12/4/22

Neural stem cells and

neuronal regeneration
: Techniques

Yung-Shu Kuan (管永恕)

NTU Institute of Biochemical Sciences
Dec. 12, 2022

Stem cells sources for neuronal regenera0on therapy

1. Embryonic:
- Embryonic SCs
- Neural crest SCs
2. Adult:
- Mesenchymal SCs
- Adult brain tissues
- Induced pluripotent SCs (iPSC)
✔
Regenera0on medicine using stem cells (checking list):
1. prolifera;on of cells
2. appropriate differen;a;on
3. isola;on and removal of any inappropriate cells
4. iden;fica;on of effec;ve cells for transplanta;on

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Advantages and disadvantages of different stem cells

Cells Neural stem cells Embryonic stem Induced pluripotent

cells stem cells

Source Primary ;ssues, (fetal, Early embryo Gene recombina;on

neonatal, and adult brain)

Advantages (1) Easy to access; (1) Strong prolifera;on (1) No ethical issues
(2) No ethical issues ability (2) No
(3) No histocompa;bility (2) Abundant sources histocompa;bility
(3) Can be passed on

Disadvantages (1) Strong immunogenicity (1) Ethical issues
(2) The mechanism of (2) The allograM
cell prolifera;on, produces a great
differen;a;on, and rejec;on reac;on
migra;on is unclear (3) Unrestrained
differen;a;on
(4) Tumorigenicity
(1) Complex opera;on
process
(2) Low reprogramming
efficiency
(3) Muta;on induc;on
(4) Tumorigenicity

Each stem cell has a specific neurogenic poten;al and can achieve certain results, but there are s;ll many problems to be solved before
they can be used for clinical applica;ons. (Neural Regen Res 15(2):242-250)

From stem cells to neuronal regenera0on

Embryonic
Adult NSCs
ES Cells
Adult
1. prolifera;on of cells 2. appropriate differen;a;on

Stem cells
Transplanta;on

Expansion
Differen;a;on
iPSCs

BMSCs and other cells

3. isola;on and removal of any inappropriate cells
(e.q. cord blood)
4. iden;fica;on of effec;ve cells for transplanta;on

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Isola0on of NSCs from Neural Tissue

1. Mice or rats were anesthe;zed by an intraperitoneal injec;on of either 230 mg/kg Sodium Pentobarbital or
400mg/kg Aver;n, followed by euthanasia.
2. Remove the brain and transfer to a 50-ml tube to a 10-cm petri dish (CLS430591) containing 20 ml of cold
Solu;on A (1XHBSS (H8264) containing 30 mM glucose (G7021), 2 mM Hepes (H0887), 26 mM NaHCO3
(S5761)). Place a small piece of filter paper onto Tissue Chopper, slightly wet the filter paper using a wet
sterile swab, and then set the brain onto the wet filter paper using curved-pointed forceps. Chop brain into
400-um coronal sec;ons and use wet sterile swab to collect the sec;ons containing SVZ (~6 sec;ons) and
hippocampus (~5 sec;ons) into a 6-cm petri dish (CLS430589) filled with 5 ml of Solu;on A.
Cri0cal step: Keep the petridish containing brain sec@ons and solu@on A on ice during the chopping and
dissec@on.
3. Dissect out the SVZ and the Dentate Gyrus (Brain Map) under a dissec;ng microscope and keep the
dissected ;ssue in separate 15-ml Falcon tubes (CLS430791), each containing 10 ml of cold Solu;on A.
Cri0cal step: This and subsequent steps are op@mized for isola@ng NSCs from the SVZ and DG prepara@on
of one mouse. If you plan to isolate cells from pooled @ssues of more than one mouse, we recommend that
you add the step of Percoll (P4937) purifica@on to remove excess myelin and other cell types
4. AMer finishing the dissec;on of all the brain sec;ons, spin down the 0ssue chunks in a low-speed centrifuge
at 200 g for 1 min at room temperature (20–25°C).
5. In the TC hood remove the supernatant and add 1 ml of 0.05% Trypsin-EDTA (59417C) to each 15-ml
conical tubes containing ;ssue chunks. Rotate the conical tubes at room temperature for 10-20 min.
Cau0on: Do not incubate longer than 30 min, as this will decrease the viability of the cells.
6. Add 1ml μl of Trypsin Inhibitor Solu0on (T7659) into each tube and rotate for another 10-20 min. Cau0on:
Do not incubate longer than 30 min, as this will reduce the viability of the cells.

