronal medium consisting of BrainPhys (StemCell Technologies), 1% N2 supplement, 2% B27 supplement, 10 ng/ml BDNF (Peprotech), and 10 ng/ ml NT-3 (Peprotech).
For quantification of Bassoon—PSD95 co-staining, images of neurons were acquired with a Zeiss LSM 780 confocal microscope with a 63× objective lens and analyzed with Zen software. Laser power and photomultiplier gain were set at the same levels for all of the samples to allow for quantitative comparisons. Five neurons per coverslip were analyzed from a total of seven coverslips from two independent experiments for each sample. Co-localizing puncta inside the MAP2 region were detected with the co-localization threshold plug-in of Fiji. We quantified the number of puncta inside the MAP2 region by using the particle analysis plug-in of ImageJ and counting puncta >0.1 μm in size to discard background.
All primary and secondary antibodies used can be found in Tables 3 and 4. For immunocytochemical analysis, cells were plated on MATRIGEL-coated 13-mm-diameter glass coverslips or in MATRIGEL-coated 24-well plates. At the indicated times, cells were washed one to three times with potassium-phosphate-buffered saline (KPBS) and fixed for 20 min at room temperature (RT) in 4% paraformaldehyde (PFA), washed three times with KPBS and blocked for 60 min in KPBS containing 0.025% Triton X-100 (TKPBS) and either 5% normal donkey serum (NDS; Millipore) or 2.5% NDS and 2.5% normal goat serum (NGS; Millipore). Primary antibody incubation was performed overnight at 4° C. in blocking solution. Cells were then washed twice for 5 min with 0.025% TKPBS and once for 5 min with blocking solution.
Incubation with secondary antibodies or phalloidin-TRITC was performed in blocking solution for 2 hr at RT. Cells were then washed once with 0.025% TKPBS for 5 min and twice for 5 min with KPBS, and coverslips were mounted using PVA:DABCO, polyvinyl alcohol (Sigma-Aldrich)-based mounting media containing DABCO (Sigma-Aldrich) anti-fading reagent.
For immunohistochemical analysis, sections were washed three times with KPBS and blocked for 60 min in 0.25% TKPBS containing 5% NDS, or 2.5% NDS and 2.5% NGS. Primary antibody incubation was performed overnight at 4° C. in blocking solution. Sections were then washed twice with 0.25% TKPBS and once with blocking solution. Incubation with secondary antibodies was performed in blocking solution for 2 hr. at RT. Sections were then washed once with 0.25% TKPBS and twice with KPBS before being mounted on slides and covered with a coverslip using PVA:DABCO. For GLAST and Cx43 staining, all washes and incubations with antibodies were done in KPBS, and the blocking step was extended to 2 h. All washes were done for 10 min, and all of the steps were performed on an orbital shaker.
For nuclear staining, 1 μg/ml Hoechst (Thermo Fisher Scientific) was used during the incubation with the secondary antibody.
Images were obtained with a Zeiss LSM 780 confocal microscope, orthogonal reconstruction was done with Zen software, and 3D rendering was done with Imaris (Bitplane).
Cells were dissociated with Accutase, pelleted for 5 min at 300 g, washed in PBS, and pelleted again for 5 min at 300 g. For CD44, cells were resuspended in 100 μl of FACS buffer containing PBS +1% FBS +0.09% sodium azide. Cells were then incubated for 30 min at RT in the dark with antibody and 7AAD for viability and then washed twice with FACS buffer and pelleted for 5 min at 300 g. Before analysis, cells were resuspended in 250 μl of FACS buffer. For VIM, after dissociation, cells were treated with a Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer's instructions. For analysis, samples were run in a BD FACS LSRFortessa analyzer, and the data were analyzed with FlowJo software.
Cells on coverslips were washed once with cold KPBS and fixed with cold methanol for 5 min. After fixing, cells were washed three times with 70% ethanol and then incubated for 30 min in 1% periodic acid (Sigma-Aldrich) diluted in 70% ethanol. After three washes with 70% ethanol, cells were stained with 0.5% Basic Fuchsin (Sigma-Aldrich) diluted in acid ethanol (8.0 parts absolute ethanol, 1.9 parts water, and 0.1 parts HCl 37%). After staining, cells were washed three times with 70% ethanol and once with KPBS, and mounted with PVA:DABCO.
In vitro cell counting was performed under a BX61 epifluorescence microscope (Olympus) in 20 random fields of view from three replicates for each condition and time point, and the counts were normalized to the number of Hoechst+ cells. In vivo quantification was performed in all sections containing iAs. The total number of GFP+ iAs and co-localization with VIM were assessed.
