Supplementary Materials for the Manuscript Titled “New
Bioengineering Insights into Human Neural Precursor Cells Behavior in
Culture”
Behnam A. Baghbaderani1, Karim Mukhida2, Murray Hong2, Ivar Mendez2 and Leo A. Behie1.
1
Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering,
University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4
2
Cell Restoration Laboratory, Faculty of Medicine, Sir Charles Tupper Medical Building,
5849 University Avenue , Dalhousie University, Halifax, Nova Scotia B3H 4H7
1. Supplementary Material and Methods
1.1 The Cell Culture and Microscopy Incubator XL-3 system
The Cell Culture and Microscopy Incubator XL-3 system (PeCon GmbH, Germany) has
been designed to maintain the cell culture conditions and monitor the culture using the
Zeiss microscope connected to a computer system with AxioVision digital image
processing software (Figure S1). The Incubator XL-3 was equipped with warm air
incubation, CO2-controller system, and CO2-incubator cover. The cells were inoculated
into a 6-wellplate or 16-wellplate Lab-Tek chamber slide and placed on a microscope
stage underneath the CO2- incubator cover. Using the AxioVision software, images were
taken from the culture in a desired time interval for a certain period of time. The imaging
software allowed tailoring all images together and generating a video that demonstrated
the cell behavior in the culture.
1.2 Time Lapse Analysis in Normal Attachment Well-Plates (Expansion Phase)
Two hours prior to incubate the cells in the Cell Culture and Microscopy Incubator XL-3
system, heating unit and CO2 controller were turned on to adjust appropriate culture
conditions (i.e. temperature at 37 oC, CO2 concentration at 5%, 95% air saturation) inside
the Incubator XL-3. Human telencephalon derived NPCs obtained at the end of P9 from
stationary culture (T-25 flask) were harvested, dissociated into single cells using
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enzymatic dissociation, and inoculated at 100,000 cells/mL into two wells of BD Falcon
6-well Plate (BD Falcon, Cat#353046) each containing 3.0 mL PPRF-h2 medium. The
well plate was made of polystyrene, which was treated by manufacturer to produce a
surface amenable for cell attachment and growth. The treatment generated a surface that
was hydrophilic, wetable and negatively charged. These well-plates were standard
normal-attachment tissue culture vessels similar to the standard T-25 tissue culture flasks
(Nalge Nunc) previously used to expand human NPCs in vitro. Each well of the 6-well
plate provided a surface area of 9.6 cm2 for the cells to grow. The depth of the culture
medium was about 0.3 cm. The medium was added to the well about 1 h before
inoculation of the cells. The sterile double distilled water was added to the surrounding
wells to minimize the extensive evaporation of the growth medium in the test-well.
Moreover, about 75% of the seam between the lid and the plate was taped to minimize
evaporation while maintaining gas transfer in the culture. The 6-well plate was then
placed in the Zeiss microscope stage underneath the incubator cover. The microscope
was properly adjusted so that photomicrographs could be taken from one of the wells
inoculated with hNPCs. The experiment was started approximately 30 min after the
inoculation of the cells. This was done to make sure the cells were settled down on the
surface of the well. The experiment was carried out for a period of 108 h (4 days and
half) in culture. Every 10 min one picture was taken by the Zeiss Axiovert 200M
microscope using AxioVision software.
Another normal attachment BD Falcon 6-well Plate containing the telencephalon
derived hNPCs inoculated at similar conditions (two wells each containing 3.0 mL PPRFh2 medium) was incubated in the humidified incubator at 37oC, 95% air saturation (20%
O2), and 5% CO2 to serve as control. At the end of the time-lapse study, the cells were
harvested from the well-plate incubated in the Incubator XL-3 and the control cultures,
underwent enzymatic dissociation, and counted using trypan-blue exclusion.
1.3 Time-Lapse Analysis in Low Attachment Well-Plates (Expansion Phase)
Three independent experiments were performed using low-attachment well-plates [i.e.
