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.
Deciphering signaling mechanisms of cartilage tissue engineered alginate scaf...Antonion Korcari
Combination of Systems Biology and Tissue Engineering approaches by creating a mechanism for 3D cartilage phenotype evaluation. More specifically, high-throughput measurements have been used to interrogate intracellular and extracellular activity of 3D cultured chondrocytes, combined with phenotypic measurements of cartilage growth (s-GAG) to correlate the mechanism of cartilage growth, either untreated or treated with different stimuli.
Microgravity is the condition in which people or objects appear to be weightless (In space). Astronauts and cosmonauts returning from long-term space missions exhibited various health problems, among them changes of the immune system, bone loss, muscle atrophy, ocular problems, and cardiovascular changes. Space biologists investigated various cell types in space to find the molecular mechanisms responsible for the observed immune disorders. Experimental cell research studying three-dimensional (3D) tissues in space and on Earth using new techniques to simulate microgravity is currently a hot topic in Gravitational Biology and Biomedicine.
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.
Deciphering signaling mechanisms of cartilage tissue engineered alginate scaf...Antonion Korcari
Combination of Systems Biology and Tissue Engineering approaches by creating a mechanism for 3D cartilage phenotype evaluation. More specifically, high-throughput measurements have been used to interrogate intracellular and extracellular activity of 3D cultured chondrocytes, combined with phenotypic measurements of cartilage growth (s-GAG) to correlate the mechanism of cartilage growth, either untreated or treated with different stimuli.
Microgravity is the condition in which people or objects appear to be weightless (In space). Astronauts and cosmonauts returning from long-term space missions exhibited various health problems, among them changes of the immune system, bone loss, muscle atrophy, ocular problems, and cardiovascular changes. Space biologists investigated various cell types in space to find the molecular mechanisms responsible for the observed immune disorders. Experimental cell research studying three-dimensional (3D) tissues in space and on Earth using new techniques to simulate microgravity is currently a hot topic in Gravitational Biology and Biomedicine.
An idea was considered as to producing an entire organ in vivo by bypassing many of the steps like cell isolation and expansion, culturing in bioreactors, scaffolds and growth factor delivery ect. involved in traditional tissue engineering. This concept was called the in vivo bioreactor (IVB).
Biomaterials were defined as “any substance, other than a drug, or a combination of substances, synthetic or natural in origin, which can be used for any period of time, as a whole or as a part of a system, which treats, augments or replaces any tissue, organ or function of the body”
Engineering bone tissue using human Embryonic Stem CellsBalaganesh Kuruba
Bone defects lead by traumatic injuries, congenital malformations and other surgical rescissions rises the immediate need for a more evolved and safer approaches in tissue repair at alarming rates for the downgrading issues with existing strategies which needs to be addressed. Currently practiced treatment methods addressing the issue with bone defects are invasive, traumatic and are not cost effective. Yet, issues of immune rejection either immediately or in the later stages have been reported claiming its ineffectiveness in some selective case studies.
Previous work by researchers carried out the "Biomimetic" approach to provide the cells with the microenvironment and in situ conditions for the cells seeded into the 3D Osteogenic scaffolds enriched with growth supplments. Here, we address the issue of non-availability of therapeutic cells - a major problem with current translational medicine by proposing the use of Human Embryonic Stem Cells in generating strong and structurally rigid bone tissue. Inducing the production of Mesenchymal Progenitor cells from Human Embryonic Stem cells in Serum supplemented expansion medium and elimination of bone Fibroblast growth factor produced high quality MPCs which were induced in osteogenic medium to result in bone differentiating cells. Culturing these MPCs produced from three different protocols into 3D Scaffold and 3D-Endoret Osteogenic Scaffold produced tissue constructs which are analysed both biochemically and Histologically to check for the Bone tissue differentiation parameters such as Bone sialoprotein deposition, Osteopontin accumulation and Collagen deposition. Matrix mineralization in these constructs were studied by uCT imaging and safety studies were conducted by studying Orthotopic implantation models in SCID mouse. And the results are expected to be optimistically affirmative which shall lay a new foundation and pioneer a whole new approach in the field of Tissue Engineering.
A presentation on Tissue Engineering made by Deepak Rajput. It was presented as a seminar requirement at the University of Tennessee Space Institute in Spring 2009.
Tissue engineering and regenerative medicine Suman Nandy
Tissue engineering is the use of a combination of cells, engineering and materials methods, and suitable biochemical and physicochemical factors to improve or replace biological tissues. Tissue engineering involves the use of a scaffold for the formation of new viable tissue for a medical purpose.
Autologous Mesenchymal Stem Cells in OrthopaedicsVladimir Bobic
Nuffield Health, The Grosvenor Hospital Chester, UK
27 June 2013. GP and Physiotherapy Seminar: Autologous Stem Cell Therapies in Orthopaedics. Moderator and Presenter: Vladimir Bobic, Chester Knee Clinic
In Vitro Analog of the Primitive Streak (ANIMATED)Nikolay Turovets
PLEASE DOWNLOAD TO SEE ANIMATION.