hWps://www.sigmaaldrich.com/technical-documents/protocols/biology/neural-stem-cell-culture-protocols.html

Isola0on of NSCs from Neural Tissue

7. Pre-wet a fire-polished glass pipeke to triturate 0ssue by pipeOng up and down 10–20 ;mes un;l there
is no ;ssue clump, and then add 8 ml of N2 medium (DMEM/F-12 (D6429), N2 supplement (SCM012),
L-glutamine (G7513) to each tube. Pellet the cells at 200 g for 5 min at room temperature.
Cau0on: Avoid genera@ng air bubbles when tritura@ng the @ssue, as this will reduce the viability of the cells.
Cri0cal step: It is important to dissociate the SVZ and the DG to single cells, as any remaining aggregates
can result in reduced yield.
8. Wash cells twice with 10 ml of N2 Medium, spin down at 200 g for 5 min at room temperature each ;me.
9. If you are isola;ng cells from pooled ;ssues of 2 or more mice, re-suspend each cell pellet with 5.5 ml of N2
medium, add 5.5 ml of Percoll/PBS solu;on, and mix by inver;ng the tubes. Pellet the cells at 400 g for 15
min at room temperature. Then wash the cells 3 ;mes with 10 ml N2 medium and spin down at 200 g for 5
min at room temperature each ;me to collect cells.
Cri0cal step: Gently remove the supernatant, as the pellet may not be firmly aWached to the boWom of the
tube. Cri@cal step: It is cri@cal to remove Percoll by washing thoroughly, as residual Percoll will affect cell
viability.
10. Wash one more 0me with 8 ml of Ini0al Prolifera0on Medium (IPM) (Neural Stem Cell Basal Medium
(SCM003), B27 (SCM013), L-glutamine (G7513), 1X Pen-Strep (P4333), 20 ng/ml of FGF-2 (F0291), and 20
ng/ml EGF (E9644)).
11. Re-suspend each cell pellet with 1 ml of IPM for DG, 2 ml of IPM for SVZ, and then plate cells into one well
of a 24-well ;ssue culture plate (CLS3527) for DG, and 2 wells for SVZ cells. Incubate cells in the CO2
incubator for 48h.
12. Change half the IPM, avoiding removing any cells. Con;nue to incubate cells for 7–14 days, changing half
the IPM every other day and monitoring the cells for the forma;on of neurospheres. Neuropheres should
form in both cultures in 1–2 weeks.

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The neurosphere assay in vitro has been widely used

to iden0fy neural stem cells

The neurosphere assay in vitro has been widely used

to iden0fy neural stem cells

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Use of hESCs in
CNS regenera0on Karyotype
RT-PCR
FACS 1. + 2.
IHC
Fron;ers in Cel & Dev Bio
August 2014 | Volume 2 |
Ar;cle 36

IHC 2.+ 3.
1. prolifera;on of cells Teratoma assay
Cell func0on assay
2. appropriate differen;a;on

3. isola;on and removal of any

inappropriate cells RT-PCR
FACS
IHC 2.+ 3.
4. iden;fica;on of effec;ve cells Teratoma assay
for transplanta;on
Cell func0on assay

Transplanata0on for 4.
Stem cell-based
therapy Pharmacology
Calcium imaging
Electrophysiology
With neural cells
Or neural precursors

Human embryonic stem cells (hESCs) as the source

Human embryonic stem cell (hESC) lines which can be commercialized and used as
candidates for cell therapies. However, hESCs are prone to instability and varia;ons.

Good quality of hESCs need to meet the following cri;cal criteria:

(1) purity (absence of any microbial contamina;on)
(2) iden;ty (corresponding to the correct profile of the cells)
(3) stability (the genotype and phenotype remain stable during passaging in vitro)

It is required to analyze the expression of several pluripotent and neuroectodermal

markers in undifferen;ated hESCs before star;ng the differen;a;on procedure.