Costar Ultra Low Attachment 6-well plate (Costar, Cat#3471)]. Each well contained 3.0
mL PPRF-h2 medium. Costar Ultra Low Attachment 6-well plate surface was comprised
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of a covalently bound hydrogel layer that was hydrophilic and neutrally charged. Since
proteins and other biomolecules can absorb to surfaces through hydrophobic and ionic
interactions, this hydrogel surface naturally inhibited non-specific immobilization via
these forces, thus inhibiting subsequent cell attachment. According to the Costar Ultra
Low Attachment 6-well plate manufacturer, a 99% reduction in the cell-surface
attachment of the anchorage-dependent cells on the Ultra Low Attachment plate could be
observed when compared to the cell-surface attachment in the standard tissue culture.
Each well of the 6-well plate provided a surface area of 9.6 cm2 for the cells to grow. The
depth of the culture medium was about 0.3 cm. The procedure of cell incubation and
acquiring time-lapse images were similar to the procedure used for normal-attachment
experiments. The following studies were conducted in the low attachment cultures:
(a) Inoculation of Single Cell Suspension of hNPCs in Low-Attachment Well-Plates
Human telencephalon derived NPCs obtained at the end of P9 from stationary culture (T25 flask) were harvested, dissociated into single cells using enzymatic dissociation, and
inoculated at 100,000 cells/mL into two wells of Costar Ultra Low Attachment 6-well
plate. To serve as control, the cells were also inoculated into two wells of a normal
attachment BD Falcon 6-well Plate at similar conditions (two wells each containing 3.0
mL PPRF-h2 medium). All cultures were incubated in a humidified incubator at 37oC,
95% air saturation (20% O2), and 5% CO2 for a period of 14 days in culture. The cultures
were fed every 5 days by replacing 40% 0f the spent medium with fresh PPRF-h2
medium. Photomicrographs were taken every day from each culture using the Leica
microscope. At the end of study, the cells were harvested from all cultures, underwent
enzymatic dissociation, and counted using trypan-blue exclusion.
(b) Inoculation of 125 mL Suspension Culture with Cell Aggregates Obtained from LowAttachment Culture
Human telencephalon derived NPCs obtained at the end of P9 from stationary culture (T25 flask) were harvested, dissociated into single cells using enzymatic dissociation, and
inoculated at 100,000 cells/mL into 6 wells of six Costar Ultra Low Attachment 6-well
plates (36 total wells). Each well contained 3 mL PPRF-h2 medium. It should be noted
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that the cells were taken from the same single cell suspension used to inoculate the Costar
Ultra Low Attachment 6-well plate in experiment (a). All cultures were incubated in a
humidified incubator at 37oC, 95% air saturation (20% O2), and 5% CO2 for a period of 8
hours, after which time the content of 34 wells were harvested and inoculated into one
125 mL spinner flask. At the same time, the content of two other wells were transferred
into two wells of a normal attachment BD Falcon 6-well Plate. The cultures were
incubated in a humidified incubator at 37oC, 95% air saturation (20% O2), and 5% CO2
for a period of 14 days in culture. The cultures were fed every 5 days by replacing 40%
of the spent medium with fresh PPRF-h2 medium. Samples (3.0 mL) were taken from the
bioreactor to take photomicrographs using the Leica microscope. The normal attachment
cultures were also monitored by Leica microscope. Two 3.0 mL samples were taken
from the spinner flask at the end of the study to perform cell counts after dissociation of
cell aggregates enzymatically into single cells. Cell counts were performed for the normal
attachment cultures in the same manner.
