- EB size affects the differentiation of hiPS cells, with larger EBs encouraging expression of the neuroectodermal marker PAX6 early on but medium EBs (500-3000 cells) best encouraging later expression of PAX6 and MiTF, a marker of ocular neuroectodermal lineage cells.
- At early timepoints, larger EBs expressed more PAX6, but at later timepoints medium EBs expressed more PAX6 and MiTF, possibly due to cell-cell interactions in larger EBs affecting PAX6 expression over time.
- The study shows EB size can be used to control differentiation of hiPS cells, with medium EBs most effectively encouraging differentiation into ocular neuroectodermal lineage cells
Endothelial Cell Mediated Delay of Blood Brain Barrier Recovery Following Tra...Arthur Stem
TBI is the leading cause of death among young adults and children in the developed world, accounting for over 50,000 deaths per year. [12] TBI results in a sleuth of poor health outcomes, including hemorrhaging, seizures, neural edema, neural inflammation, and cognitive and emotional disabilities. All of these outcomes are a direct result of fundamental degradation of the BBB over a time course post TBI. [1] [12] The BBB is an integral structure that forms around the microvascular of the cerebral cavity. Endothelial cells form the basal membrane through which strictly controlled movement of molecules is observed between the extravascular and intravascular space across this basal membrane. This basal membrane is maintained by endothelial cells, having tight junctions between them to make up the pores through which transport of molecules can occur between the brain and microvasculature. These tight junctions are maintained through cross-talk between the endothelial cells and supporting neurons such as astrocytes and pericytes. [2] A multitude of proteins make up the tight junctions between the endothelial cells, including six main scaffolding structures Claudins 1, 3, and 5, ZO-1, Occludins, and Cadherins. [3] VEGF release following trauma induces endothelial cells to release matrix metalloproteinases (MMPs), in particular MMP9, which can catalyze the N-terminal amino acids that compose the tight junction protein ZO-1. [10] [11] MMP9 when in circulation is also known to activate tumor necrosis factor alpha (TNFɑ) which in turn upregulates transcription of MMP9, creating a positive feedback loop. [11] The management of MMP production is three fold, transcription, proenzyme activation, and substrate inhibition. [11] In our study, it is proenzyme activation via TP that is the focus and how that affects the overall transcription levels of the tight junction proteins within the endothelial cells and astrocytes.
Endothelial Cell Mediated Delay of Blood Brain Barrier Recovery Following Tra...Arthur Stem
TBI is the leading cause of death among young adults and children in the developed world, accounting for over 50,000 deaths per year. [12] TBI results in a sleuth of poor health outcomes, including hemorrhaging, seizures, neural edema, neural inflammation, and cognitive and emotional disabilities. All of these outcomes are a direct result of fundamental degradation of the BBB over a time course post TBI. [1] [12] The BBB is an integral structure that forms around the microvascular of the cerebral cavity. Endothelial cells form the basal membrane through which strictly controlled movement of molecules is observed between the extravascular and intravascular space across this basal membrane. This basal membrane is maintained by endothelial cells, having tight junctions between them to make up the pores through which transport of molecules can occur between the brain and microvasculature. These tight junctions are maintained through cross-talk between the endothelial cells and supporting neurons such as astrocytes and pericytes. [2] A multitude of proteins make up the tight junctions between the endothelial cells, including six main scaffolding structures Claudins 1, 3, and 5, ZO-1, Occludins, and Cadherins. [3] VEGF release following trauma induces endothelial cells to release matrix metalloproteinases (MMPs), in particular MMP9, which can catalyze the N-terminal amino acids that compose the tight junction protein ZO-1. [10] [11] MMP9 when in circulation is also known to activate tumor necrosis factor alpha (TNFɑ) which in turn upregulates transcription of MMP9, creating a positive feedback loop. [11] The management of MMP production is three fold, transcription, proenzyme activation, and substrate inhibition. [11] In our study, it is proenzyme activation via TP that is the focus and how that affects the overall transcription levels of the tight junction proteins within the endothelial cells and astrocytes.
