U.S. patent application number 16/329659 was filed with the patent office on 2019-06-27 for methods and compositions for spinal cord cells.
This patent application is currently assigned to CEDARS-SINAI MEDICAL CENTER. The applicant listed for this patent is CEDARS-SINAI MEDICAL CENTER. Invention is credited to Alexis J. KERL, Sam SANCES, Dhruv SAREEN, Clive SVENDSEN, Gad VATINE.
Application Number | 20190194606 16/329659 |
Document ID | / |
Family ID | 61241755 |
Filed Date | 2019-06-27 |
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United States Patent
Application |
20190194606 |
Kind Code |
A1 |
VATINE; Gad ; et
al. |
June 27, 2019 |
METHODS AND COMPOSITIONS FOR SPINAL CORD CELLS
Abstract
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. When seeded with endothelial cells, interaction
further enhanced neural function, elicited vascular-neural
interaction, niche gene expression with 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.
Inventors: |
VATINE; Gad; (Los Angeles,
CA) ; SANCES; Sam; (Santa Monica, CA) ;
SVENDSEN; Clive; (Pacific Palisades, CA) ; SAREEN;
Dhruv; (Porter Ranch, CA) ; KERL; Alexis J.;
(Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CEDARS-SINAI MEDICAL CENTER |
Los Angeles |
CA |
US |
|
|
Assignee: |
CEDARS-SINAI MEDICAL CENTER
Los Angeles
CA
|
Family ID: |
61241755 |
Appl. No.: |
16/329659 |
Filed: |
August 29, 2017 |
PCT Filed: |
August 29, 2017 |
PCT NO: |
PCT/US2017/049193 |
371 Date: |
February 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15352289 |
Nov 15, 2016 |
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16329659 |
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PCT/US2016/057724 |
Oct 19, 2016 |
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15352289 |
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62380780 |
Aug 29, 2016 |
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62380780 |
Aug 29, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/15 20130101;
C12N 2500/38 20130101; C12N 2539/00 20130101; C12N 5/069 20130101;
C12N 2535/10 20130101; C12N 2531/00 20130101; C12N 2501/155
20130101; B01L 2300/044 20130101; C12N 2501/727 20130101; C12N
2501/41 20130101; B01L 3/502715 20130101; C12N 5/0623 20130101;
C12N 2506/45 20130101; C12M 23/16 20130101; C12N 2501/385 20130101;
C12N 2501/999 20130101; B01L 2300/123 20130101; B01L 2300/0858
20130101; C12M 25/02 20130101; C12N 5/0619 20130101; A61K 35/30
20130101; C12N 2502/088 20130101; C12N 5/0068 20130101; C12N
2501/13 20130101 |
International
Class: |
C12N 5/00 20060101
C12N005/00; C12N 5/0797 20060101 C12N005/0797; C12M 3/06 20060101
C12M003/06; B01L 3/00 20060101 B01L003/00; C12M 1/12 20060101
C12M001/12; C12N 5/071 20060101 C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 19, 2016 |
US |
PCT/US2016/057724 |
Claims
1. A method of generating spinal neural progenitor cells (spNPCs)
comprising: 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.
2. The method of claim 1, wherein the iPSCs are cultured in neural
induction media for a period of about 5-7 days.
3. The method of claim 1, wherein the replated neural ectodermal
cells are cultured in differentiation media for a period of about
5-7 days.
4. The method of claim 1, wherein the neural induction media
comprises one or more of: LDN193189, SB431542, and CHIR99021.
5. The method of claim 1, wherein the cell culture substrate is
matrigel.
6. The method of claim 1, wherein the differentiation media
comprises one or more of: ascorbic acid, retinoic acid, SAG.
7. The method of claim 1, further comprising freezing the
spNPCs.
8. A cryopreserved solution of spNPCs made by the method of claim
7.
9. A cell culture supplement, comprising: 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.
10. A cell culture media comprising: the cell culture supplement of
claim 9, and a base media.
11. The cell culture media of claim 10, wherein the base media
comprises DMEM/F12.
12. A method of generating spinal neural progenitor cells (spNPCs)
comprising: 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.
13. The method of claim 12, wherein the neural induction media,
differentiation media, or both, comprise a supplement comprising:
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.
14. The method of claim 12, wherein the neural induction media
comprises one or more of: LDN193189, SB431542, and CHIR99021.
15. The method of claim 12, wherein the cell culture substrate is
matrigel.
