Patent Application
20190194606
The invention relates to culturing neuronal lineage cells, particularly spinal cord progenitors, together with endothelial cells in a fluidic device under conditions whereby the cells mimic the structure and function of developmental structures.
Human stem cell derived models of neurodegenerative diseases are challenged by immaturity in vitro. Developing neurons in vivo interact with multiple non-neuronal cell types as they mature. Microphysiological systems (MPS), also known as Organ-Chips, possess novel microvolume culture geometries that also enable co-culture of distinct cell types relevant for organ function. Brain Microvascular endothelial cells (BMECs) share many common signaling pathways with neurons early in development, however their contribution to human neuronal maturation is largely unknown. There is a great need in the art to develop differentiation systems that faithfully mirror in vivo developmental structures and processes.
Described here are systems and methods for deriving both spinal motor neurons and brain microvascular endothelial cells from induced pluripotent stem cells using distinct methods and combining them in a chip format. Neurons cultured alone in chip microvolume displayed increased calcium transient function and chip-specific gene expression compared to 96 well plates. BMECs seeded into a distinct channel in the chip directly contacted developing neural cultures and this interaction further enhanced neural function and elicited vascular-neural interaction and niche genes. Transcriptomic comparison to fetal and adult spinal cord tissue revealed enhanced in vivo-like signatures arising from the chip co-cultures. Development of novel media formulations further allow for improved readout of differentiation process, by eliminating additives that otherwise confound differentiation processes and resulting phenotypes.
Described herein is a method of generating spinal neural progenitor cells (spNPCs) including providing induced pluripotent stem cells (iPSCs), differentiating iPSCs into neural ectodermal cells by culturing in neural induction media, and dissociating neural ectodermal cells, replating on a cell culture substrate and further culturing the replated neural ectodermal cells in differentiation media to generate spNPCs. In other embodiments, the iPSCs are cultured in neural induction media for a period of about 5-7 days. In other embodiments, the replated neural ectodermal cells are cultured in differentiation media for a period of about 5-7 days. In other embodiments, the neural induction media comprises one or more of: LDN193189, SB431542, and CHIR99021. In other embodiments, the cell culture substrate is matrigel. In other embodiments, the differentiation media comprises one or more of: ascorbic acid, retinoic acid, SAG. In other embodiments, the method further includes freezing the spNPCs.
Also described herein is a cryopreserved solution of spNPCs made by is a method of generating spinal neural progenitor cells (spNPCs) including providing induced pluripotent stem cells (iPSCs), differentiating iPSCs into neural ectodermal cells by culturing in neural induction media, and dissociating neural ectodermal cells, replating on a cell culture substrate and further culturing the replated neural ectodermal cells in differentiation media to generate spNPCs.
Described herein is a cell culture supplement, including one or more first additives selected from the group consisting of: Albumin, Bovine Serum, Sodium Bicarbonate, L-Ascorbic Acid, Putrescine, D(+)-Galactose, Holo-transferrin, Catalase, L-Carnitine, Glutathione (reduced), Sodium Selenite, Ethanolamine, and T3 (triiodo-L-thyronine), and one or more second additives selected from the group consisting of: Corticosterone, Linoleic Acid, Linolenic Acid, Lipoic Acid (thioctic acid), Progesterone, Retinol Acetate, a-Tocopherol (vitamin E), and a-Tocopherol acetate. Further described herein is a culture media including the cell culture supplement and a base media. In other embodiments, the base media comprises DMEM/F12.
Also described herein is a method of generating spinal neural progenitor cells (spNPCs) including providing induced pluripotent stem cells (iPSCs), differentiating iPSCs into neural ectodermal cells by culturing in neural induction media for about 6 days, and dissociating neural ectodermal cells, replating on a cell culture substrate and further culturing the replated neural ectodermal cells in differentiation media for about 6 days to generate spNPCs. In other embodiments, the neural induction media, differentiation media, or both, comprise a supplement including: one or more first additives selected from the group consisting of: Albumin, Bovine Serum, Sodium Bicarbonate, L-Ascorbic Acid, Putrescine, D(+)-Galactose, Holo-transferrin, Catalase, L-Carnitine, Glutathione (reduced), Sodium Selenite, Ethanolamine, and T3 (triiodo-L-thyronine), and one or more second additives selected from the group consisting of: Corticosterone, Linoleic Acid, Linolenic Acid, Lipoic Acid (thioctic acid), Progesterone, Retinol Acetate, a-Tocopherol (vitamin E), and a-Tocopherol acetate. In other embodiments, the neural induction media comprises one or more of: LDN193189, SB431542, and CHIR99021. In other embodiments, the cell culture substrate is matrigel. In other embodiments, the differentiation media comprises one or more of: ascorbic acid, retinoic acid, SAG. Also described herein is a quantity of spNPCs, made by the aforementioned method. In other embodiments, the method further includes freezing the spNPCs. Further described herein is a cryopreserved solution of spNPCs made by the aforementioned method.