In vitro analog of the primitive streak: efficient derivation of highly enriched populations of hepatocytes from various types of human pluripotent stem cells.
May, 2011
An idea was considered as to producing an entire organ in vivo by bypassing many of the steps like cell isolation and expansion, culturing in bioreactors, scaffolds and growth factor delivery ect. involved in traditional tissue engineering. This concept was called the in vivo bioreactor (IVB).
Biomaterials were defined as “any substance, other than a drug, or a combination of substances, synthetic or natural in origin, which can be used for any period of time, as a whole or as a part of a system, which treats, augments or replaces any tissue, organ or function of the body”
Engineering bone tissue using human Embryonic Stem CellsBalaganesh Kuruba
Bone defects lead by traumatic injuries, congenital malformations and other surgical rescissions rises the immediate need for a more evolved and safer approaches in tissue repair at alarming rates for the downgrading issues with existing strategies which needs to be addressed. Currently practiced treatment methods addressing the issue with bone defects are invasive, traumatic and are not cost effective. Yet, issues of immune rejection either immediately or in the later stages have been reported claiming its ineffectiveness in some selective case studies.
Previous work by researchers carried out the "Biomimetic" approach to provide the cells with the microenvironment and in situ conditions for the cells seeded into the 3D Osteogenic scaffolds enriched with growth supplments. Here, we address the issue of non-availability of therapeutic cells - a major problem with current translational medicine by proposing the use of Human Embryonic Stem Cells in generating strong and structurally rigid bone tissue. Inducing the production of Mesenchymal Progenitor cells from Human Embryonic Stem cells in Serum supplemented expansion medium and elimination of bone Fibroblast growth factor produced high quality MPCs which were induced in osteogenic medium to result in bone differentiating cells. Culturing these MPCs produced from three different protocols into 3D Scaffold and 3D-Endoret Osteogenic Scaffold produced tissue constructs which are analysed both biochemically and Histologically to check for the Bone tissue differentiation parameters such as Bone sialoprotein deposition, Osteopontin accumulation and Collagen deposition. Matrix mineralization in these constructs were studied by uCT imaging and safety studies were conducted by studying Orthotopic implantation models in SCID mouse. And the results are expected to be optimistically affirmative which shall lay a new foundation and pioneer a whole new approach in the field of Tissue Engineering.
A presentation on Tissue Engineering made by Deepak Rajput. It was presented as a seminar requirement at the University of Tennessee Space Institute in Spring 2009.
Tissue engineering and regenerative medicine Suman Nandy
Tissue engineering is the use of a combination of cells, engineering and materials methods, and suitable biochemical and physicochemical factors to improve or replace biological tissues. Tissue engineering involves the use of a scaffold for the formation of new viable tissue for a medical purpose.
Autologous Mesenchymal Stem Cells in OrthopaedicsVladimir Bobic
Nuffield Health, The Grosvenor Hospital Chester, UK
27 June 2013. GP and Physiotherapy Seminar: Autologous Stem Cell Therapies in Orthopaedics. Moderator and Presenter: Vladimir Bobic, Chester Knee Clinic
In Vitro Analog of the Primitive Streak (ANIMATED)Nikolay Turovets
PLEASE DOWNLOAD TO SEE ANIMATION.
In vitro analog of the primitive streak: efficient derivation of highly enriched populations of hepatocytes from various types of human pluripotent stem cells.
May, 2011
Titulo: Green Build
Alunos:ANA PAULA BEZERRA,Carlos Henrique Studenroth,Cibele,Geisy Teixeira,Leda Maria Nolasco de Lima,
Cidade: Faria Lima
Disciplina: Aquisições
Turma: GEEP02
Data:11-09-2015
Hora:23:43
Comentarios:
Publico até ápos a correção
Titulo: BeautyNow Versão Final 27082015
Alunos:Benedito Borges,Fernando Ribeiro Rodrigues ,João Vitor Boschetti,Laura Antonia Sauer,Letícia Dal Bello,LÚCIO KLEBER LUDWIG,Mônica Furtado,Vanessa Lauermann,
Cidade: Porto Alegre
Disciplina: Aquisições
Turma: GP33
Data:27-08-2015
Hora:23:22
Comentarios:Boa Noite Professor Marco,
Encaminho novamente o Gerenciamento de Aquisições do nosso projeto BeautyNow para sua validação. Ontem havia encaminhado o arquivo com os títulos incorretos. Este último é a versão atualizada final.
Aguardo sua confirmação de recebimento.
Obrigada.