16. The method of claim 12, wherein the differentiation media
comprises one or more of: ascorbic acid, retinoic acid, SAG.
17. A quantity of spNPCs made by the method of claim 12.
18. The method of claim 12, further comprising freezing the
spNPCs.
19. A cryopreserved solution of spNPCs made by the method of claim
18.
Description
FIELD OF THE INVENTION
[0001] 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.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
SUMMARY OF THE INVENTION
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1. Depiction of protocol for cellular differentiation,
including use of SLDM media. Sareen lab differentiation media
(SLDM) is a new supplement method made in the Inventors' lab, while
IMDM/F12 is the traditional method of making the spinal cord cells.
Whereas differentiating iPSCs for 6 days using IMDM/F12 method
could lead to sample loss due to cell death after dissociating and
re-seeding on this day, SLDM improves adherence and viability of
differentiating cells.
[0009] FIG. 2. Comparative study of media including IMDM vs SLDM.
Day 6, 5.times. magnification. At day 6, neuroepithelia are
formed.
[0010] FIG. 3. Comparative study of media including IMDM vs SLDM.
Day 6, 10.times. magnification. Seen here are cells cultured in
IMDM possessing folds and curves that are indicative of inability
to survive dissociation and re-seeding.
[0011] FIG. 4. Comparative study of media including IMDM vs SLDM.
Day 6, 20.times. magnification.
[0012] FIG. 5. Comparative study of media including IMDM vs SLDM.
Day 12, 5.times. magnification. At day 12, motor neuron precursors
are formed. Here, cells in SLDM method look very uniform at this
day 12 stage.
[0013] FIG. 6. Comparative study of media including IMDM vs SLDM.
Day 12, 10.times. magnification.
[0014] FIG. 7. Comparative study of media including IMDM vs SLDM.
Day 12, 20.times. magnification.
[0015] FIG. 8. Comparative study of media including IMDM vs SLDM.
Day 12, 5.times. magnification.
[0016] FIG. 9. Comparative study of media including IMDM vs SLDM.
Day 12, 10.times. magnification.
[0017] FIG. 10. Comparative study of media including IMDM vs SLDM.
Day 12, 20.times. magnification.
[0018] FIG. 11. Comparative study of media including IMDM vs SLDM.
Day 18, 5.times. magnification.
[0019] FIG. 12. Comparative study of media including IMDM vs SLDM.
Day 18, 10.times. magnification.
[0020] FIG. 13. Comparative study of media including IMDM vs SLDM.
Day 18, 20.times. magnification. Cells/neurons on day 18 in
IMDM/F12 are in tight clumps, preventing effective imaging
analysis.
[0021] FIG. 14. Comparative study of media including IMDM vs SLDM.
Day 18, 5.times. magnification.
[0022] FIG. 15. Comparative study of media including IMDM vs SLDM.
Day 18, 10.times. magnification.
[0023] FIG. 16. Comparative study of media including IMDM vs SLDM.
Day 18, 20.times. magnification.
[0024] FIG. 17. Comparative study of media including IMDM vs SLDM.
Day 25, 5.times. magnification.
[0025] FIG. 18. Comparative study of media including IMDM vs SLDM.
Day 25, 10.times. magnification.
[0026] FIG. 19. Comparative study of media including IMDM vs SLDM.
Day 25, 20.times. magnification.
[0027] FIG. 20. Comparative study of media including IMDM vs SLDM.
Day 25, 5.times. magnification.
[0028] FIG. 21. Comparative study of media including IMDM vs SLDM.
Day 25, 10.times. magnification. Using traditional IMDM method, a
greater number of non-neuronal "flat" cell types can be seen in the
background using this method, when compared to SLDM culturing.
[0029] FIG. 22. Comparative study of media including IMDM vs SLDM.
Day 25, 20.times. magnification.
[0030] FIG. 23: spNPCs survive and mature in the chip
microenvironment. (a) Schematic of Spinal neural progenitor (spNPC)
differentiation from induced pluripotent stem cell (iPSC) cultures.
Cells were fated to neural ectoderm (NE) using WNT agonist
CHIR99021 and SMAD inhibitors LDN193189 and SB431542 for 6 days
then patterned to ventral spinal neurons using Retinoic Acid (RA)
and Sonic hedgehog agonist (SAG) in 6-well plates. At day 12,
spNPCs were frozen, banked and thawed for experiments (Cryo-bank).
spNPCs were seeded into the top channel of the SC-chip (dotted
line) and incubated for 6 days. (b) Schematic of dual-channel organ
chip seeded with spNPCs in top channel. (c) Phosphorylated
neurofilament heavy chain (SMI32) is enriched in spinal motor
neurons (spMNs) and expressed in cells populating entire top
channel. Scale bar=200 microns. (d) Immunostaining of main channel
of the chip of 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), scale=200 microns (left), 40 microns (right).