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089 (1996 December); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
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.
Some abbreviations are used herein. For example, “MN” refers to motor neurons. The letter “i” indicates “induced.” Thus, “iMN” indicates induced motor neurons, i.e. motor neurons that were induced or generated from other cells, e.g. stem cells. “diMN” indicates direct induced motor neurons. “iMNP” indicates induced motor neuron progenitor cells, which are not fully differentiated into mature neurons.
There are many ways to evaluate the integrity and physiology of an in vitro system that mimics the blood brain barrier. Two of the most common methods are Transepithelial Electric Resistance (TEER) and Lucifer Yellow (LY) rejection. Importantly, manipulations must be
performed using aseptic techniques in order for the cells to remain in culture without contamination. TEER measures the resistance to pass current across one or more cell layers on a membrane. The measurement may be affected by the pore size and density of the membrane, but it aims to ascertain cell and/or tissue properties. The TEER value is considered a good measure of the integrity of the cell monolayer.
Lucifer Yellow (LY) travels across cell monolayers only through passive paracellular diffusion (through spaces between cells) and has low permeability. Therefore it is considerably impeded in passing across cell monolayers with tight junctions. Permeability (Papp) for LY of <5 to 12 nm/s has been reported to be indicative of well-established monolayers.
A wide variety of human tissues can be generated from human induced pluripotent stem cells (iPSCs). However, cells generated in vitro in most culture systems remain fetal-like in nature, thereby limiting their utility for research and disease modeling. Comparative analysis of developing spinal cord tissue and iPSC-derived motor neuron cultures have revealed that even with prolonged time in culture, spinal motor neurons generated from iPSCs do not possess functional and molecular maturation signatures beyond 4-6 gestational weeks. Novel culture systems that aim to enhance the function and maturation of iPSC-derived motor neurons (MNs) in vitro are of considerable interest, especially in order to study MN dysfunction in adult-onset disorders such as amyotrophic lateral sclerosis (ALS).
Unlike conventional cell culture plates, Organ-on-Chip systems offer 3-dimensional microengineered platforms that aim to better recapitulate the cellular microenvironment including cell-cell interactions at the microscale. Due to reduced media volume and non-convection geometry, microengineered cultures may provide greater concentration of soluble signals compared to 96-well plates. While groups have described these microvolume effects on various cell types, the neurogenic effects of microvolume culture on iPSC-derived neurons remain unknown, prompting us to explore them here.
Developing spMNs rely on other cell types to provide signaling cues critical to maturation. Neuromuscular junction formation and astrocyte emergence begin at 9 and 15 weeks' post-fertilization, respectively, and astrocytes continue to proliferate into post-natal development. While the importance of the interaction of these cell types in improving MN function has been demonstrated in vitro, this is not sufficient to fully mature MNs. Of note, preceding astrocyte emergence, brain microvascular endothelial cells (BMECs) invade the neural tube from the perineural vascular plexus beginning at 4 weeks post-fertilization and form a primitive blood brain barrier that directly interacts with developing neural tube progenitors and neurons. During development, vascular angiogenesis and axon neurite outgrowth share common morphogenic mechanisms known as angioneurins. In the adult context, BMECs have also been shown to influence neurogenesis. The concordant developmental timing of the two cell types, as well as shared developmental signaling pathways prompted the Inventors' hypothesis that iPSC-derived BMECS could interact with and mature iPSC-derived MN cultures in vitro. In order to explore this hypothesis, the Inventors used an Organ-Chip system that provide unique co-culture paradigms to allow distinct cell types to interact in a controlled manner.
In the current study, the Inventors use ventralized spinal neural progenitors, generated from human iPSCs in a Spinal Cord-Chip system. Compared to 96-well plate, cultures in the Chip had increased neural activity and expressed enhanced neural differentiation genes. When iPSC-derived BMECs were included, the Inventors observed further significant increases in neuronal activity, induction of vascular-neuron interaction genes, and developmental gene expression profile that indicated a more in vivo-like signature. This platform provides insight into endogenous signaling of spinal neural cultures and BMEC interaction during early human development. It also provides a novel vascularized in vitro model of motor neuron differentiation from iPSCs to better understand motor neuron-related diseases.