Characterization of embryoid bodies formed with different protocols 使用不同培養方式形...Honey Cheng
That's part of my first year master researching in 2011, National Chung Hsing-University, Taiwan. Mice embryonic stem cells differentiating with embryoid bodies in a unattached formed, so I summarized a slides review. 這是2011年在中興就讀研究所第一年時所研究的方向. 胚胎幹細胞能夠在懸浮狀態形成類胚體與分化, 所以為此我整理了一份簡報, 介紹不同方式形成類胚體之特性.
Effect of stemregenin1 and sb431542 small molecules on ex vivo expansion of u...Liberty University (LU)
Cord blood Hematopoietic stem cells (HSCs) with several advantages including low chance of viral contamination and low rate of Graft versus host disease (GVHD) are appropriate candidate for vast medical applications such as transplantation. The main obstacle of cord blood HSCs is the low number cells. To improve ex-vivo expansion of umbilical cord HSCs we introduced a new culture system. Isolated HSCs were seeded in Three-dimensional(3D) on Polyethersulfone(PES) scaffolds and Twodimensional(2D) culture conditions and treated with SB431542 and Stemregenin1(SR1) small molecules. On the fifth and tenth days the expanded cells in different groups were investigated for expression of specific markers by flow cytometry, expression of some stemness genes by qRT-PCR and colony formation by methocult medium. SR1 molecule significantly increased expansion of CD34+ cells while SB431542 induced more CD34+/38+ cells. Also SB431542 treated cells showed higher colony formation capacity. SR1 increased the expression of c-Myc, HOXB4 and SALL4 while SB431542 seemed to inhibit HOXB4 expression and increase SALL4.Together all, this study introduced a new ex vivo culture setting for further medical application of HSCs. Our data showed simultaneous use of these two small molecules can provide appropriate outcome for HSCs transplantation includes both of engraftment and repopulation.
Human amniotic fluid cells (hAFCs) may differentiate into multiple cell lineages and thus have a great potential to become a donor cell source for regenerative medicine. The ability of hAFCs to differentiate into germ cell and oocyte-like cells has been previously documented. Herein we report the potential use of hAFCs to help restore follicles in clinical condition involving premature ovarian failure.
Characterization of embryoid bodies formed with different protocols 使用不同培養方式形...Honey Cheng
That's part of my first year master researching in 2011, National Chung Hsing-University, Taiwan. Mice embryonic stem cells differentiating with embryoid bodies in a unattached formed, so I summarized a slides review. 這是2011年在中興就讀研究所第一年時所研究的方向. 胚胎幹細胞能夠在懸浮狀態形成類胚體與分化, 所以為此我整理了一份簡報, 介紹不同方式形成類胚體之特性.
Effect of stemregenin1 and sb431542 small molecules on ex vivo expansion of u...Liberty University (LU)
Cord blood Hematopoietic stem cells (HSCs) with several advantages including low chance of viral contamination and low rate of Graft versus host disease (GVHD) are appropriate candidate for vast medical applications such as transplantation. The main obstacle of cord blood HSCs is the low number cells. To improve ex-vivo expansion of umbilical cord HSCs we introduced a new culture system. Isolated HSCs were seeded in Three-dimensional(3D) on Polyethersulfone(PES) scaffolds and Twodimensional(2D) culture conditions and treated with SB431542 and Stemregenin1(SR1) small molecules. On the fifth and tenth days the expanded cells in different groups were investigated for expression of specific markers by flow cytometry, expression of some stemness genes by qRT-PCR and colony formation by methocult medium. SR1 molecule significantly increased expansion of CD34+ cells while SB431542 induced more CD34+/38+ cells. Also SB431542 treated cells showed higher colony formation capacity. SR1 increased the expression of c-Myc, HOXB4 and SALL4 while SB431542 seemed to inhibit HOXB4 expression and increase SALL4.Together all, this study introduced a new ex vivo culture setting for further medical application of HSCs. Our data showed simultaneous use of these two small molecules can provide appropriate outcome for HSCs transplantation includes both of engraftment and repopulation.
Human amniotic fluid cells (hAFCs) may differentiate into multiple cell lineages and thus have a great potential to become a donor cell source for regenerative medicine. The ability of hAFCs to differentiate into germ cell and oocyte-like cells has been previously documented. Herein we report the potential use of hAFCs to help restore follicles in clinical condition involving premature ovarian failure.