(e) Representative image of SC-chip neurons treated with Fluo-4
calcium activated dye and acquired live in FITC channel. (f)
Florescence of individual active neurons normalized to baseline
florescence and plotted over time (dF/F) (g) Raster plot showing
detected events of corresponding traces.
[0031] FIG. 24: Co-culture of iPSC derived MNs and BMECs in Spinal
Cord-Chip. (a) Schematic of dual-differentiation and seeding
paradigm in EC/Spinal Cord-Chip. Both spNPCs and BMECs are
generated from human iPSCs and seeded into top and bottom channels
respectively. Transverse section of the EC/Spinal Cord-Chip (right)
shows two compartments separated by porous membrane. (b)
Immunostaining of whole Spinal Cord-Chip at 6-days incubation
expressing SMI32 in top channel and ISL1 expressed by BMECs on
bottom channel. (c) Maximum projection images cropped along Z-axis
to show top and bottom compartments of seeding end of EC/Spinal
Cord-Chip immunostained with MN markers SMI32 and islet 1 (ISL1),
and tight junction marker zona occludens 1 (ZO-1), Scale bar=400
microns (left) 40 microns (right). (d) Confocal optical
reconstruction at along Z axis (top) of Spinal Cord-Chip exhibits
distinct separation of cultures at 6 days separated by porous PDMS
membrane. Perspective view (bottom) exhibits confluent layer of
BMECs surrounding entire bottom channel, Scale bar=100 microns. (e)
Representative images of cells attached to porous membrane in the
main channel (top and bottom planes). Spinal Cord-Chip was seeded
either with spNPCs alone in the top channel (Spinal Cord-Chip) or
with the addition of non-GFP BMECs into the bottom compartment
(EC/Spinal Cord-Chip). (f) High magnification of top compartment
show GFP negative, ISL1 positive clusters indicating BMEC
infiltration. (g-h) Quantification of GFP and ISL1 populations in
top compartment. Error bars represent standard deviation. Plots
were determined from 6 individual chips from three culture rounds
(dots). No significant difference cultures determined by t-test
(ns).
[0032] FIG. 25: Spinal Cord-Chip environment increases spontaneous
neural activity. (a) Individual Representative calcium transient
activity of spNPC (top) and BMEC (bottom) cultures acquired at the
seeding compartments where cultures do not interact. Change in
florescence intensity over background (dF/F) plotted with respect
to time totaling 60 seconds. (b) Calcium transient activity plots
of 30 representative neurons derived from 83iGFP spNPC cultures in
each culture condition. (c) Transient frequency of 194-297 neurons
spanning three separate replicates per condition. Neuron activity
was completely ablated with the administration of tetrodotoxin
(TTX). (d) Immunocytochemistry staining of islet1 (ISL1) and smi32
(right) of site previously acquired for live calcium transients
using fluro-4 dye (left). ISL1.sup.+ neurons (arrows) superimposed
to determine MN firing specificity. (e) Mean transient frequency of
representative active neurons derived from 00iCTR spNPC cultures
compared to ISL1 overlaid MNs. (g-h) 30 Representative transient
events from ISL1 SMI32 double-positive neurons quantified and
displayed as raster plot and plotted as frequency in hertz (Hz).
Means represented by black bars and error bars represent standard
error of the mean. Significance calculated by one way ANOVA, **
denotes P<0.01, ***=P<0.005, and ****=P<0.001.
[0033] FIG. 26: Spinal Cord-Chip induces neural differentiation and
vascular interaction gene expression. (a) GFP-MN isolation
schematic: nuclear GFP expressing spNPCs seeded on top channel with
isogenic non-GFP expressing BMECs on bottom. Fluorescence-assisted
cell sorting (FACS) conducted on FITC to purify spMNs after BMEC
co-culture. (b) Single population histogram of chips seeded only
with non-GFP BMECs (black) or GFP-MNs (green) that defined negative
and positive fractions respectively. Number of events displayed on
y axis and FITC intensity on x-axis (c) Mean RPKM data of purified
Spinal Cord-Chip and EC/Spinal Cord-Chip cultures normalized to
RPKM data from iPSC derived BMECs cultured alone. Canonical markers
claudin 5 (CLDNS), occluding (OCLN), Tight junction protein 1
(TJP1), Glut-1 (SLC2A1), von willebrand factor (VWF), Tie2 receptor
(TEK), endoglin (ENG), and melanoma cell adhesion molecule (MCAM).