Described herein is a method of generating spinal neural progenitor cells (spNPCs) including providing induced pluripotent stem cells (iPSCs), differentiating iPSCs into neural ectodermal cells by culturing in neural induction media, and dissociating neural ectodermal cells, replating on a cell culture substrate and further culturing the replated neural ectodermal cells in differentiation media to generate spNPCs. In other embodiments, the iPSCs are cultured in neural induction media for a period of about 5-7 days, including about 6 days. In other embodiments, the replated neural ectodermal cells are cultured in differentiation media for a period of about 5-7 days, including about 6 days. In other embodiments, the neural induction media comprises one or more of: LDN193189, SB431542, and CHIR99021. In other embodiments, the cell culture substrate is matrigel. In other embodiments, the differentiation media comprises one or more of: ascorbic acid, retinoic acid, SAG. In other embodiments, the method further includes freezing the spNPCs. In various embodiments, the concentration of LDN193189 is 0.02 to 2 uM, SB431542 is 0.1 to 100 um, and CHIR99021 is 0.3 uM to 30 uM. In various embodiments, the concentration of retinoic acid is 0.01 um to 2 uM, cAMP is 0.01 uM to 5 uM, SAG is 0.01 uM to 5 uM, GDNF is 1 to 50 ng/mL, BDNF is 1 to 50 ng/mL.
For example, iPSCs mechanically passaged at low density, can be differentiated for 6 days to drive neural ectodermal cells in neural induction media consisting of IMDM/F12, B27, N2, 1% NEAA, 0.2 uM LDN193189, 10 uM SB431542, 3 uM CHIR99021. Alternatively, SLDM, without B27, N2, 1% NEAA, 0.2 uM LDN193189, 10 uM SB431542, 3 uM CHIR99021 may be used. Cells are then passaged with enzymatic dissociation, such as using Accutase and reseeded onto a cell culture substrate such as matrigel. In some instances, one may pattern the neural ectodermal cells to form spNPCs in Stage 2 media consisting of IMDM/F12, B27, N2, 1% NEAA, 200 ng/ml ascorbic acid, 0.1 uM retinoic acid, 1 uM SAG for an additional 6 days. Alternatively, SLDM, without B27, N2, 1% NEAA, 200 ng/ml ascorbic acid, 0.1 uM retinoic acid, 1 uM SAG may be used. spNPC cells can then be dissociated and cryogenically frozen for later use. Upon thaw, MNs were cultured in Stage 3 media consisting of IMDM/F12, B27, N2, 1% NEAA, 200 ng/ml ascorbic acid, 0.5 uM retinoic acid, 0.1 uM cAMP, 0.1 uM SAG, 10 ng/ml GDNF, 10 ng/ml BDNF, 1% PSA and fed every two days. Alternatively, SLDM without B27, N2, 1% NEAA, 200 ng/ml ascorbic acid, 0.5 uM retinoic acid, 0.1 uM cAMP, 0.1 uM SAG, 10 ng/ml GDNF, 10 ng/ml BDNF, 1% PSA can be used. At day 12 of culture cells are labeled ventral spinal neuron progenitors (SNP) when cultured using the method described in this and the above paragraph. At day 12, SNPs may be frozen, banked and then thawed when used for seeding chips or used to directly seed chips.
Also described herein is a cryopreserved solution of spNPCs made by is a method of generating spinal neural progenitor cells (spNPCs) including providing induced pluripotent stem cells (iPSCs), differentiating iPSCs into neural ectodermal cells by culturing in neural induction media, and dissociating neural ectodermal cells, replating on a cell culture substrate and further culturing the replated neural ectodermal cells in differentiation media to generate spNPCs.
Described herein is a cell culture supplement, including one or more first additives selected from the group consisting of: Albumin, Bovine Serum, Sodium Bicarbonate, L-Ascorbic Acid, Putrescine, D(+)-Galactose, Holo-transferrin, Catalase, L-Carnitine, Glutathione (reduced), Sodium Selenite, Ethanolamine, and T3 (triiodo-L-thyronine), and one or more second additives selected from the group consisting of: Corticosterone, Linoleic Acid, Linolenic Acid, Lipoic Acid (thioctic acid), Progesterone, Retinol Acetate, a-Tocopherol (vitamin E), and a-Tocopherol acetate. Further described herein is a culture media including the cell culture supplement and a base media. In other embodiments, the base media comprises DMEM/F12. For example, a cell culture supplement including one or more first additives, and one or more second additives includes all of the additives listed in Table 1. In various embodiments, the additives are at a concentration of 0.01-10, 10-100, 100-500, 500 or more ug/mL. In other embodiments, the additives are at a concentration of 1-10, 10-50, 50-100, 100 or more mg/mL. Such supplement may be added in a base media, including DMEM/F12 and other additives as shown in Table 2.