RNA isolation was performed with the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. To prevent DNA contamination, RNA was treated with DNase I (Qiagen). The yield of RNA was determined with a Nanodrop ND-1000 spectrophotometer (Saveen & Werner). One microgram of RNA was reverse-transcribed with the qScript cDNA Synthesis Kit (Quantabio). For mouse and human NFIB and SOX9 analysis, RT-qPCR was carried out with Fast SYBR Green Master Mix (Thermo Fisher Scientific) and specific primers for each gene (Table 5) with a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Other RT-qPCRs were carried out with TaqMan Universal PCR Master Mix and TaqMan assays (Table 6) with an iQ5 Real-Time PCR detection system (Bio-Rad). For single-cell qR-PCR analyses, viable (as determined by DRAQ7 staining) iAs or primary astrocytes were sorted through a FACSAria II (BD Biosciences) to individual wells of 96-well plates, and mRNA levels were quantified with the Fluidigm Biomark dynamic array system as previously described (Yang, N. et al. Nat. Methods 14:621-628 (2017)). Data can be presented as heat maps for example as generated by the Morpheus online tool (clueDOTio/Morpheus/).
Cells cultured in MATRIGEL-coated 35-mm glass-bottom Petri dishes (Ibidi) were loaded for 30 min with 2 μM Fluo-4 (calcium indicator; Life Technologies) prepared according to the manufacturer's instructions. Cells were washed with medium once and imaged immediately. Live fluorescence imaging was done with a Zeiss LSM 780 confocal microscope, and images were taken every 1.94 s with a 10× objective for 5 min before and 15 min after the addition of ATP (100 μM or 30 μM in AxD calcium-imaging assays). For quantification of the change in intensity over time, astrocytes were outlined as regions of interest (ROIs) and analyzed with Zen software (Zeiss). For each ROI, the change in fluorescence intensity over time was plotted. The percentage of activated cells was counted manually, with ROIs displaying transients (increasing fluorescence values at least 50% of the basal value) in each video compared with the total number of ROIs.
Cells were plated on days 6-7 in MATRIGEL-coated six-well plates and cultured until day 14 or 21. After 30 min of incubation in Hank's balanced salt solution (HBSS) buffer without calcium and magnesium (Gibco), cells were incubated for 3 h in HBSS with calcium and magnesium (Gibco) containing 100 μM glutamate. Samples of medium were collected after 3 h and analyzed with a colorimetric glutamate assay kit (Sigma-Aldrich) used according to the manufacturer's instructions.
Cytokine Stimulation.
Induced astrocytes differentiated for 14 or 21 days were incubated for 8 h with or without 10 ng/ml IL-1β (Peprotech) in fresh Maturation medium. After stimulation, RNA isolation and real-time qPCR were performed as described above.
Co-cultures of iNs with mAs or iAs-SB, or without glia, were grown on coverslips (see above) and transferred to the recording chamber for in vitro recordings. During electrophysiological recordings, coverslips were constantly perfused with carbogenated artificial cerebral spinal fluid (ACSF; 119.0 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 2.5 mM CaCl2, 26.0 mM NaHCO3, 1.25 mM NaH2PO4, and 11.0 mM glucose, pH ˜7.4) at 34° C. Whole-cell patch-clamp recordings from iNs were performed using a cesium-based intracellular solution (135.0 mM CsCl, 10.0 mM HEPES, 10.0 mM NaCl, 2.0 mM Mg-ATP, 0.3 mM Na-GTP, and 5.0 mM QX314) to detect sPSCs. Biocytin (1-3 mg/ml; Biotium) was dissolved in the pipette solution for post hoc identification of recorded iNs. Wholecell patch-clamp recordings were performed with a HEKA double patch-clamp EPC10 amplifier, with PatchMaster used for data acquisition. Data were analyzed offline with IgorPro and NeuroMatic (Rothman, J. S. & Silver, R. A. Front. Neuroinform. 12:14 (2018)). sPSCs were detected by NeuroMatic event detection, and each event was checked and identified as an sPSC manually. The identification of bursts was done manually. Bursts were characterized as a higher occurrence of sPSCs in a short time span followed by a period with a lower frequency of sPSCs.
Diffusion through gap junctions was visualized by filling of single iAs that were either grown on coverslips or implanted to the brains of adult mice. iAs on coverslips were grown as monocultures (described above).