(c) Inoculation of Low-Attachment Well-Plates with Cell Aggregates Taken from
Suspension Culture
Two samples of human telencephalon derived NPCs (2.0 mL each) were taken as
aggregates of cells on day 5 from one 125 mL suspension bioreactor at P7, and inoculated
at approximately 1.5×105 cells/mL into two wells of two ultra-low attachment 6-well
plates. Prior to the addition of cell suspension, 1.0 mL fresh PPRF-h2 medium was added
to each well, and the well-plate incubated at 37oC, 95% air saturation (20% O2), and 5%
CO2 for 1 h. One of the ultra-low attachment 6-well plates was then placed in the Zeiss
microscope stage underneath the incubator cover. The microscope was adjusted so that
photomicrographs could be taken from one of the wells inoculated with hNPCs. Timelapse experiment was started approximately 30 min after the inoculation of the cells. The
experiment was carried out for a period of 100 h (about 4 days) in culture. Every 10 min
one picture was taken by the Zeiss microscope using AxioVision software.
A second low attachment culture containing hNPCs taken from the same
suspension bioreactor was prepared and placed in the humidified incubator (at 37oC, 95%
air saturation, and 5% CO2) to serve as control to the culture used in the time-lapse study
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and ensure the culture conditions in the Incubator XL-3 were comparable to the
humidified incubator. In addition, samples were taken from the same suspension culture
to inoculate two wells of normal-attachment 6-well plate exactly at the same conditions
as mentioned for the ultra-low attachment culture. Double distilled water was added to
the surrounding wells of each well-plate to avoid evaporation of the growth medium in
the test wells. Photomicrographs were taken every day from the cells grown in parallel in
suspension culture and control cultures. The cells were harvested from all cultures,
underwent enzymatic dissociation, and counted using trypan-blue exclusion at the end of
study.
1.4 Time-Lapse Analysis of Human Telencephalon NPC Differentiation
The time-lapse study was performed using the Cell Culture and Microscopy Incubator
XL-3 system. Two hours prior to start the time-lapse study, heating unit and CO2
controller were turned on to adjust the temperature at 37oC and CO2 concentration at 5%
inside the Incubator XL-3. Then, human telencephalon derived NPC aggregates were
taken on day 10 of P13 from stationary culture, dissociated into single cells, and plated at
20,000 cells/well in two wells of poly-D-Lysine/laminin precoated surface of a 16 wellplate Lab-Tek chamber slide. Each well contained 200 μL cytokine-free PPRF-h2
medium. The medium was added to each well about 1 h before inoculation of the cells.
Sterile double distilled water was added to the surrounding wells to avoid extensive
evaporation of the growth medium in the test-well. Moreover, about 75% of the seam
between the lid and the plate was taped to minimize evaporation while maintaining gas
transfer in the culture. The chamber-slide was then placed on the stage of the Zeiss
Axiovert 200M microscope underneath the incubator cover. The microscope was
adjusted so that photomicrographs could be taken from one of the wells inoculated with
hNPCs. The experiment was started approximately 30 min after the inoculation of the
cells. This was done to make sure the cells were settled down on the surface of the well.
The experiment was carried out for a period of 64 h (about 2.5 days) in culture. Every 10
min one picture was taken by the Zeiss Axiovert 200M microscope using AxioVision
software. Another Lab-Tek chamber-slide inoculated with the single cell suspension of
telencephalon derived hNPCs at similar conditions (two wells each containing 200 μL
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cytokine-free PPRF-h2 medium) was incubated in the humidified incubator at 37oC, 95%
air saturation (20% O2), and 5% CO2 to serve as control.
1.5 Florescent Activated Cell Sorting (FACS) Analysis
FACS analysis was performed using a FACSCalibur (BD Biosciences, USA) to
investigate the population of hNPCs positively staining for a number of cell surface
markers including CD13, CD14, CD19, CD24, CD29, CD34, CD44, CD45, CD90,
CD133, and CD166. Human-telencephalon derived NPCs previously obtained at the end
of passage level 10 from 125 mL suspension bioreactors and cryopreserved in PPRFh2/DMSO (10%) were used in this study. One cryopreserved vial of the cells was thawed
and underwent two additional passages (each passage lasted 14 days) in stationary culture
(T-25 flask) before performing FACS. Human NPC aggregates were then dissociated into
single cells, and fixed for 20 min in ice-cold 2% formaldehyde. After washing with 1X
PBS solution (three 5 min), the pellet remaining from the last washing step was
resuspended in 10 mL of blocking solution comprised of 1x PBS solution supplemented
with 3% fetal bovine serum (FBS), and incubated in the dark at 4-8 oC for 30 minutes.