Also described herein is a method of generating spinal neural progenitor cells (spNPCs) including providing induced pluripotent stem cells (iPSCs), differentiating iPSCs into neural ectodermal cells by culturing in neural induction media for about 6 days, and dissociating neural ectodermal cells, replating on a cell culture substrate and further culturing the replated neural ectodermal cells in differentiation media for about 6 days to generate spNPCs. In other embodiments, the neural induction media, differentiation media, or both, comprise a supplement including: one or more first additives selected from the group consisting of: Albumin, Bovine Serum, Sodium Bicarbonate, L-Ascorbic Acid, Putrescine, D(+)-Galactose, Holo-transferrin, Catalase, L-Carnitine, Glutathione (reduced), Sodium Selenite, Ethanolamine, and T3 (triiodo-L-thyronine), and one or more second additives selected from the group consisting of: Corticosterone, Linoleic Acid, Linolenic Acid, Lipoic Acid (thioctic acid), Progesterone, Retinol Acetate, a-Tocopherol (vitamin E), and a-Tocopherol acetate. In other embodiments, the neural induction media comprises one or more of: LDN193189, SB431542, and CHIR99021. In other embodiments, the cell culture substrate is matrigel. In other embodiments, the differentiation media comprises one or more of: ascorbic acid, retinoic acid, SAG. Also described herein is a quantity of spNPCs, made by the aforementioned method. In other embodiments, the method further includes freezing the spNPCs. Further described herein is a cryopreserved solution of spNPCs made by the aforementioned method.
Two human induced pluripotent stem cell lines (83iCTR and 00iCTR) used in this study were previously derived from non-diseased control patient fibroblasts. Spinal motor neuron derivation was achieved using iPSCs mechanically passaged at low density, then differentiated for 6 days to drive neural ectodermal cells in neural induction media consisting of IMDM/F12 (Gibco), B27, N2, 1% NEAA, 0.2 uM LDN193189, 10 uM SB431542, 3 uM CHIR99021 (Cayman Chemical). Cells were then passaged using Accutase (Sigma-Aldrich) and reseeded onto matrigel and patterned to form spNPCs in Stage 2 media consisting of IMDM/F12 (Gibco), B27, N2, 1% NEAA, 200 ng/ml ascorbic acid, 0.1 uM retinoic acid, 1 uM SAG for an additional 6 days. Cells were then dissociated and cryogenically frozen for later use. Upon thaw, MNs were cultured in Stage 3 media consisting of IMDM/F12 (Gibco), B27, N2, 1% NEAA, 200 ng/ml ascorbic acid, 0.5 uM retinoic acid, 0.1 uM cAMP, 0.1 uM SAG, 10 ng/ml GDNF, 10 ng/ml BDNF, 1% PSA and fed every two days. At day 12 of culture cells are labeled ventral spinal neuron progenitors (SNP) when cultured using the method described in this and the above paragraph. At day 12, SNPs may be frozen, banked and then thawed when used for seeding chips or used to directly seed chips.
For BMEC Differentiation, cells are thawed (or dissociated fresh) and seeded into the chip at day 8-9 (in the case of BMECs differentiation) and at various points in neural differentiation.
The Inventors developed an improved serum-free reformulation of B27. The Inventors found that B27 included high levels of proteins like catalase and superoxide dismutase, which may confound disease phenotypes related to oxidative stress and mitochondria. For example, neurons differentiated in the Inventors' SLDM vs IMDM media from C9orf72 mutant ALS patient neurons made higher level of dipeptide repeats (disease phenotype of mutant C9orf72 ALS neurons) in the SLdiMN media. Utilizing the SLDM media formulation, the Inventors were able to achieve reliable differentiation of spinal motor neurons across multiple different donor iPSCs, with improved plating and seeding of cells at day 6 (minimal # of fails). See
The following instructions will make 200 mL of 50× supplement. Each 10 mL aliquot can supplement 500 mL of differentiation medium.
7) Combine 140 uL of Sodium Selenite; 10 uL of Ethanolamine; and 10 uL of T3 with 8 mL of DMEM/F12.
Prepare this solution in a 15 mL conical tube while the powdered components are dissolving.
8) Add 100 uL each of Corticosterone, Linoleic Acid (LA), Linolenic Acid (LnA), Lipoic Acid (LpA), Tocopherol; and Tocopherol Acetate to the secondary solution.
9) Add 50 uL of Retinol Acetate and 40 uL of Progesterone to the secondary solution
10) Add the secondary solution to the primary solution.
11) Bring the total solution up to 200 mL
12) Let sit overnight at 4 C.
13) Gently stir, then aliquot twenty aliquots of 10 mL each. Label and store at −20 C.
1) Add SLDM supplement to DMEM/F12 in a stericup
4) Add 543 uL of Superoxide dismutase
6) Mix and vacuum filter
7) Store at 4 C covered in aluminum foil
Using SLDM media and supplement, the Inventors observed higher survivability of cells after dissociation and re-plating. This media further allowed for cryo-preservatio and reliable thawing at day 12. The cryopreservation, using CryoStore CS10, capability allows for banking of large number of spinal cord progenitor cells for screening and regenerative applications in the future.