For GFP+ iAs-SB that were implanted into adult mice, acute coronal brain slices were prepared. Mice were anesthetized and decapitated, brains were removed, and 250-μm coronal brain slices were prepared as previously described (Oki, K. et al. Stem Cells 30:1120-1133 (2012)). Coverslips and acute brain slices were constantly perfused with carbogenated ACSF. Individual iAs were patched with a potassium-gluconate-based intracellular solution (122.5 mM potassium gluconate, 12.5 mM KCl, 10.0 mM HEPES, 2.0 mM Na2ATP, 0.3 mM Na2-GTP, and 8.0 mM NaCl), using the whole-cell configuration for biocytin filling. Biocytin (1-3 mg/ml; Biotium) was dissolved in the pipette solution prior to recording. iAs were filled with biocytin for 10-15 min before the patch pipette was carefully removed and the cells were fixed with 4% PFA for 20 min or overnight for cells grown on coverslips or acute slices, respectively.
To detect biocytin-filled iAs grown on coverslips, Alexa Fluor 568-conjugated streptavidin (Thermo Fisher Scientific) diluted 1:500 in 0.025% TKPBS with 5% NDS was added for 1 h at RT.
For detection of biocytin-filled iAs-SB in acute brain slices, samples were rinsed three times for 10 min in KPBS, blocked for 1 h at RT with 1% TKPBS+10% NDS, incubated with primary antibody in blocking solution overnight at room temperature, rinsed three times for 10 min in 1% TKPBS, incubated for 2 h with secondary antibodies in 1% TKPBS+5% NDS, rinsed 3 times for 10 min in KPBS, placed on glass slides and dried overnight at room temperature before being mounted with DABCO:PVA and a coverslip.
hESCs were dissociated with Accutase, and 300,000 cells were transfected using FuGene (Active Motif), with 1 μg of each px462 Cas9n vector (Addgene #62987) carrying two different sgRNAs targeting the GFAP sequence near the site of the R239C mutation (Supplementary Table 7) and a 120-bp donor ssDNA carrying the R239C mutation and a C>T change in the PAM sequence of one of the sgRNAs (Supplementary Table 6). After transfection, cells were plated in MATRIGEL-coated six-well plates with mTeSR1 medium containing 10 μM ROCK inhibitor. Two days after transfection, a 48-h selection with 1.25 ng/ml of puromycin was performed. The surviving cells were dissociated after 7-10 days, and 100 cells were replated in six-well plates coated with laminin-521 (BioLamina), according to the manufacturer's instructions, with mTeSR1 medium containing 10 μM ROCK inhibitor. Subsequently, colonies were picked, plated, and expanded in a 24-well plate with mTesR1 medium containing 10 μM ROCK inhibitor. Clones were analyzed after DNA extraction with a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. Using specific primers (Table 7), we amplified a 279-bp region of the GFAP gene to check for incorporation of the mutation into the genome by sequencing. For off-target effects, genomic locations of interest were amplified with specific primers (Table 8). Sequence data were analyzed using SnapGene (GSL Biotech).
tetO-GFP cotransduced cells were dissociated with Accutase 7-10 days after induction, pelleted at 300 g for 5 min, and resuspended in Cytocon Buffer (43.25 g of myo-inositol, 5 g of polyvinyl alcohol, and 200 ml of PBS in 800 ml of distilled water) containing doxycycline (2.5 μg/ml) at 20,000-40,000 cells/μl. NSG mice at postnatal day 2-3 were anesthetized by hypothermia and placed on a chilled stereotaxic platform, and one unilateral injection of 1 μl was delivered with a 26-gauge Hamilton syringe into the forebrain at the following coordinates (from bregma and brain surface): anterior—posterior, 0-0.5 mm; medial-lateral, 1.5 mm; dorsal-ventral, −0.5-1.0 mm. Adult mice were anesthetized with isoflurane (3% during induction, 1.5-1.8% during maintenance) and placed into a stereotaxic frame. The mice received two injections of approximately 37,000-50,000 cells resuspended in 1 μl of Cytocon Buffer at the following coordinates: anterior-posterior, +0.38 mm; medial-lateral, ±1.5 mm; and dorsal-ventral −0.75 and −2.5 mm. For both newborn and adult implanted mice, 4-13 weeks after implantation, the mice were deeply anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg per kg body weight) and perfused with saline (0.9% NaCl) followed by 4% PFA; the brains were removed and post-fixed overnight with 4% PFA at 4° C., cryoprotected in 30% sucrose cut into 30-μm coronal serial sections on a sliding microtome (Leica) and either processed for immunohistochemistry or stored in antifreeze solution at −20 ° C. All procedures were conducted in accordance with the European Union Directive and were approved by the ethical committee for the use of laboratory animals at Lund University and the Swedish Board of Agriculture.