Determination and comparison rate of expression markers of osteoblast derived...IJERD Editor
Nowadays high accident rates, fractures leading to permanent bone disorders and the impossibility of bone transplant have made scientists to look for new methods of repairing injured bones. Considering the application of stem cells in bone tissue engineering, there exists the necessity to investigate various culture methods and suitable fields and scaffolds. Thus, we decided to induce adipose-derived stem cells into osteoblast cells in two systems of pellet culture and monolayer and compare osteogenic markers. Methods: Stem cells have been separated via mechanical and enzymatic methods and cultured in monolayer and pellet culture models with osteogenic medium. Then, RNA was separated from differentiated cells, complementary DNA (cDNA) was synthesized and amplified. Polymerase chain reaction (PCR) product was transferred to electrophoresis gel. The intensity of the bands was measured by Image-J software and analyzed by SPSS.
Induced Pluripotent Stem-Like Cells Derived from Ban, a Vietnamese Native Pig...AI Publications
Induced pluripotent stem cells (iPSc) is a promising technology for applying in bio-medicine and biodiversity conservation. In the present study, we isolate and culture fibroblasts from Ban – a Vietnamese native pig breed and transfer episomal plasmid containing genes Oct3/4, Sox2, Klf4, l-Myc, LIN28 and EBNA1 in order to reprogram cells. We isolated, cultured and cryopreserved successfully 9 primary fibroblast lines from Ban (culture percentage is 90.0%). Plasmids was successfully transferred into Ban fibroblasts with high efficiency. Changes in morphology of fibroblasts into pluripotent stem-like cells showed that they had been reprogrammed under the effect of transferred genes. The pluripotency signal was further proved by in vitro differentiation by formation of embryoid body in all 3 transfected cell lines. The results showed that pluripotent stem-like cells has successfully derived in Ban pigs.
PowerPoint giving a summary on research in stem cells (brief historical overview), and the explanatory component of the papers which changed the game of stem cell research Yamanka's Nuclear Reprogramming.
1. Combat Casualty Care
P R O T E C T
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INSTITU
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OF SURGICAL RE
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EARCH
Effect of Embryoid Body Size on Differentiation of iPS Cells into
Neuroectodermal-Lineage Ocular Cells
O.A. Creasey1, A. Muñiz2, M.L. Plamper2, H-C.H. Wang2
1Summer Intern, Pittsburgh Tissue Engineering Initiative, 2United States Army Institute of Surgical Research
Department of Ocular Trauma, Fort Sam Houston, TX 78234-6315
Results (Continued)Abstract
Cell death in ocular tissues such as the retina and retinal pigment
epithelium (RPE) due to trauma, disease, and aging is a major cause of
blindness. Potential therapies to halt or reverse vision loss may require
replacement of damaged cells with stem cell derived tissues. Current
differentiation methods for human induced pluripotent stem cells (hiPS
cells) and human embryonic stem cells have allowed the derivation of
neuroectodermal cells such as retinal neurons and RPE cells [1, 2, 3], but
only at low efficiencies. Higher efficiency differentiation methods are
essential if these cell-based therapies are to be applied to human patients.
Past research has shown that embryoid body (EB) size can affect the
efficiency with which stem cells differentiate into germ layers [4]. This
study investigates the effect of EB size on efficiency of differentiation of
iPS cells into neuroectodermal cells.
In preparation for embryoid body formation, hiPS (IMR-90-1) cells were
cultured on matrigel. Different sized EBs were formed using AggreWell
plates according to manufacturer’s instructions. The EBs were
subsequently plated on matrigel in six-well plates and fed with
differentiation medium. At Day 6, Day 17, and Day 29 RNA was collected
from the EBs and RT-PCR was performed to determine expression of the
PAX6 gene, a marker specific to neuroectodermal cells. In addition, RT-
PCR was run on the Day 17 and Day 29 RNA to determine expression of
the MiTF gene, a marker specific to development of ocular cells of
neuroectodermal lineage [3, 5].