The cell suspension was then centrifuged at 300g for 5 minutes. After removing the
supernatant, cell aliquots (1 × 106 cells) were incubated (on ice-cold in the dark for 30
minutes) with mouse anti-human CD14-IgG1-FITC (SeroTec, Cat# MC2185F), mouse
anti-human CD19-IgG1-FITC (SeroTec, Cat# MCA1940F), mouse anti-human CD90IgG1-FITC
(SeroTec,
Cat#
MCA90F),
mouse
anti-human
CD166-IgG1-FITC
(Fitzgerald, Cat# RDI-CD166-3FT), mouse IgG1 isotype-FITC (SeroTec, Cat#
MCA928F) (isotype control), mouse anti-human CD44-IgG2a-FITC (SeroTec, Cat#
MCA89F), mouse IgG2a isotype-FITC (SeroTec, Cat# MCA929F) (isotype control),
mouse anti-human CD13-IgG1k-PE (BD Biosciences, Cat# 555394), mouse anti-human
CD24-IgG1-PE (SeroTec, Cat# MCA1379), mouse anti-human CD34-IgG1k-PE (BD
Biosciences, Cat# 550822), mouse anti-human CD45-IgG1k-PE (BD Biosciences, Cat#
555483), mouse anti-human CD29-IgG1k-PE (Beckman Coulter, Cat# 4B4-RD1), mouse
IgG1k isotype-PE (BD Biosciences, Cat# 555749) (isotype control), mouse anti-human
CD133/2-PE (Miltenyi Biotec, Cat#130-090-853), and mouse IgG2b isotype-PE
(Miltenyi Biotec, Cat# 130-092-215) (isotype control). In addition, a negative control was
Page 6 of 17
prepared by incubating a single cell suspension of the cells (1 × 106 cells) in only
blocking solution and treating in the same condition as other samples. The samples were
then washed 1X PBS solution (three 5 min) prior to perform FACS analysis.
1.6 Cell Counts and Viabilities
The contents of duplicate T-25 flasks, wells of 6-well plates, or samples taken from
duplicate bioreactors were used to measure the viable cell density and viability in
stationary culture or suspension culture, respectively. Prior to cell counts, hNPC
aggregates were enzymatically dissociated using 0.25% Trypsin-EDTA solution. Cell
counts were performed in duplicate using a hemocytometer (VWR) and 0.1% trypan blue
dye (Sigma). Samples were diluted, if necessary, using Ca2+- and Mg2+-free phosphatebuffered saline (PBS) (Invitrogen).
1.7 Statistical Analysis
Comparison of the immunocytochemical analysis was conducted using one-way ANOVA
(analysis of variance). Moreover, one-way ANOVA was used to compare the cell-fold
expansion achieved for hNPCs grown in different culture conditions. The results were
considered statistically significant at p ≤ 0.05.
2. Supplementary Results and Discussion
2.1 Time-Lapse Analysis of Cell Expansion in Low-Attachment Culture
When human NPCs were inoculated as single cells in low-attachment culture, small
aggregates of cells were formed in relatively faster period of time when compared to the
aggregate formation in normal-attachment culture (Figure S2). Eight hours post
inoculation [Fig. S2 (B)], small aggregates of 70-80 μm could be observed in the lowattachment culture. Over time, these aggregates were further aggregated to one another
and formed very large aggregates of cells [Fig. S2 (C)]. By day 14, only a few large
aggregates of about 2 mm in size could be observed in the low-attachment culture [Fig.
S2 (D)].