Differentiating spinal cord neurons can be maintained in this media up to 90 days, with the ability to manipulate and modify individual components that are packaged into commercially and widely used B27/N2.
The Chip was fabricated by using modified methods for Chip microfabrication as previously described. Briefly, PDMS pre-polymer was mixed at a 10:1 ratio of PDMS base to curing agent, wt/wt using a planetary mixer (Thinky ARE-310). PDMS pre-polymer was then cast onto molds forming the microchannels of the upper layer (1,000 μm wide×1,000 μm high) and lower layer (1,000 μm wide×200 μm high). The membrane was cast onto a silicon mold that was fabricated using photolithography and deep reactive ion etching, resulting in 7 um pores. The components were cured overnight and removed from the mold. The upper layer, membrane, and lower layer were permanently bonded via plasma bonding to form the complete Chip. Chips then were treated with plasma in 100% oxygen for 2:00 minutes and immediately coated with Matrigel for the neural channel and a mixture of collagen IV (Invitrogen), Fibronectin (Invitrogen), and diluted in water in a ratio of 1:4:5 for the vascular channel. Coated chips were then incubated overnight at 37° C. and 5% CO2. Chips were seeded with BMECs at a concentration of 25,000 cells/μL and allowed to attach to the membrane by turning the chip over and incubating for 3 hours. Later spNPCs were thawed and seeded at a density of 6000 cells/μL and incubated overnight before flushing with fresh media. Media was then replaced every other day with approximately 254, in main channel and an extra 25μ in reservoir tips on either side.
Fetal tissue was received from the Birth Defects Research Laboratory at the University of Washington under their approved Institutional review board (IRB), consent and privacy guidelines. Protocols were performed in accordance with the Institutional Review Board's guidelines at the Cedars-Sinai Medical Center under the auspice IRB-SCRO Protocol Pro00021505.
Spinal cord samples arrived with estimated age and as partially intact spinal columns which were partitioned into approximated cervical, thoracic and lumbar sections. Fetal tissue was subsequently fixed in 4% paraformaldehyde for 48 hr, and placed in 30% sucrose for an additional 24 hour. Finally, spinal cords were embedded in Tissue-Tek OCT (VWR) and sectioned at 25 μm using a cryostat (Leica) at −20° C. and directly mounted on glass slides (Fisher Scientific). Tissue sections were permeabilized in cold MeOH for 20 minutes, and blocked in PBS containing 5% normal donkey serum (Sigma, D9663) and 0.25% Triton-X for 1.5 hour, then transferred to primary antibody solution containing mouse anti-GLUT1 (R&D Systems, 1:100) SMI-32P-100 (Covance, 1:1,000), and goat anti-Islet-1 (R&D, AF1387, 1:500), and rabbit anti-NFH (Sigma, N4142, 1:1000), rabbit anti-SIRT (Sigma, SAB4502861, 1:100) and incubated overnight at 4° C. Samples were then incubated for 1 h at room temperature in donkey anti-mouse Alexa Fluor 488 and donkey anti-goat 594 secondary antibodies (Life Technologies, A21202 and A21289, 1:1,000 each). Fetal samples were mounted in Fluoromount-G (Southern Biotech, 0100-01) and acquired at 20× using automated stitching on a Leica DM 6000 microscope for whole mount image.
iPSC-derived cultures were fixed in 4% paraformaldehyde, and rinsed with PBS. Cultures were permeabilized in 10% Triton X at RT for 10 min and blocked in 5% donkey serum and 0.1% Triton-X at RT for 1 h. Samples were incubated overnight at 4° C. in primary antibody solution containing the following antibodies: mouse anti-SMI-32 (Covance, SMI-32P-100, 1:1,000), rabbit anti-ZO-1 ( ), mouse anti-GLUT1 (GLUT1), mouse anti-Occludin ( ), mouse anti-Claudin5 ( ), rinsed in PBS, and incubated for 1 h at room temperature in donkey anti-mouse Alexa Fluor 488, donkey anti-goat 594, and donkey anti-goat 647 secondary antibodies (Life Technologies, A21202 and A21289, 1:1,000 each).
Confocal images were acquired using an A1 confocal microscope (Nikon) using a Plan Apo 10× objective at 1-micron increments.