PAX6 was expressed in cells from all four EB sizes at Day 6, Day 17, and
Day 29. At Day 6, PAX6 expression increased with EB size. At both Day
17 and Day 29, EBs containing 500 and 3000 cells expressed more PAX6
than EBs containing 200 or 15000 cells. MiTF was also expressed at Day
17 and Day 29 in all four EB sizes. At Day 17 expression was higher in
the 200, 500, and 3000 EBs than in the 15000 cell EBs. At Day 29
expression was highest in the 500 cell EBs.
These results show that gene expression differs between the cells of
different EB sizes. They also show that although larger EBs have the
highest expression of PAX6 at early timepoints and therefore could be
optimal for differentiation into neuroectodermal cells, medium-sized EBs
(500 and 3000 cells) are best for differentiation of neuroectodermal-
lineage ocular cells as indicated by co-expression of PAX6 and MiTF at
later timepoints. We speculate that the loss of PAX6 expression in larger
EBs at later timepoints is due to cell-cell interactions between increasingly
confluent cells.
Objectives
The objective of this study is to determine the effect of embryoid body
size on differentiation of neuroectodermal cells from human iPS cells.
Methods
Culture and Maintenance of iPS cell cultures: Human iPS (IMR-90-1
cells) from WiCell were cultured on matrigel (BD Biosciences) coated six
well plates and maintained in mTeSR1 media (Stem Cell Technologies).
The medium was changed daily until cells were ready for passage (approx
4-5 days).
Conclusions
Acknowledgements
We would like to acknowledge Joan Schanck and LaShon Jackson of PTEI for
coordinating support and thank PTEI for making this research opportunity available.
We also acknowledge Dr. David Baer and Dr. Robert Christy of the United States
Army Institute of Surgical Research and all of the other USAISR and BAMC staff
for their help in coordinating this opportunity.
• EB size affects the differentiation pathway of hiPS cells.
• Larger EB sizes encourage expression of the neuroectodermal marker
PAX6 at early timepoints.
• Medium EB sizes, especially the 500 cell EBs, encourage expression of
PAX6 and MiTF at later timepoints.
• Cell-cell interactions between increasingly confluent cells could affect
expression of PAX6 at later timepoints.
• In the future EB size will be used to control hiPS cell differentiation.
References
[1] Buchholz, D. E., S. T. Hikita, et al. (2009). "Derivation of functional retinal
pigmented epithelium from induced pluripotent stem cells." Stem Cells 27(10):
2427-2434.
[2] Idelson, M., R. Alper, et al. (2009). "Directed differentiation of human
embryonic stem cells into functional retinal pigment epithelium cells." Cell Stem
Cell 5(4): 396-408.
[3] Meyer, J. S., R. L. Shearer, et al. (2009). "Modeling early retinal development
with human embryonic and induced pluripotent stem cells." Proc Natl Acad Sci U S
A 106(39): 16698-16703.
[4] Bauwens, C. L., R. Peerani, et al. (2008). "Control of human embryonic stem
cell colony and aggregate size heterogeneity influences differentiation trajectories."
Stem Cells 26(9): 2300-2310.
[5] Vaajasaari, H., T. Ilmarinen, et al. (2011). "Toward the defined and xeno-free
differentiation of functional human pluripotent stem cell-derived retinal pigment
epithelial cells." Mol Vis 17: 558-575.
Disclaimer: The opinions or assertions contained herein are the private views of the
author and are not to be construed as official or as reflecting the views of the
Department of the Army or the Department of Defense.
Methods (Continued)
Results
Figure 1: iPS cells in Culture. The cells have a small & round
phenotype, a characteristic of pluripotent stem cells. (magnification
100x) IPS cells were cultured on matrigel in mTeSR1 at 37˚C and 5%
CO2.
Figure 2: EB formation in AggreWell plates. Each microwell
contains A. 200 cells, B. 500 Cells, C. 3000 Cells. Cells were
incubated 24 hours at 37º C and 5% CO2 for the formation of EBs.