When human NPC aggregates were taken from the low-attachment culture at 8
hours postinoculation and inoculated into the normal attachment culture [Fig. S2 (E-H)]
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or 125 mL suspension bioreactor [Fig. S2 (I-L)], the aggregates size gradually increased
over time. Moreover, the number of aggregates formed in the normal attachment culture
and suspension culture was significantly higher than the number of aggregates formed in
the low attachment culture. It is also important to note that high level of cell debris could
be observed in the low attachment culture two days after inoculation until the end of the
study. In contrast, the normal attachment cultures and suspension culture were clean, and
cell debris was not observed in these cultures.
The results of cell counts for the cells placed in low attachment well-plates
exhibited an average cell-fold expansion of 2.7 after 14 days, which was significantly
lower than the cells-fold expansion of 15.8 in normal-attachment culture (P-value <
0.0001) and 17.2 in suspension culture (P-value < 0.0001). Moreover, an average
viability of 76% was determined for the cells in low attachment culture, whereas the
viability of the cells in normal attachment and suspension cultures was above 93%. These
results indicated that absence of cell-surface attachment might have negatively affected
on human NPC growth in the low-attachment culture.
To further investigate the role of cell-surface attachment on hNPC aggregate
formation and observe the cell behavior in the absence of cell-surface attachment, it was
decided to take hNPC aggregates from suspension culture and plate in low attachment
culture. Figure S3 illustrates photomicrographs of the hNPC aggregates taken from
suspension culture on day 5 and added to the low attachment culture. A time-lapse video
was prepared from this study (supplemental online video 2). Upon inoculating the cell
aggregates into low-attachment culture, the majority of aggregates (with an average size
of about 200 µm) clumped together and formed a very big aggregate with a size of over 1
mm after 14 h in culture. A smaller aggregate can also be found besides the larger
aggregate. Approximately 34 h postinoculation, single cells and / or cell debris started to
shed from the periphery of the aggregates, and the amount of debris increased over time.
Cell debris cannot be observed around the large aggregates in Figure S3 because the size
of aggregate was very big and the aggregate was not in the same focal plan with smaller
aggregate. However, when both wells of the low-attachment culture were observed under
the Zeiss microscope at the end of the study, large amount of debris were observed
around all the aggregates formed in the low-attachment culture in both wells (Figure S4).
Page 8 of 17
Cell debris surrounding the aggregates of cells could be an indication of extensive cell
death in this culture.
In contrast to the low attachment culture, hNPCs continued growing and forming
new aggregates in suspension culture. In the normal-attachment stationary culture, the
aggregates grew in size but did not agglomerate to one another. Figure S5 demonstrates
hNPC aggregates grown in suspension culture from the beginning of the time-lapse study
(day 5) until the end of the time-lapse study (day 9) [Fig. S5 (A-C)], and the aggregates
grown in standard normal-attachment culture over that same period of time [Fig. S5 (DF)]. The size of human NPC aggregates increased over time in both suspension and
standard stationary cultures without observing formation of very large aggregates or
production of significant amount of cell debris in the culture medium. Large agglomerate
of cell aggregates could be observed in the low-attachment culture (control lowattachment culture), which was placed in the humidified incubator next to the normal
attachment culture and suspension culture [Fig. S5 (G-I)]. The morphology of the
aggregates in the control low-attachment culture was comparable to that observed in the
time-lapse video.