(d) Principle component analysis plots of first two principle
components (PC) (top) and PCs 2, and 3 (bottom). Arrows indicate
weighting along the axis of each respective PC. (e) Top 200 ranked
genes from each PC displayed as Z-score calculated across all
conditions for each row and indicated by colorimetric scale. Each
PC was entered into DAVID ontology pathway analysis and top 7
categories listed for each PC. The number of genes (count) in each
category are displayed with corrected significance values from
DAVID analysis (Bonferonni). (f,g) Differentially expressed genes
contributing to the "Response to Protein Stimulus" and "Neural
Differentiation" DAVID terms enriched in PC2. RPKM data averaged
across sample replicates and normalized to 96W control. (h,i)
Differentially expressed genes contributing to "Vascular
Development" and "Extracellular Matrix" DAVID terms enriched in
PC3.
[0034] FIG. 27: BMECs induce vascular interaction gene expression
in Chip. (a) PCA comparing expression data of differentially
expressed genes from PC2 and PC3 and including in vivo adult laser
captured MNs (green), in vivo fetal spinal cord (purple), and in
vitro experimental data. (b) Heatmap of top ranking genes from
Dev-PC2 that describe variance in fetal gene expression. Z-score
calculated from Log.sub.e RPKM data per gene row and displayed by
colorimetric scale. (c) Mean Somatostatin (SST) expression plotted
for each condition, error bars represent standard deviation. (d)
Representative images of 96 well (96W) and Spinal Cord-Chips
co-cultured with BMECs (EC/Spinal Cord-Chip) immunostained with SST
and MN markers SMI32 and ISL1, scale bars=40 microns. (e) Whole
mount image of day 67 human fetal spinal cord (top) immunostained
with SST, SMI32 and ISL1. Higher magnification of ventral horn ISL1
positive MN pool (box) co-expressing SST (bottom).
[0035] FIG. 28: (a) Schematic of relative emergence of cell types
that directly interact with developing human MNs in vivo. (b) Days
53 and 67 human fetal spinal cords immunostained with neuronal
marker neuofilament heavy chain (NFH), BMEC marker glucose
transporter 1 (GLUT1), and transcription factor ISL1. High
magnification of ventral horns (box) show ISL1 positive MN pools
(yellow dotted line). (c) Images of iPSC-derived BMECs cultured in
96-well plate for 6 days and immunostained with GLUT1, ISL1, and
tight junction markers zona occludens 1 (ZO-1) and Occludin
(OCLDN). (d) Images of spNPCs cultured in 96W plate and
immunostained with 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).
[0036] FIG. 29: (a) Calcium transient activity plots of 30
representative neurons derived from 00iCTR spNPC cultures in each
culture condition. (b) Quantification of at least 3 chips per
condition from one experimental replicate.
[0037] FIG. 30: (a) Density plots of 10,001 genes for each sample
for transcriptomic analysis. Quantile normalized data (right). (b)
Pierson correlation coefficient analysis of normalized RPKM
datasets displayed by colorimetric scale. (c) Notch pathway genes
differentially expressed in the Spinal Cord-Chip conditions.
[0038] FIG. 31: (a) Density plots of 10,001 genes of developmental
comparison containing adult laser captured MNs (green), fetal
Spinal cord (purple) and sorted iPSC-derived neurons (black).
Quantile normalized data (bottom). (b) PCA analysis of variance
across all samples and all 10,001 genes.
DETAILED DESCRIPTION
[0039] 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 22.sup.nd 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 3.sup.rd ed.,
revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith,
March's Advanced Organic Chemistry Reactions, Mechanisms and
Structure 7.sup.th 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 2.sup.nd ed., Cold
Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Kohler 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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).
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
Example 1
Pluripotent Stem Cell Culture, Directed Differentiation into spMNs
and BMECs
[0054] 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.
[0055] 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.
Example 2
Improved Media Formulation
[0056] 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
FIGS. 1-22. The SLDM media with supplements can fully serve as a
replacement to IMDM/F12, and B27.
[0057] The following instructions will make 200 mL of 50.times.
supplement. Each 10 mL aliquot can supplement 500 mL of
differentiation medium.