(magnification 100x)
Figure 3: Different sized EB-derived colonies on Day 2 of
differentiation. A. 200 cells, B. 500 Cells, C. 3000 Cells, D. 15,000
Cells. Cells were incubated for two days at 37º C and 5% CO2 in
differentiation medium after the EBs were plated on matrigel-coated
plates. (magnification 20x)
Figure 4: Expression of PAX6 and MiTF at Day 17 of
differentiation. All four EB sizes showed expression of PAX6. The
500 and 3000 cell EBs show higher PAX6 expression than that of the
200 and 15000 cell EBs..All four EB sizes showed expression of
MiTF with higher levels in the 200, 500, and 3000 cell EBs. The
15000 EBs showed the lowest levels of MiTF. The error bars show
standard error.
A B
C D
Results (Continued)
BA
C -1.20
-0.88
-0.87
-1.19
0.00
-1.01
-0.97 -0.94
-1.45
0.00
-1.6
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
200 500 3000 15000
Log10(RelativeQuantitation)
EB Size
Expression of PAX6 and MiTF at Day 17
PAX6
MiTF
-0.49
-0.20
0.05
0.29
0.00
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
200 500 3000 15000
Log10(RelativeQuantitation)
EB Size
Expression of PAX6 at Day 6
PAX6
Figure 4: Expression of PAX6 at Day 6 of differentiation. All four
EB sizes showed expression of PAX6 and expression increased with
increasing EB size. The 200 and 500 cell EBs had a lower expression
of PAX6 than ARPE-19 cells while the 15000 cell EBs had a higher
expression of PAX6 than ARPE-19 cells. Expression of PAX6 in the
3000 EBs was roughly the same as in ARPE-19 cells. The error bars
show standard error.
Formation of Embryoid bodies: Embryoid bodies (EBs) consisting of
200, 500, 3000 and 15000 cells were prepared using AggreWell 400 &
800 plates. Briefly, an iPS single cell suspension was prepared in
AggreWell Medium and seeded in AggreWell plates at a density that
would yield the desired number of cells per microwell. The AggreWell
plates were then centrifuged at 100 x g for 3 minutes to capture the cells in
the microwells . The plates were then incubated at 37 ºC and 5% CO2 for
24 hours to allow formation of EBs. The EBs were then collected and
seeded in matrigel coated six well plates in AggreWell Medium for 24
hours. To initiate the differentiation protocol the AggreWell Medium was
then replaced with differentiation medium consisting of 10% Knockout
serum, 0.1mM β-Mercaptoethanol, 0.1 mM nonessential amino acids, 2.0
mM Glutamine and 10 µg/ml gentamicyn in DMEM/F12.
RNA Extraction and RT-PCR: RNA was extracted from the cell cultures
using an RNeasy Plus Mini Kit (Qiagen). Briefly, cells were either lysed
directly in the six-well plates using Buffer RLT Plus (Qiagen) or removed
from the plate with accutase and re-suspended in Buffer RLT Plus after
washing. The lysate was then homogenized using QIAshredder spin
columns (Qiagen) and the RNA was extracted using RNeasy Plus Mini Kit
protocol. The RNA was translated to cDNA using a High Capacity
RNA-to-cDNA Kit (Applied Biosystems) and then PCR was run using
Taqman Gene Expression Assays for PAX6 and MiTF on a 7300 Real
Time PCR System (Applied Biosystems). 18S served as the internal
control. The data was then analyzed using the ΔΔ CT method. The RQ of
PAX6 and MiTF in ARPE19 cells are made to equal to 1 and are used as
the baseline for analysis. The data is plotted as the log10 of the RQ.
-1.12
-0.51
-0.73
-1.12
0.00
-1.01
-0.66
-0.90
-0.82
0.00
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
200 500 3000 15000
Log10(RelativeQuantitation)
EB Size
Expression of PAX6 and MiTF at Day 29
PAX6
MiTF
Figure 5: Expression of PAX6 and MiTF at Day 29 of
differentiation. All four EB sizes showed expression of PAX6 and
MiTF. The highest PAX6 expression is seen in the 500 cell EBs with
somewhat lower expression in the 3000 cell EBs and even lower
expression in the 200 and 15000 cell EBs. The highest expression of
MiTF was also seen in the 500 cell EBs with lower expression in the
3000 and 15000 cell EBs and the lowest expression in the 200 cell
EBs. The error bars show standard error.