Human NPC expansion in suspension culture, low-attachment stationary culture
and normal-attachment stationary culture were investigated at the end of the time-lapse
study (Figure S6). Cell-fold expansion was calculated over a course of 4 days as ratio of
the viable cell density at the end of the time-lapse study to the viable cell density at the
beginning of the study. These data showed that the cells in the suspension bioreactor and
normal-attachment stationary culture reached significantly higher cell-fold expansion of
approximately 2.0 after 4 days in each respective culture. In contrast, the cells in the low
attachment cultures exhibited significantly lower cell-fold expansion of about 0.6 over
that same period (P-value < 0.001). It is important to note that the cells in suspension
culture exhibited an overall cell-fold expansion of 8.8 after 9 days based on the
inoculation cell density of approximately 100,000 cells/mL on day 0 of the suspension
culture. The expansion achieved in suspension culture was concordance with the results
of hNPC expansion in our previous studies 1. The viability of the cells in the normal
attachment culture (approximately 95%) and suspension culture (96%) were higher than
the viability of the cells in the low attachment culture (about 73%). Some of the cells in
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the low attachment culture were completely destroyed and released into the culture in the
form of cell debris (Figure S4). Therefore, the actual level of viability in the lowattachment culture might have been overestimated because trypan-blue exclusion method
only accounts for the intact cells.
2.2 Rationale behind using Surface markers:
Table S1 summarizes the results of our FACS analysis, roles or functions of the cell
surface markers used over the course of these studies, and some of the literature studies
highlighting these roles and functions.
CD24 and CD133 (Panel I)
CD24 and CD133 are two surface markers that have been used to detect neural
stem/progenitor cells isolated from the mammalian CNS
2, 3
. CD24 is a surface protein
that is present on some of the ependymal cells, and on the neuroblasts in the SEZ of the
mammalian brain 2. Depend on the region of isolation, neural precursor cells positively
stained for CD133 may also be CD24-IR
2, 3
. CD133 is a surface marker protein
selectively expressed by neurosphere initiating cells, and has been shown to be expressed
by human NPCs derived from the forebrain 4. CD133 has been also suggested as a
potential surface marker for neural stem cells
2, 3
. Our FACS analysis showed that
approximately 90% of the telencephalon derived hNPCs were positively expressing
CD24 and CD133 surface markers (see Table S1).
CD29, CD44, CD90, and CD166 (Panel II)
CD29 or integrin β1, the protein encoded by the ITGB1 gene, is a member of
heterodimeric integral membrane cell adhesion molecules, integrins, and a fibronectin
receptor involved in a variety of biological functions including cell-matrix adhesion, cell
adhesion, protein binding, protein heterodimerisation, cell signaling activities for
regulation of progenitor cell proliferation, survival, clonogenic growth, and maintenance
during in vitro culture
5, 6
. CD44 is a cell adhesion molecule and a receptor for collagen,
osteopontin, hyaluronic acid (a major component of extracellular matrices), and matrix
metalloproteinases. CD44 is involved in diverse functions including organization and
Page 10 of 17
metabolism of extracellular matrix, engaging the cytoskeleton and co-ordination of the
signaling events to enable the cell response to changes in the environment
7-9
. CD90 or
Thy-1 is a glycosylphosphatidylinositol-anchored glycoprotein expressed on human
fibroblasts, neurons, blood stem cells, and endothelial cells, as well as murine T cells.
CD90 is an important regulator of cell-cell and cell-matrix interactions involved in a
widespread biological functions including inhibition of neurite outgrowth, apoptotic
signaling, leukocyte and melanoma cell adhesion and migration, tumor suppression, and
fibroblast proliferation and migration
10
. CD166 or ALCAM (activated leukocyte cell
adhesion molecule) belongs to Ig superfamily and is involved in axon guidance,
hematopoiesis, immune response and tumor metastasis
11
. Our FACS analysis showed
that the majority of hTNPCs were positively stained for a number of cell surface markers
involved in cell-ECM and cell-cell interactions including CD29 (96%), CD90 (57%), and
CD44 (56%). About 19% of the cells were CD166-positive.
CD13, CD14, CD19, CD34, CD45 (Panel III)
CD13, CD14, CD19, CD34, and CD45 are cell surface markers that have been used to
check possible contamination of the CNS derived NPCs with non-CNS derived cells
2, 3
.