The undifferen;ated hESCs :

✔ Express : OCT-3/4 and Nanog, SSEA-4, SSEA-1, TRA-1-60, CD24 (pluripotent markers)
✗ No expression : CD133, NCAM, NF70, nes;n (NP markers)

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Neural stem cell / neural precursor markers

Differen0ated neural marker

GFAP

b3-Tubulin

MAP2

MBP

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Marker expression analysis of hESCs

CCTL14 line
FACS pluripotent
markers

Fron@ers in Cel & Dev Bio

August 2014 | Volume 2 |
Ar@cle 36

Induced pluripotent stem cells (iPSC)

Reprograming
factors

hWp://images.slideplayer.com/19/5848059/slides/slide_3.jpg

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Human iPSC induc0on factors

O=Oct4, S=Sox2, K=Klf4, M=c-Myc, L=Lin28, N=Nanog

Cord Adipose-
Cell Fibroblasts Hepato- Kera;no- Neural Pancrea;c Amnio;c
blood derived
Type cytes cytes stem cells β-cells cells
stem cells stem cells

Reprog- OKSM, OKSM, OKSM, OKSM OKSM, O OKSM OKSM,

ramming OSLN, OS OKS OKS OSN
Factors OKS

Oct4: Transcrip;on factor expressed in undifferen;ated pluripotent embryonic stem cells and germ cells during
normal development. Together with Sox2 and Nanog, is necessary for the maintenance of pluripotent poten;al.

Sox2: Transcrip;on factor expressed in undifferen;ated pluripotent embryonic stem cells and germ cells during
development. Together with Oct-4 and Nanog, is necessary for the maintenance of pluripotent poten;al.

Klf4: Zinc-finger-containing transcrip;on factor Krüppel-like factor 4; used for genera;on of human/mouse ES cells.
c-Myc: bHLH-containing transcrip;on factor, an proto-oncogene used for genera;on of human/mouse ES cells.
Lin28: ; Zinc-finger CCHC-containing RNA binding protein, used for genera;on of human ES cells.
Nanog: Homeodomain-containing transcrip;on factor essen;al for maintenance of pluripotency and self renewal in
embryonic stem cells. Expression is controlled by a network of factors including Sox2 and the key pluripotency
regulator Oct-4.

Differen0a0on of Human iPSCs into NSCs

Two commonly used methods:

1. Uses two SMAD inhibitors, Noggin (SRP4675) and SB431542 (S4317), to drive the rapid
differen;a;on of ES/iPS cells into a highly enriched popula;on of NPCs.

2. Use small molecule-based differen;a;on method, which relies on three small molecules to
inhibit GSK-3β, CHIR99021 (SML1046), TGFβ, SB431542 (S4317), and Notch, Compound E
(565790) signaling pathways, along with human LIF (LIF1010). This new small molecule-based
neural differen;a;on protocol increased neural differen;a;on kine;cs and allowed the
deriva;on of truly mul;potent neural stem cells that respond to regional pakerning cues
specifying forebrain, midbrain, and hindbrain neural and glial subtypes.

*LIF=Leukemia Inhibitory Factor

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Neural stem cell culture characteris0cs

Culture of NSCs in 2D Monolayer Culture

1. propaga;on and differen;a;on of NSCs, dilute laminin (L2020) to final concentra;on of 5 μg/ml.
Use 2 mL volume for 3.5 cm plates, 3-5 mL volume for 6 cm plates and 7-10 mL volume for 10-cm plates
and T75 flasks. Incubate in a humidified 37C incubator for at least 1 hour. Coated plates and flasks can be
stored in the laminin solu;on at 2-8C for 3 weeks or at -20C for 6-8 months. Just before use, bring the
coated plates or flasks up to room temperature and aspirate the laminin solu;on. Rinse the plates once
with 1X PBS (806552) before use.
2. Remove the vial of Neural Progenitor Cells (SCC007, SCC008, SCR055, SCC035) from liquid nitrogen and
incubate in a 37C water bath. Closely monitor un;l the cells are completely thawed. Maximum cell viability
is dependent on the rapid and complete thawing of frozen cells.
IMPORTANT: Do not vortex the cells.
3. As soon as the cells are completely thawed, disinfect the outside of the vial with 70% ethanol. In a laminar
flow hood, use a 1 or 2 mL pipeke to transfer the cells to a sterile 15 mL conical tube. Be careful to not
introduce any bubbles during the transfer process.
4. Using a 10 mL pipeke, slowly add dropwise 9 mL Neural Expansion Medium (SCM005, SCM004)
(pre-warmed to 37C) to the 15 mL conical tube.
IMPORTANT: Do not add the whole volume of medium at once to the cells. This may result in decreased
cell viability due to osmo@c shock.
5. Gently mix the cell suspension by slow pipexng up and down twice. Be careful to not introduce any
bubbles. IMPORTANT: Do not vortex the cells.
6. Centrifuge the tube at room temperature at 200 x g for 3-5 minutes to pellet the cells.