Data are presented as the mean±s.e.m. unless otherwise stated in the respective figure legends. Statistical analyses (Prism) were performed using different tests as appropriate. For RT-qPCR results, unpaired two-tailed t-tests with Welch's correction for unequal variances were performed. For cytokine stimulation, statistical analysis was carried out by one-tailed ratio-paired t-test comparing iAs with and without treatment. For synapse formation, normal distribution was assessed with a Shapiro-Wilk test and an ordinary one-way ANOVA comparing all groups. In the case of AxD calcium imaging response, a multiple t-test was performed to compare WT and AxD iAs values at each time point. For the peak comparison, data did not show a normal distribution according to Shapiro-Wilk test, so an unpaired Mann-Whitney test was used. In the case of the electrophysiology experiments, a Mann-Whitney test or an unpaired t-test was used when appropriate. Significance was set at P<0.05.
CaCl2: Dissolve at 2.5M in sterile water. Aliquot and store at −20° C.
Doxycycline: 25 mg/mL dissolved in water (10000× stock). Sterilize with a 0.22 μm filter and store at −20° C. Protect from light.
CNTF: Reconstitute at 10 μg/ml in sterile 10 mM sodium phosphate containing 0.1% BSA. Aliquot and store at −20° C.
bFGF: Reconstitute at 1 mg/ml in sterile 5 mM Tris, pH 7.6 containing 0.1% BSA. Aliquot and store at −20° C.
BMP4: Reconstitute at 10 μg/ml in sterile 4 mM HCl containing 0.1% BSA. Aliquot and store at −20° C.
dbcAMP: Reconstitute at 100 mg/ml in sterile water. Aliquot and store at −20° C. Protect from light.
N-acetyl-cysteine: Reconstitute at 50 mg/ml in sterile water. Aliquot and store at −20° C. Heparin-binding EGF-like growth factor: Reconstitute at 50 μg/ml in sterile PBS containing 0.1% BSA. Aliquot and store at −20° C.
Expansion Medium:
FGF Medium:
Maturation Medium:
Lentiviruses were produced in HEK 293T cells by co-transfecting pMD2.G, pRSV-Rev and pMDLg/pRRE helper vectors together with the vector for one transcription factor using 2.5 M CaCl2. 75 μg of transcription factor plasmids was used together with 30 μg of PMDLg/pRRE, 22 μg of pMD2.G and 15 μg of pRSV-Rev plasmids for two T175 flasks. Medium was changed 16 hours after transfection and viruses were harvested 48 hours after transfection, pelleted by centrifugation (20,000×g for 2 hours at 4° C.) resuspended in 100 μl DMEM, aliquoted and kept at −80° C.
Day −2: Dissociate hESC or hiPSC with Accutase and replate 5×105 cells in MATRIGEL-coated 6-well plates with mTeSR1 containing 10 μM Rock inhibitor.
Day −1: Aspirate medium and add 2 ml of fresh mTeSR1 containing 8 μg/ml of Polybrene per well. Add 1 μl of each virus per well.
Day 0: Aspirate medium and add 2 ml of fresh mTeSR1 containing 2.5 μg/ml of Doxycycline per well.
Days 1 and 2: Aspirate medium and add 2 ml of Expansion Medium containing 2.5 μg/ml of Doxycycline, 1.25 μg/ml of Puromycin and 200 μg/ml of Hygromycin per well.
Day 3: Aspirate medium and add 2 ml of 3:4 of Expansion Medium and 1:4 of FGF Medium containing 2.5 μg/ml of Doxycycline and 200 μg/ml of Hygromycin per well.
Day 4: Aspirate medium and add 2 ml of 1:1 of Expansion Medium and FGF Medium containing 2.5 μg/ml of Doxycycline and 200 μg/ml of Hygromycin per well.
Day 5: Aspirate medium and add 2 ml of 1:4 of Expansion Medium and 3:4 of FGF Medium containing 2.5 μg/ml of Doxycycline and 200 μg/ml of Hygromycin per well.
Day 6: Aspirate medium and add 2 ml of FGF Medium containing 2.5 μg/ml of Doxycycline.
Day 7: Dissociate cells with Accutase and pellet at 300 g for 5 min. Re-plate cells in MATRIGEL-coated coverslips, petri dishes or wells according to desired output. Use the necessary amount of FGF Medium containing 2.5 μg/ml of Doxycycline.
Day 8: Aspirate medium and add fresh FGF Medium containing 2.5 μg/ml of Doxycycline.
Day 10: Aspirate half of the medium and add the same amount of Maturation Medium containing 2.5 μg/ml of Doxycycline. From here, change half of the medium every 2-3 days.
In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and/or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.
Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the present invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may 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 deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term “about.” As used herein, the term “about” means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.
The terms “a,” “an,” “the” and similar referents used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. 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 herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the present invention so claimed are inherently or expressly described and enabled herein.
All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/072065 | 8/16/2019 | WO |