CD13 is a myeloid differentiation molecule expressed on committed myeloid progenitors,
granulocytes, monocytes, and leukemic cells of myeloid origin. It is also expressed on
nonhemopoietic cells including fibroblasts, renal proximal tubule, and small intestine
brush-border membrane
12
. CD14 is abundantly expressed on the surface of mature
monocytes and in trace amounts on granulocytes 13. Neutrophil Marker CD19 is generally
considered a positive B-cell response regulator that governs intrinsic and stimulantdependent signaling thresholds in B cells. CD19 is a member of the Ig superfamily
expressed only on B cells and follicular dendritic cells 14. CD34 is a common marker for
endothelial cells
3, 15
, and CD45 is a common marker for blood cells (i.e. Leukocyte
common antigen) 3, 15. In agreement with the results of literature
3, 15
, our FACS analysis
showed that no CD13-, CD14-, CD19-, CD34-, and CD45-IR cells were observed among
the population of human NPCs, suggesting unlikelihood of contamination with nonneural cells (see Table S1).
Page 11 of 17
Table S1 Summary of the cell surface markers used to analyze hNPCs. These markers can be
classified into three groups: (I) neural precursor cells surface markers (shaded in grey), (II) cell
adhesion molecules involved in a variety of biological functions including cell-cell and cellECM interactions (shaded in yellow), and (III) non-CNS derived cells surface markers (white).
Surface
Marker
% of Immunoreactive
hNPCs
CD24
95.98
CD133
86.48
CD29
96.78
CD44
56.20
CD90
55.67
CD166
19.25
CD13
0
CD14
0
CD19
0
CD34
CD45
0
0
Role / Function
A surface protein present on ependymal
cells and neuroblasts in the SEZ of the
brain
A surface protein selectively expressed by
neurosphere initiating cells and the
forebrain-derived hNPCs
A cell adhesion molecule and a
fibronectin receptor involved in multi
biological functions
A cell adhesion molecule and a receptor
for collagen, osteopontin, hyaluronic acid,
and matrix metalloproteinases.
A glycosylphosphatidylinositol-anchored
glycoprotein and an important regulator
of cell-cell and cell-matrix interactions
and widespread biological functions
ALCAM (activated leukocyte cell
adhesion molecule) or CD166 belongs to
Ig superfamily and is involved in axon
guidance, hematopoiesis, immune
response and tumor metastasis
A myeloid differentiation molecule
expressed on committed hematopoietic
progenitor cells
Macrophage, Monocyte,
Neutrophil Marker
A member of the Ig superfamily
expressed only on B cells and follicular
dendritic cell
A marker for endothelial cells
A marker for blood cells
Page 12 of 17
Reference
2
2-4
5, 6
7-9
10
11
12
13
14
3, 15
3, 15
Figure S1 Photographs of the Cell Culture and Microscopy Incubator XL-3 system. (A)
shows experimental set-up for the Cell Culture and Microscopy Incubator XL-3 system:
(1) computer system connected to a Zeiss Axiovert 200M microscope, (2) the Zeiss
microscope, (3) Incubator XL-3, (4) digital temperature controller, (5) heating unit, and
(6) CO2 controller. (B) shows a closer view of the Incubator XL-3: (1) a 6-well plate
containing the cells, (2) incubator cover, (3) a bottle containing distilled water to provide
humidity for the cells under the cover, and (4) connection tubes provided to pass CO2 gas
through the bottle of water and supply to the culture.