hWps://www.sigmaaldrich.com/technical-documents/protocols/biology/neural-stem-cell-culture-protocols.html

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Culture of NSCs in 2D Monolayer Culture

7. Decant as much of the supernatant as possible.

8. Resuspend the cells in a total volume of 2 mL of Neural Expansion Medium (prewarmed to 37C) containing
FGF-2, 20 ng/mL (F0291). Plate the cell mixture onto a poly-L-ornithine and laminin-coated 3.5-cm ;ssue
culture plate.
IMPORTANT: For op@mal growth, thawing the cells on @ssue culture plates that are larger than a 3.5-cm
@ssue culture plate is not recommended.
9. Incubate the cells at 37C in a 5% CO2 humidified incubator.
10. The next day, exchange the medium with fresh Neural Expansion Medium (prewarmed to 37C) containing
FGF-2 (20 ng/mL). Exchange with fresh medium every other day thereaMer.
11. When the cells are approximately 90 - 100% confluent, they can be dissociated with AccutaseTM (A6964)
and passaged or alterna;vely frozen for later use. The cells should be maintained at high cell density at all
;mes and thus the recommended passaging is at 1:2 to 1:6.

Culture of NSCs in 3D Neurosphere Culture

1. AMer 7–12 days of neurosphere culture (step 1-15 above), collect all primary spheres without
disturbing the akached cells; spin at 200 g for 5 min at room temperature.
2. Carefully remove the medium and add 1 ml of 0.05% trypsin/EDTA (59417C) to each tube.
Dissociate the spheres using a 1-ml blue ;p by pipexng up and down 20 ;mes to digest the
spheres within 2 min. Add 1 ml of Trypsin Inhibitor Solu;on (T7659) to each tube and pipeke up
and down 10 more ;mes. Add 5 ml of IPM to each tube, mix by inver;ng the tubes a few ;mes,
and pellet the cells at 200 g for 5 min at room temperature.
3. Re-suspend the DG cell pellet in 1 ml of IPM and the SVZ cell pellet in 2 ml of N2 medium. Dilute
a 10-μl aliquot from each sample in 10 μl of 0.5% Trypan blue (T8154) and count viable cells using
a hemocytometer.
4. Plate the DG cells into one well of a 24-well plate, and the SVZ cells into one well of 6-well plate
and incubate cells.
5. Replace half the medium with fresh IPM for the DG cells and fresh N2 medium for the SVZ cells
every other day for 1 week. Avoid taking any cells out when changing out half the medium.
AMer the first passage, maintain the DG and the SVZ NSCs in N2 medium and passage NSCs by
seeding at 3X104 -1X105 cells/ml every 2–3 days.

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Cell therapy approved in Taiwan (in 2019)

hkps://www.liver.org.tw/journalView.php?cat=62&sid=801&page=1

The principle of mesenchymal stem cell therapy

1. Harvest mesenchymal stem cells (MSCs) from pa;ent’s iliac bone.

2. The MSCs are incubated in the Cell Processing Center for about two weeks.
3. About 100 million MSCs are packed for a product.
4. The pa;ent receives an intravenous drip contains MSCs, which takes 30–60 minutes.

Dr. Osamu Honmou, Sapporo University, Japan

hkps://advances.tri-kobe.org/en/feature/62/spinal-cord-treatment-using-stem-cells

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Stem cell therapy for spinal cord injury (SCI)

Cell & Bioscience 10, Ar;cle number: 112 (2020)

Mechanisms of ac;on of MSCs in ameliorate SCI

Mechanisms of ac;on of MSCs in ameliorate SCI. MSC transplanta;on

promotes the spinal cord regenera;on by di eren;a;ng into neural and
glial cells, secrete paracrine factors and microvesicles, reduce inflamma;on
and oxida;ve stress, promote survival of remaining neurons and angiogenesis
as well as inhibit gliosis.
Cell & Bioscience 10, Ar;cle number: 112 (2020)

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Classifica0on of AIS grading

ASIA (American Spinal Injury Associa;on) Impairment Scale (AIS)

Cell & Bioscience 10, Ar;cle number: 112 (2020)

Comparison between different MSCs

1. BMSCs 2. UC-MSCs 3. ADSCs

Cell & Bioscience 10, Ar;cle number: 112 (2020)

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Factors affect the efficacy of MSC therapy to treat SCI

Cell & Bioscience 10, Ar;cle number: 112 (2020)

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