BMECs were seeded in endothelial cell media into either a T75 flask, or the bottom channel of the SC-chip. SNPs were thawed and seeded into either the 96 well plates or the top channel of the Chip and incubated overnight. The following day, media was replaced with Stage 3 media in all conditions. BMEC flask was washed 2× with minimal neural media (IMDM:F12 0.5% N2, 1% B27, 0.5×NEAA, 1×PSA). 24 hours later BMEC media was collected, centrifuged and filtered. BMEC conditioned media was then supplemented with remaining ingredients for Stage 3 media. Media for all conditions was replaced every two days. At 6 days post-seeding, samples were dissociated with Accutase (Sigma-Aldrich). 3 Chip or 3 wells of a 96 well plate were pooled for each experimental replicate for a total of three replicates. Pooled samples were washed in PBS, resuspended in cold MACS buffer (Miltenyl Biotech), and filtered using a 40-micron screen. Cells were sorted using an Influx FACS sorter (BD). GFP positive gating was established using SC-chip seeded exclusively with 83iGFP MNs for a positive control, and 83iCTR BMECs for a negative control. Samples were sorted using this established gating. Positive fraction cell pellets were frozen at −80° C. until processing. For population counts at day 30, both channels were dissociated as before, and one Chip per replicate was quantified by number of GFP positive events.
Fluo-4 calcium dye (Invitrogen), was prepared at 10 mM in 50% Pluronic solution and DMSO, and diluted to a final concentration of 20 μM in ECS. Tissue cultures were incubated at RT for 30 minutes, then washed in fresh ECS and incubated an additional 30 minutes before acquiring. After a 2-minute burn in phase, 16 bit videos were acquired for 3 minutes at 20 Hz on an Eclipse Ti microscope (Nikon) using a Plan Flor 20× objective (Nikon) equipped with an Orca-Flash4.0LT digital camera (Hamamatsu). As no difference in event detection was determined between 16- and 8-bit data, all datasets were down-sampled to 8-bit (ImageJ). Calcium activity was counted by tracing ROI in blinded fashion, i.e. the treatment conditions were not associated with the results, and creating masks for use in extracting intensity data (ImageJ). 20-50 neurons were counted per site, and at least 3 Chip or wells were included for each condition. A total of 148-400 neurons per condition were analyzed using MATLAB. dF/F traces were extracted through FluoroSNNAP. Automated calcium event detection was accomplished through template libraries described previously at a threshold of 0.05. Events were then curated manually by a blinded counter, in other words a person evaluated the events without knowing the associated treatment conditions.
SNPs (fresh or thawed) were seeded into the top channel of the SC-chip and incubated for 6 days. SNPS or MNs were cultured in Stage 3 media (after day 12) consisting of IMDM/F12 (Gibco), B27, N2, 1% NEAA, 200 ng/ml ascorbic acid, 0.5 uM retinoic acid, 0.1 uM cAMP, 0.1 uM SAG, 10 ng/ml GDNF, 10 ng/ml BDNF, 1% PSA and fed every two days.
After 6 days, cultures underwent immunostaining of cells in the main channel of the chip, including but not limited to markers of spMNs SMI32, nuclear marker islet1 (ISL1), Beta 3 tubulin (TUBB3), NKX6.1, neurofilament marker microtubule-associated protein 2 (MAP2), and synaptic marker synaptophysin (SYNP).
Tall channel microphysiological systems (Emulate Inc.) were treated with plasma in 100% oxygen for 2:00 minutes and immediately coated with matrigel for the neural channel and a mixture of collagen IV (Invitrogen), Fibronectin (Invitrogen), and diluted in water in a ratio of 1:4:5 respectively, and incubated overnight at 37° C. and 5% CO2. Chips were seeded with MNs and BMECs on the same day sequentially by flipping the Chip and allowing cells to attach to the membrane by gravity. Chips were fed with approximately 25 uL of Stage 3, with an extra 25 uL in reservoir tips and media was replaced every other day.