Figure S2 Expansion of the telencephalon human neural precursor cell (hNPC)
aggregates formed in low-attachment surface culture and transferred into standard normal
attachment and suspension cultures (P10). Shown are photomicrographs of (A) hNPC
single cells immediately following inoculation into low-attachment well plates, (B) hNPC
aggregates formed 8 h post-inoculation, (C) hNPC aggregate formed 4 days postinoculation, and (D) hNPC aggregates formed 14 days post-inoculation. Also shown are
(E) hNPC aggregates taken at 8 h from the low-attachment culture, (F) hNPC aggregates
one hour (total period of 9 h) after inoculation into the normal attachment culture, (G)
hNPC aggregates 4 days after inoculation into the normal attachment culture, and (H)
hNPC aggregates 14 days after inoculation into the normal attachment culture. Also
shown are (I) hNPC aggregates taken at 8 h from the low-attachment culture, (J) one hour
(total period of 9 h) after inoculation into suspension culture, (K) hNPC aggregates 4
days after inoculation into suspension culture, and (L) hNPC aggregates 14 days after
inoculation into the suspension culture. Scale bar represents 250 µ. Abbreviations: P10,
passage level 10.
Figure S3 Time-lapse analysis of the telencephalon human neural precursor cell (hNPC)
aggregate taken from suspension culture and inoculated into low-attachment culture (P7).
The telencephalon hNPCs were taken on day 5 from suspension culture and inoculated at
1.5×105 cells/mL into two wells of a low-attachment 6-well Plate. Photomicrographs
were taken every 10 min from one of the wells placed on the Zeiss microscope stage
underneath the incubator cover. The experiment was carried out for a period of 100 h
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(about 4 days) in culture. Shown are selected photomicrographs of the time-lapse video at
0, 0.33, 0.67, 1.0, 2.0, 4.0, 8.0, 16.0, 20.0, 34.0, 42.0, and 47.0 h post-inoculation into the
low attachment culture. Scale bar represents 100 μm. Abbreviations: P7, passage level 7.
Figure S4 Cell debris surrounding the aggregates of the telencephalon human neural
precursor cells (hNPCs) in the low-attachment well-plate (P7) indicating extensive cell
death (end of time-lapse study in low-attachment culture). Photomicrographs show (A)
low-power view (5× objective) of hNPC aggregates on day 4 of the study in the well
under the surveillance, (B) high-power view (20× objective) of the smaller aggregate
shown by arrow in A, (C) low-power view (5× objective) of hNPC aggregates on day 4
of the study in the second well of the low attachment well-plate, and (D) high-power
view (10× objective) of the smaller aggregates shown by arrow in C. Photomicrographs
were taken by the Zeiss microscope. Scale bars: (A) and (C), 400 μm; (B), 100 μm; (D),
200 μm. Abbreviations: P7, passage level 7.
Figure S5 Expansion of the telencephalon human neural precursor cells (hNPCs) in
suspension bioreactors (P7) and transferred into the low attachment and normal
attachment cultures (control cultures parallel to the time-lapse experiment). Shown are
photomicrographs of (A) hNPC aggregates 5 days after inoculation into suspension
culture, (B) hNPC aggregates 7 days post-inoculation, (C) hNPC aggregates 9 days postinoculation. Also shown are (D) hNPC aggregates taken on day 5 from suspension
culture and placed in the normal attachment culture, (E) hNPC aggregates 2 days after
placing in the normal attachment culture, and (F) hNPC aggregates 4 days after placing in
the normal attachment culture. Also shown are (G) hNPC aggregates taken on day 5 from
suspension culture and placed in the low attachment culture, (H) hNPC aggregates 2 days
after placing in the low attachment culture, and (I) hNPC aggregates 4 days after placing
in the low attachment culture. Scale bar represents 250 µ. Abbreviations: P7, passage
level 7.
Page 14 of 17
Figure S6 Cell-fold expansion and viability of the telencephalon human neural precursor
cells (hNPCs) grown in the low-attachment, normal attachment, and suspension cultures
(P7). Human NPCs were taken on day 5 from suspension culture and inoculated at
1.5×105 cells/mL into two wells of a low-attachment 6-well plate and two wells of a
normal-attachment 6-well plate for a period of 4 days. The cell aggregates were then
harvested, enzymatically dissociated into single cells, and underwent cell counts. Shown
are (A) the cell-fold expansion and (B) viability (%) of the cells grown in either culture
condition 4 days postinoculation. Abbreviations: P7, passage level 7.
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