TABLE-US-00001 TABLE 1 Supplement Concentrate [Stock] [Supplement]
200 mL Reagent DMEM/F12 1x 1x Up to 200 mL Albumin, Bovine Powder
125 mg/mL 25 g Serum Sodium Bicarbonate Powder 27.15 mg/mL 5.43 g
L-Ascorbic Acid Powder 3.2 mg/mL 640 mg Putrescine Powder 805 ug/mL
161 mg D(+)-Galactose Powder 750 ug/mL 150 mg Holo-transferrin
Powder 250 ug/mL 50 mg Catalase Powder 125 ug/mL 25 mg L-Carnitine
Powder 125 ug/mL 20 mg Glutathione (reduced) Powder 50 ug/mL 10 mg
Sodium Selenite 1 mg/mL 0.7 ug/mL 140 uL Ethanolamine 1 g/mL 50
ug/mL 10 uL T3 (triiodo-L- 2 mg/mL 0.1 ug/mL 10 uL thyronine)
Ethanolic Stocks Corticosterone 2 mg/mL 1 ug/mL 100 uL Linoleic
Acid 100 mg/mL 50 ug/mL 100 uL Linolenic Acid 100 mg/mL 50 ug/mL
100 uL Lipoic Acid (thioctic 4.7 mg/mL 2.35 ug/mL 100 uL acid)
Progesterone 3.2 mg/mL 0.32 ug/mL 20 uL Retinol Acetate 20 mg/mL 5
ug/mL 50 uL a-Tocopherol (vitamin 100 mg 50 ug/mL 100 uL E)
a-Tocopherol acetate 100 mg 50 ug/mL 100 uL
Procedure:
1) Dissolve 25 g of Bovine Serum Albumin in 160 mL of DMEM/F12.
[0058] Stirring is necessary and dissolving the solution on ice may
be impossible. Perform at room temperature with the stir speed set
as low as possible. 2) Dissolve 5.43 g of Sodium Bicarbonate in the
solution. [0059] Weigh out and prepare the other components while
the initial components are dissolving. 3) Dissolve 640 mg of
L-Ascorbic acid in the solution.
4) Dissolve 161 mg of Putrescine.
5) Dissolve 150 mg of Galactose.
6) Add 50 mg of Holo-transferrin; 25 mg of Catalase; 20 mg of
Carnitine; and 10 mg of Glutathione.
[0060] 7) Combine 140 uL of Sodium Selenite; 10 uL of Ethanolamine;
and 10 uL of T3 with 8 mL of DMEM/F12.
[0061] 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.
TABLE-US-00002 TABLE 2 Sareen Lab Differentiation Media (SLDM)
including supplement Reagent [Stock] Location [Final] 500 mL
DMEM/F12 1x 4.degree. C., Gomez -- 500. mL SLDM 50x -20.degree. C.,
1x 10. mL Supplement Wednesday PSA 1x 4.degree. C., Gomez, 1% 5. mL
(Anti-anti) or Fester Glutamax 100x 4.degree. C., Gomez 1x 5. mL
Superoxide 2.5 mg/mL -20.degree. C., 2.5 .quadrature.g/mL 500.
.quadrature.L Dismutase Fester, SODM box Insulin 10 mg/mL 4.degree.
C., Gomez 4 ug/mL 200. .quadrature.L
Procedure:
[0062] 1) Add SLDM supplement to DMEM/F12 in a stericup
2) Add 5 mL of PSA
3) Add 5 mL of Glutamax
[0063] 4) Add 543 uL of Superoxide dismutase
5) Add 200 uL of Insulin
[0064] 6) Mix and vacuum filter 7) Store at 4 C covered in aluminum
foil
[0065] 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.
[0066] 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.
Example 3
Chip Microfabrication and Culture
[0067] 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
.mu.m wide.times.1,000 .mu.m high) and lower layer (1,000 .mu.m
wide.times.200 .mu.m 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.degree. C. and 5% CO.sub.2. Chips were seeded with
BMECs at a concentration of 25,000 cells/.mu.L 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/.mu.L and incubated overnight before flushing with fresh
media. Media was then replaced every other day with approximately
254, in main channel and an extra 25.mu. in reservoir tips on
either side.
Example 4
Immunohistochemistry
[0068] 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.
[0069] 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 .mu.m
using a cryostat (Leica) at -20.degree. 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.degree. 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.times. using automated stitching on a
Leica DM 6000 microscope for whole mount image.