To study the neurogenic consequences of Organ-Chip microenvironment, a rapid protocol was developed to derive first neural ectoderm ventralized spinal neural progenitor cells (spNPC) from healthy control human fibroblast-derived iPSCs in 12 days that could be frozen and banked for future experiments (
Spontaneous neuronal activity drives developmental programs and is essential for the formation of mature neuronal circuits in the central nervous system (CNS) (Moody and Bosma 2005). To determine if the Spinal Cord-Chip culture was amenable to spontaneous neuronal activity, cultures were treated with calcium-activated dye Fluo-4 and florescent activity was acquired at 20 Hz for 3 mins (
To recreate vascular-neural interactions in vitro, BMECs from the same 83CTR iPSC line were derived using an established protocol and seeded into the bottom channel of the Spinal Cord-Chip (
The Inventors further discriminated the infiltration of both cell types in the Spinal Cord-Chip co-culture. The 83iCTR iPSC line was engineered to constitutively express green florescent protein (83iGFP) with a nuclear localization signal by inserting the cassette into the AAVS1 locus using a zinc finger nuclease. 83iGFP iPSCs were then differentiated into spNPCs and seeded into the top channel as before, while BMECs were generated from the wildtype iPSC line and seeded into the bottom channel (
The Inventors next asked whether exposure of spNPC cultures to either the chip microenvironment and/or BMEC led to enhanced MN physiological function compared to culture in a 96-well (96W) plate. Live imaging and calcium-activated dye Fluo-4 were used to assess neuronal activity in these density-matched 83iCTR spNPC culture environments. Calcium transient signatures of BMECs analyzed in isolation at the seeding end were characterized by gradual influx and efflux that was distinct from the neural compartment (
As iPSC-derived MN cultures commonly contain a mixture of both interneurons and excitatory MNs, the Inventors next wanted to determine if MNs themselves were specifically activated in the chips. To achieve population identification of calcium imaging data, cultures from the 00iCTR activity study were immunostained for motor neuron markers SMI32 and ISL1 and merged with the same site of live calcium acquisition (
While calcium imaging is an important marker of neuronal activity, it does not capture certain aspects of neuronal maturation and cell-cell interaction. To address these, the Inventors performed RNA sequencing on spNPC cultures in the Spinal Cord-Chip alone or with BMEC co-culture. Day 12 GFP-positive spNPC cultures were thawed and then cultured for 6 days in 4 conditions: 96W, 96W with BMEC conditioned media (96CM), Spinal Cord-Chip seeded with spNPCs alone (Chip) or with the addition of BMECs (Chip+BMEC). After 6 days, they were dissociated and the GFP-positive population (neural) was separated from the GFP-negative population (BMEC) by florescence-assisted cell sorting (FACS) (
To discover signaling pathways activated in each condition, principal component analysis (PCA) was performed on all samples, and the top 200 ranked genes from each analysis were then entered into the network-based pathway analysis platform DAVID (FIG. 25d-e). The greatest variance (PC1) separated the day 12 spNPCs from day 18 MN stage in all other conditions (
Because PC2 elicited “Neural Differentiation” and “Response to Protein Stimulus” DAVID gene ontology categories, the Inventors further investigated Spinal Cord-Chip induced genes relating to neural maturation. Relative expression of genes contributing to both categories were compared by calculating a Z-score for each gene across all samples (
Angioneurin signaling pathways related to VEGF, notch and axon guidance mechanisms are known to play heavily on both neuronal and vascular maturation in vivo. These pathways were therefore probed to determine if BMEC activated these pathways when co-cultured with neurons in the Spinal Cord-Chip. Vascular endothelial growth factor (VEGF) is a major contributor to sprouting angiogenesis in response to hypoxia and has been implicated in neural maturation. VEGFA receptor, KDR was induced in response to BMEC contact (
Within the “Vascular Development” category, cell surface proteins important for both vascular infiltration and neuronal outgrowth were expressed specifically in response to BMEC co-culture. Thrombospondin domain-containing Semaphorins 3A and 5A transcripts increased 2 and 5-fold respectively compared to Spinal Cord-Chip. Semaphorins have opposing effects on angiogenesis in vivo, and in neurons are required for axon guidance, synaptogenesis, and neural maturation. Chemokine receptor (CXCR4) and ephrin B2 (EFNB2) are cell surface guidance molecules that are both required for motor neuron axon outgrowth.
Extracellular matrix proteins were among the most significantly induced in response to BMEC interaction and contained proteins that are implicated in neural maturation. Thrombospondin 1 (THBS1) of astrocyte origin has been shown to facilitate synaptogenesis and maturation derived of iPSC-derived neurons when co-cultured in vitro. THBS1 is cleaved by A disintegrin and metalloproteinase with thrombospondin moteifs 1 (ADAMTS1), which was also significantly induced. While ADAMTS18 activity in the neurodevelopmental context is unknown, ADAMTS1 is expressed in motor neurons during spinal cord injury. Extracellular matrix component Tenacin-C (TNC) was also significantly induced and has been shown to mediate motor neuron axon outgrowth in zebrafish and avian models. Collagen is an important constituent of vascular basement membrane and neurovascular niche. Expression of collagen family members COL5A2, COL12A1, and most significantly COL14A1 (over 10-fold expression) were significantly expressed by neural cultures in response to BMEC inclusion into the Spinal Cord-Chip.
