|
|
|
BME395F 2018 BIOMEDICALSYSTEMSENGINEERINGIICELLSANDTISSUES E...
BME395F_2018_BIOMEDICALSYSTEMSENGINEERINGIICELLSANDTISSUES_E.pdf
Showing 5 out of 9
BME395F 2018 BIOMEDICALSYSTEMSENGINEERINGIICELLSAN...
BME395F_2018_BIOMEDICALSYSTEMSENGINEERINGIICELLSANDTISSUES_E.pdf-UNIVERSITY OF TORONTO FACULTY OF APPLIED
BME395F 2018 BIOMEDICALSYSTEMSENGIN...
BME395F_2018_BIOMEDICALSYSTEMSENGINEERINGIICELLSANDTISSUES_E.pdf-UNIVERSITY OF TORONTO FACULTY OF APPLIED
Page 5
Figure 2
Neuralization of hES cells by dual-
SMAD inhibition permits a pre-rosette, neural
stem cell with dopaminergic and motoneuronal
potential. (a-c) The PAX6
neural tissue (green)
expressed rosette markers (red) Nestin (a), PLZF
(b), 701
(
c).
(
d) Rosettes are formed when PAX6
tissue is passaged to conditions promoting
rosettes
(BASF)
confirmed by K167 (green) and
phospho histone H3 (red) expression,
evidence of interkinetic nuclear migration.
(e,f)
In the absence of factors that confer regional
I
luminal
neuronal specificity, the PAX6
neural tissue
(green) expressed OTX2 (e) and FOXG1
(
f),
indicating that the tissue defaults to forebrain
specification.
(g-j)
Neural crest could be
identified on the periphery of the
PAX6*
tissue
(green) based on AP2
(g),
HNK1 (h), PAX7 (I),
and p75 expression (I) (red). (k,I) Upon passage,
the neural crest cells gave rise to pigmented cells
I
51545)
(k) that expressed HMB45 (I; green), indicating
melanosome synthesis. (m) Dopaminergic
neuronal patterning was initiated with the
addition of super sonic on days 5-9, followed
by the addition of BDNF, ascorbic acid, sonic
hedgehog and FGF8 or days 9-12. Dopaminergic
I
cells were matured on days 12-19 with BDNF,
ascorbic acid, GDNF, TGFb3 and cAMP.
Motoneuronal patterning was initiated at day 5
with the addition of BDNF, ascorbic acid, sonic
19
hedgehog, and retinoic acid. Cells were passaged
on day 11. (no) Without passage, TH
cells
could be observed by day 19.
(p)
When passaged
en bloc
on day 12, more mature processes from
TH
cells were observed.
(q,r)
For motoneuron
HB9
induction, nuclear expression of the motoneuron
markers ISLi (q) and HB9 (r) were observed
I
within a total of 19 d of differentiation from hES
cells. Scale bars, 100 pm (a ce-jo-r); 50
im
-
"
(d),
200 pm
(kIn).
TH neuron:
SHH
BASF
Motoneuron:
BASR
Passage
Xft
PAX6
Pigment
ted
PAXS
SB43 1542
-
Noggin
-
Day:
TUJ1
T1 I, TUJ1
TUJ1
ISLi
FAX
Melanosome (HI
I
12
5
9
II
12
'KSR
.:..-.
.-
LETTERS
0
.T1
1
4
We previously reported the isolation of rosette neural stem (R-NS)
cells
and neural crest stem (NCS) cells
from hES cells. We next
sought to determine the lineage relationship of the early PAX6
neuroectodermal cells observed in the dual-SMAD-inhibition protocol
to the R-NS and NCS cell populations described previously. Immuno
cytochemical analysis showed that, similar to R-NS cells, PAX6
neuroectodermal cells express general neural stem cell markers, such
as Nestin, and R-NS cell markers, including promyelocytic leukemia
zinc finger (PLZF;
Fig. 2a,b;
day 11 of differentiation). However,
cyto arch itecture and Z01 expression indicated that neuroepithellal
cells under these conditions were nonpolarized, exhibiting a more
primitive ES cell—like cytoarchitecture. These nonpolarized areas
were interspersed with R-NS cell—like areas composed of polarized
columnar epithelial cells (Fig. 2c). The developmental hierarchy of
these two cell populations was further explored upon subsequent
passage. Early neuroepithelial cells spontaneously converted into
rosette structures with apical ZOl expression and evidence of
interkinetic nuclear migration after cell passage (Fig.
2d).
These
data suggest that the Noggin/5B431542 protocol yields an early
l'AX6 neuroepithelial population capable of rosette formation. The
early PAW cells may therefore represent the most primitive
hES cell derived neural precursor stage isolated to date. R-NS cells
have been shown to acquire anterior CNS markers by default 8.
NATURE BIOTECHNOLOGY
VOLUME 27 NUMBER
3
MARCH 2009
PAX6 neuroepithelial cells generated by
means of the dual-SMAD-inhibition protocol
exhibited an anterior CNS character, as evidenced by expression of
0o2 and FoxGtB
(Fig.
2e,f),
similar to R-NS cells
8
. Notably, PAX6
cells under these conditions co expressed markers of neural crest,
including AP2, HNKt, PAX7 and p75 (NGFR)
(Fig. 2g
j).
Manipula-
tion of the initial hES cell plating density skewed the ratio of PAX6
CNS and PAX6 neural crest—like cells. High plating densities resulted
in near-exclusive differentiation toward PAW cells, whereas low
densities promoted neural crest—like differentiation (Supplementary
Fig. 5 online). The presence of large numbers of neural crest like cells
before rosette formation suggested that dual-SMAD inhibition yields
an early neural crest population distinct from R-NS cell—derived NCS
cells'. Supporting the notion of an early neural crest population with
distinct lineage potential, cells could be readily enriched for pigmented
cells co-expresssing the melanosome marker, HMB45 (Fig. 2k,l). In
contrast, R-NS cell—derived NCS cells typically do not yield pigmented
cells under similar conditions
3
. However, not all
HMB45+
cells co-
expressed the neural crest marker SOXtO, suggesting the presence of
other pigmented cell populations, including retinal pigment epithelial
cells arising from the PAW domain (data not shown). It will be
interesting in the future to compare differential fate potential of early
versus R-NS cell derived neural crest precursors in our hES-cell
system with the fate potential of different neural crest precursor
populations
in VIVO.
277
Ace your assessments! Get Better Grades
Browse thousands of Study Materials & Solutions from your Favorite Schools
University of Toronto
University_of_Toronto
School:
Biomedical_Engineering_II-_Cells_and_Tissues
Course:
Great resource for chem class. Had all the past labs and assignments
Leland P.
Santa Clara University
Introducing Study Plan
Using AI Tools to Help you understand and remember your course concepts better and faster than any other resource.
Find the best videos to learn every concept in that course from Youtube and Tiktok without searching.
Save All Relavent Videos & Materials and access anytime and anywhere
Prepare Smart and Guarantee better grades
Know more
Students also viewed documents
lab 18.docx
lab_18.docx
Course
Course
3
Module5QuizSTA2023.d...
Module5QuizSTA2023.docx.docx
Course
Course
10
Week 7 Test Math302....
Week_7_Test_Math302.docx.docx
Course
Course
30
Chapter 1 Assigment ...
Chapter_1_Assigment_Questions.docx.docx
Course
Course
5
Week 4 tests.docx.do...
Week_4_tests.docx.docx
Course
Course
23
Week 6 tests.docx.do...
Week_6_tests.docx.docx
Course
Course
106