[0070] 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.degree. 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).
Example 5
Imaging and Population
[0071] Confocal images were acquired using an A1 confocal
microscope (Nikon) using a Plan Apo 10.times. objective at 1-micron
increments. FIG. 24d was generated from auto-stitching three
confocal stacks at 10.times. (Nikon Elements). Images were rendered
with maximum projection and quantified using Imaris (Bitplane). For
population and ISL1 nuclei size analysis, images were cropped to
only top channel and max projected using IMARIS software. Sites
were prioritized as containing maximal GFP positive nuclei in the
frame. GFP positive nuclei were quantified using spots algorithm
and ISL1 positive nuclei were quantified by filtering by GFP
co-localization.
Example 6
Transcriptomic Analysis
[0072] 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.times. with minimal neural media (IMDM:F12 0.5% N2, 1% B27,
0.5.times.NEAA, 1.times.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.degree. 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.
Example 7
Live Calcium Transient Imaging and Analysis
[0073] Fluo-4 calcium dye (Invitrogen), was prepared at 10 mM in
50% Pluronic solution and DMSO, and diluted to a final
concentration of 20 .mu.M 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.times. 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.
Example 8
Seeding Chips Using Day 12 Spinal Neural Progenitor (SNP) Cells
[0074] 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.
[0075] 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).
Example 9
Seeding Chips Using Day 12 Spinal Neural Progenitor (SNP) Cells and
BMECs in SC-Chip
[0076] 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.degree. C. and 5% CO.sub.2. 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.
Example 10
Neurons in Spinal Cord-Chip Develop and are Spontaneously
Active
[0077] 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 (FIG. 23a).
spNPCs were seeded into the top channel of the dual-channel Spinal
Cord-Chip constructed of polydimethylsiloxane (PDMS) (Emulate Inc.)
(FIG. 23b). Top and bottom channels of the chip are separated by a
50-micron thick membrane perforated by 7-micron diameter pores
spaced 40-microns apart from center to center. Within 6 days of
incubation in the top channel, mixed neural cultures expressed
markers of developing spinal motor neurons (spMN), including motor
neuron progenitor markers NKX6.1 and TUBB3, and spMNs as indicated
by co-expression of the markers islet-1 (ISL1) and phosphorylated
neurofilament heavy chain (SMI32) (FIG. 23c,d). Neural cultures
also stained positive for MAP2 and synaptophysin, indicating that
synaptogenesis was initiated within the Chip.
[0078] 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 (FIG. 23e). By plotting the change in
fluorescence with respect to time (dF/f), neuron-specific calcium
transient events were characterized by fast onset and slow decay,
consistent with developing neurons in vitro (FIG. 23f). Calcium
transient event detection in the Spinal Cord-Chip showed extensive
activity in the relatively early cultures, providing evidence of
accelerated development of neural activity and connectivity (FIG.
1g).
Example 11
BMECs Infiltrate into Top Channel of Spinal Cord-Chip and Form a
Barrier
[0079] 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
(FIG. 24a). After 6 days in culture, immunostaining with GLUT-1 and
SMI32 revealed clear separation of the two cultures in the top
(neural) and bottom (endothelial) channels along the entire Chip
(FIG. 24b-d). BMECs exhibited cobblestone morphology typical of
endothelial culture in vitro and expressed endothelial tight
junction proteins zona occludens 1 (ZO-1), glucose transporter 1
(GLUT-1), and Claudin 5 (FIG. 24b-d, S1b). BMEC cultures also
expressed nuclear ISL1, previously unreported in human vascular
endothelium (FIG. 24c). To confirm vascular ISL1 expression in
vivo, human fetal spinal cord sections at post-conception days 53
and 67 were immunostained for ISL1 and GLUT-1 and found to be
co-expressed within infiltrating vasculature (FIG. 248). While
SMI32-positive soma were not found in the bottom channel,
ZO-1-positive SMI32-negative regions in the top channel revealed
infiltration of BMECs (FIG. 24c-d). Confocal 3D reconstruction of
the main channel displayed clear distinction of the two cultures
with confluent BMECs surrounding the entire bottom channel that
formed a continuous vessel-like tube (FIG. 24d).
[0080] 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 (FIG. 24e). This
allowed for clear distinction of GFP-positive MN cultures from GFP
negative, ISL1-positive expressing BMECs. 83iGFP spNPCs seeded into
the top channel without BMECs in the bottom channel readily
infiltrated through the membrane pores into the bottom channel. In
contrast, when 83iCTR-BMECs were included in the bottom channel,
GFP-positive spNPC infiltration was effectively blocked (FIG. 24e).