The pathways induced in response to BMEC co-culture in the Spinal Cord-Chip are known to be present in the developing CNS, however, cited studies were primarily generated using animal models. To ensure that induced pathways reflected those in the human developing spinal cord and to determine the extent to which these activated genes contributed to in vivo maturation, whole transcript data from the culture conditions were compared to datasets generated from primary human spinal cord tissue. Published transcriptomic profiles of fetal spinal cord (purple) and adult laser captured spinal motor neurons of a non-diseased cohort (green) were included and normalized to the 10002 genes that were analyzed in the culture experiments (
Dev-PC2 provided a ranked list of genes that contributed to the in vivo fetal signature along the most positively weighed genes. The Inventors therefore, sought to determine specific genes induced by Spinal Cord-Chip culture that promoted an in vivo-like spinal cord signature. The 50 highest-weighted Dev-PC2 genes were compared across all iPSC-derived cultures as before (
In addition to BMEC's appreciated purpose as the gatekeepers to the brain, this model demonstrates the functional effects of iPSC-derived endothelial cells on neural development in vitro. Attempts to study the interplay of vascular endothelium on developing neural tissue have been challenged by inherent limitations to traditional approaches to co-culture. Transwell type cultures have been useful in the study of soluble signaling between primary murine BMECs from the adult neurovascular niche which report pro-proliferative effects through media conditioning that enhance neurogenic potential of primary cortical neural stem. However, soluble factors secreted by BMECs were not a major driver of proliferation, transcript expression, and spontaneous neural function in developing iPSC-derived cultures within the Chips. Whether this difference in soluble signaling is a result of iPSC origin and differentiation, or whether BMECs in the adult neurovascular stem cell niche are functionally distinct from the developing brain microvasculature context, or whether the difference is due to the specific advantage offered by the Organ-Chip system remains unclear. Prominent expression of Isl1 in both BMECs and MNs were observed in human spinal cord and iPSC-derived spMNs. Though BMEC ISL1 activity on vascular development and interaction with the developing neural tube is unknown, ISL1 knockout mice with ablated MN pools also displayed compromised vasculature indicating ISL1 requirement in vascular and MN development.
The advantages of a more functional system generated from including more of the cellular diversity of the spinal cord will likely increase physiological relevance further. Astrocytes are a critical component of the blood brain barrier and have been shown to increase neuronal maturation in co-culture. In addition, microglia colonization of the spinal cord begins at 9 weeks in human development is critical for synaptic pruning during development, and is implicated in neurodegenerative disease pathology such as C9Orf72 mutant ALS. Methods to derive astrocytes and microglia from human iPSCs have been described and could offer additional parameters to determine cell type contribution to and protection from disease. The multi-channel design of the chip system may allow for additional cell types to be seeded in a controlled manner to achieve an increased level of in vivo-like physiology.
The instability of cell attachment in PDMS chips remain a significant challenge for long term culture that if resolved could allow for additional maturation of iPSC-derived neural tissues. The hydrophobic nature of PDMS material represents unique challenges that could be mediated by altering surface properties through chemical treatments and refined ECM substrates. Additionally, the 7 uM size of the membrane pores was ideal for interaction of BMECs to neural cells, however reduced diameters may be necessary to combat pronounced BMEC infiltration into the top channel for application of this system to test BBB penetrance of therapeutics into neural tissue. Constitutive media flow has been shown to be required for proper organ development in other tissues and could represent a novel parameter for accelerated development.
The function of SST in human developing spinal cord MNs remains largely unknown. In a separate study based on meta-analysis of published microarray data, SST was determined to be a major contributor to spinal cord maturity. This suggests SST could be a critical neuropeptide in human motor neuron development. Somatostatin receptor family member 1 is abundantly expressed in the mouse spinal cord and could mediate SST action, however intracellular SST as observed by immunostaining could also have non-canonical effects on MN development. The expression of SST as a result of Spinal Cord-Chip culture and in response to co-culture with BMECs indicates that the chip microenviroment and paradigm for co-culture may help increase physiological relevance of iPSC-derived neurological models which show great promise in elucidating key functional and pathological mechanisms of the human CNS.
The various methods and techniques described above provide a number of ways to carry out the invention. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. A variety of advantageous and disadvantageous alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several advantageous features, while others specifically exclude one, another, or several disadvantageous features, while still others specifically mitigate a present disadvantageous feature by inclusion of one, another, or several advantageous features.
Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.
Although the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the invention extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.
Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are spinal cord cells, methods and compositions for production of such cells, including media formulations, and other variations of the invention. Various embodiments of the invention can specifically include or exclude any of these variations or elements.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention 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 the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the invention (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. 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. 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.
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 thus fulfilling the written description of all Markush groups used in the appended claims.
Preferred embodiments of this invention are described herein, including the best mode known to the inventor for carrying out the invention. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the invention can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this invention include 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 elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.
In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that can be employed can be within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present invention are not limited to that precisely as shown and described.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/057724 | Oct 2016 | US | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/049193 | 8/29/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62380780 | Aug 2016 | US | |
| 62380780 | Aug 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15352289 | Nov 2016 | US |
| Child | 16329659 | US | |
| Parent | PCT/US2016/057724 | Oct 2016 | US |
| Child | 15352289 | US |