In the top channel, defined regions of GFP negative/ISL1-positive
nuclei were observed consistent with ZO-1 staining showing BMEC
infiltration from the bottom channel (FIG. 24c). To determine the
influence of BMEC contact on spMN culture development, top channel
GFP positive nuclei were quantified and assessed for ISL1 positive
expression. There was no significant difference in GFP.sup.+ spMN
nuclei in the presence of BMECs (FIG. 24f-h). In addition, the
percent ISL1-positive nuclei were not significantly different,
indicating BMECs did not significantly affect MN
differentiation.
Example 12
Chip and BMECs Increase Spontaneous Activity of Spinal Neural
Cultures
[0081] 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 (FIG. 25a). To ensure that only neuron activity was
analyzed in all conditions, putative BMEC activity was filtered out
by requiring events to return to baseline within 20 seconds.
Transient events for 148-300 neurons from each condition were
plotted and quantified (FIG. 25b). Results showed that there was a
significant increase in the average number of spontaneous neuronal
transients in the Spinal Cord-Chip cultures compared to 96-wells
(FIG. 25c). Of note, neurons cultured with BMECs in the Spinal
Cord-Chip had a significantly increased frequency of calcium
transients compared to culture in the Spinal Cord-Chip alone.
Transient fluorescence was effectively blocked in the presence of
voltage-gated sodium channel antagonist tetrodotoxin (TTX),
indicating fluorescence specificity to channel activity. These
findings were reproduced using an additional control iPSC line,
00iCTR (FIG. 29a-b).
[0082] 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 (FIG. 25d). 114
SMI32.sup.+/Isl1.sup.+ MNs were compared to 232 representative
active neurons from the same acquisition site.
ISL1.sup.+/SMI32.sup.+ MNs had significantly less activity when
compared to overall neuronal activity (FIG. 25e). Calcium events
were detected in ISL1.sup.+/Smi32.sup.+ MNs for each culture
environment (FIG. 25f). Interestingly, increased activity in
SMI32.sup.+/Isl1.sup.+ MNs was consistent with overall increased
activity observed in chip cultures when compared 96-well plates
(FIG. 29e). Rare populations of MNs were highly active in the BMEC
containing chips, however no overall increase in average transient
frequency in MNs was observed compared to spNPCs cultured in Chip
alone. This indicated that MNs within the mixed culture were more
functional compared to the 96-well plate, and that MN activity with
the addition of BMECs was more diverse.
Example 13
Chip Microenvironment and BMEC Co-Culture Induce Distinct
Transcriptomic Signatures
[0083] 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) (FIG. 26a-b). A lack of BMEC-specific marker expression in
the FACS sorted samples chips containing BMECs indicated that BMECs
were effectively removed through FACS (FIG. 26c). Thawed spNPCs
were also included to determine differences in gene expression
resulting from the 6 days in culture. 10002 genes expressing at
least 0.5 RPKM in at least one condition were included in the
dataset and RPKM data was quantile normalized across all samples
(FIG. 30a). Pearson correlation analysis showed high
reproducibility among sample replicates (FIG. 30b).
[0084] 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 (FIG. 25d). Day 18 MNs
had higher expression of gene pathways involved in neural
differentiation, neurogenesis, and axonal growth (FIG. 25c). The
next greatest variance (PC2) separated chip culture conditions
(Chip and Chip+BMEC) from the two 96-well cultures (96W and 96CM)
and were driven by genes associated with neural differentiation
pathways that were specifically up-regulated in the chip
microenvironment. However, PC2 did not show a notable difference
between the 96W and 96CM conditions suggesting only a small, and
possibly antagonistic effect of conditioned media on neural
cultures grown in 96-well plates. Finally, PC3 was defined by genes
that were differentially expressed when spNPCs were directly
co-cultured with BMECs within the chips (Chip+BMEC). DAVID analysis
highlighted genes associated with extracellular matrix, secreted
signaling peptides, and endothelial cell interaction pathways.
Example 14
The Chip Microenvironment Enhances Neurogenic Programs in spNPC
Cultures
[0085] 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 (FIG. 26f,g). Genes associated with
"Response to Protein Stimulus" were upregulated in both Spinal
Cord-Chip and EC/Spinal Cord-Chips, suggesting increased endogenous
signaling activity resulting from chip microenvironment. Among the
induced genes, early growth response gene 1 (EGR1) is known to be
rapidly transcribed in response to environmental stimulation and
neural activation. Cyclin D1 (CCND1) has been shown to promote MN
neurogenesis in the developing spinal cord in a cell
cycle-independent manner, acting trough a Notch-signaling effector
pathway (Lukaszewicz and Anderson 2011). Within the "Neural
Differentiation" category, transcription factors involved in spinal
cord fate specification were also induced (FIG. 26g). PAX6 is an
intermediate neural progenitor differentiation marker that
regulates neuronal cell cycle exit in the spinal cord. GLI2 and
GLI3 were also induced and are act to regulate dorsal-ventral
patterning in the spinal cord. Additionally, Up-regulation of ISL1
expression (FIG. 26g) confirmed staining in Spinal Cord-Chips and
further supported increased differentiation resulting from the chip
microenvironment.
Example 15
BMECs Induce Angioneurin and Extracellular Matrix Gene Expression
in Developing Neural Cultures in Spinal Cord-Chip
[0086] 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 (FIG. 26h). The Notch signaling pathway is
heavily implicated in both angiogenesis and neural patterning
during development in vivo. Inhibition of the Notch pathway through
small molecule .gamma.-secretase inhibitors have been used to
enhance iPSC-derived MN differentiation in vitro leading to further
investigation into Notch pathway activity. (FIG. 30c).
Significantly increased expression of DLK1, a potent inhibitor of
notch signaling, and down regulation of Notch activating ligands
JAG1, DLL1, DLL3, and DLL4 was observed in response to BMEC
co-culture in the Spinal Cord-Chip.
[0087] 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.
[0088] 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.
Example 16
Spinal Cord-Chip Induces Genes that are Associated with Fetal
Development
[0089] 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 (FIG. 31a). PCA performed on
the 10,002 genes analyzed in the culture experiments (FIG. 30b)
illustrated a minor advancement towards in vivo fetal spinal tissue
development and no differential effects among culture conditions.
However, a more focused PCA based on the top and bottom 200 ranked
gene loadings from PC2 and PC3 distinguished the culture conditions
(FIG. 25d). Dev-PC1 distinguished iPSC-derived neural cultures,
fetal and adult samples in a similar fashion to the previous
experiment (FIG. 27a), and Dev-PC2 positively weighted genes
corresponded to expression that reflected the human fetal spinal
cord. In this reduced dataset, iPSC-derived conditions separated
into discrete clusters and the Spinal Cord-Chip (blue) and
EC/Spinal Cord-Chip (orange) conditions registered more positively
along the Dev-PC2 axis and closer to the fetal samples, indicating
that this condition induces gene expression changes relevant to and
promoting in vivo spinal tissue development.
Example 17
The Spinal Cord Chip Environment Induces Somatostatin Expression in
iPSC-Derived spMNs
[0090] 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 (FIG. 27b). Id
proteins (Id2/Id3) induced in the Spinal Cord-Chips were also
highly expressed in the Fetal-SC samples. Angioneurin gene SEMA5A
approached similar levels to in vivo samples, however, expression
could not be confirmed by antibody staining (data not shown). The
6.sup.th ranking gene in PC2 Somatostatin (SST) was highly
expressed in the fetal Spinal Cord samples. In the brain in vitro
and in vivo, SST has been shown to have trophic factor activity,
influence synaptogenesis and axonal pathfinding and regulate
cortical circuit maturation. Compared to 96W samples, Spinal
Cord-Chip and EC/Spinal Cord-Chip environment induced 9 and 15-fold
SST expression, respectively. To confirm protein expression in
vitro, 96W and EC/Spinal Cord-Chips were cultured as before and
stained with SST antibody along with MN marker ISL1 and SMI32.
Pronounced SST staining was observed in the Spinal Cord-Chip both
in the absence of BMECs, and to a greater extent with BMEC
co-culture (FIG. 27d). Expression was co-localized with SMI32
positive staining and proximal to ISL1 nuclear staining in the
mixed culture, providing evidence that SST expression was specific
to iPSC-derived spMNs. To determine if MN localization of SST was
consistent with developing spMN pools in vivo, a day 67 human fetal
spinal cord section was immunostained for SST and MN markers ISL1
and SMI32 as before (FIG. 27e). ISL1 positive spMN pools could be
visualized in the ventral horn that again contained positive
staining for SST, confirming MN specificity in vivo.
Example 18
Discussion
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
* * * * *