Publico até ápos a correção
Cuckoo Search Optimization of Blebs in Human Embryonic Stem CellsIJMERJOURNAL
ABSTRACT: The main aim of this project is to segment the bleb from human embryonic stem cells (hESC). The behavior of bleb can be used to distinguish apoptotic bleb from the healthy bleb. The health of the human embryonic stem cells can be determined using the portion of bleb formed on the surface of the stem cells. The complete bleb formation contains bleb extraction and retraction. This paper uses the active contour algorithm for the segmentation of bleb from human embryonic stem cells. The output of the segmentation, input video and area of bleb can be used as an input to the optimization process. The cuckoo search algorithm is utilized for optimization, which inspired from the brooding parasitism will enhance the segmentation result. The proposed method attains the quick and accurate analysis in the bleb extraction process
Exosomes are smallest extracellular vesicles of size 30 to 100 nm originated from late endosomes. These are released by broad array of cells including B‐ cells, cells, dendritic cells (DCs), T‐cells, epithelial cells,
platelets and many more.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Chlorella Vulgaris Alleviates Lead-induced Testicular Toxicity Better than Zi...Prof. Hesham N. Mustafa
Natural products are studied to combat reproductive alterations of Lead. The current work aim to disclose the efficacy of Chlorella vulgaris and Zingiber officinale to alleviate lead acetate induced toxicity. Sixty adult male Wistar rats were distributed into four groups. Group 1 was considered control, group 2 received 200 mg/l PbAc water, group 3 received 50 mg/kg/rat of C. vulgaris extract and 200 mg/l PbAc water, and group 4 received 100 mg/kg/rat of Z. officinale and 200 mg/l PbAc water for 90 days. Testis samples were subjected to ultrastructural examination. It is observed that PbAc caused degenerative alterations in the spermatogenic series in many tubules, with a loss of germ cells and vacuoles inside the cytoplasm and between the germ cells. Mitochondria exhibited ballooning, with lost cristae and widening of the interstitial tissue, destructed nuclear envelopes of primary spermatocytes, and distortion of the axonemes of the mid-pieces of the sperms. With the treatment with C. vulgaris or Z. officinale, there were noticeable improvements in those modifications. It has concluded that both C. vulgaris and Z. officinale represent convincing medicinal components that may be used to ameliorate testicular toxicity in those exposed to lead in daily life with superior potentials revealed by C. vulgaris due to its chelating action. in XXIV International Symposium on Morphological Sciences on 2nd- 6th September, 2015 Oral Presentation [O-02].at Prof. Dr. Cemil Bilsel Congress Hall, Faculty of Science, Istanbul University, Istanbul, Turkey.
4. 4Fig. 1. Blastocysts contain pluripotential stem cells.
Human - 6~7 day
Mouse - 3.5 day
(Terese et al., 2001)
Cleavage stage embryo Cultured
blastocyst
Isolated inner cell mass
Mouse fibroblast
feeder cells
Established ES cell cultures
Cells dissociated
and replated
Mouse fibroblast
feeder cells
5. 5
For most ES cell lines, this occurs via the
formation of three-dimensional aggregates
called embryoid bodies (EBs).
Ectoderm →nervous system, reproductive tract
etc.
Endoderm →digestive system, respiratory system
and most gland
Mesoderm →muscle, blood vessels, reproductive
system, urinary system , skeletal
system.
6. Major Problems of during EB formation
Agglomeration
6
2hr8hr
(Dang et al., 2001)
7. • Agglomeration of EBs that may have
negative effects on proliferation and
differentiation in the mouse model. (Dang et
al., 2001)
• Because of agglomerated large EBs revealed
extensive cell death and eventually large
necrotic centers due to mass transport
limitations. (Nir et al., 2003) 7
10. Static Suspension culture
10
Single ES cells
37 ℃
CO2 4.5~5%
Cultured 24~48 hrsSingle ES cellsES colonies
0.25%Trypsin, 20~30 sec
1000 rpm for 3~5 mins
Count the cell number
Fig. 2. A rough flow chart about suspension culture.
(Gomes et al., 2010)
Petri dish
11. Hanging drop
11
top bottom
3~4 ml PBS
37 ℃
CO2 4.5~5%
Cultured 24~48 hrs
Single ES cells
Fig. 3. A rough flow chart about hanging drop.
(Ao et al., 2011)
Petri dish
12. 12
Entrapment
Single ES cells
37 ℃
CO2 4.5~5%
Cultured 24~48 hrs
96 well dish
104~105 ES/well
Fig. 4. A rough flow chart about entrapment for
formation of EBs. (Ao et al., 2011)
13. Commercial - AggreWell
13
Fig. 5. AggreWell™ contains microwells to make uniform cell
aggregates.
(A) AggreWell™400 plates contain microwells 400 μm in diameter.
Photo taken at 40x magnification.
(B) AggreWell™800 plates contain microwells 800 μm in diameter.
Photo taken at 40x magnification. (StemCell Tech.)
14. Bioreactor
14
Fig. 6. Bioreactors of stirred
suspension system: (A)slow
turning lateral vessel (STLV)
and (B)high aspect rotating
vessel (HARV).
(Rungarunlert et al., 2009)
STLV culture(A)
central gas transfer cord
HARV culture(B)
Cultured 12~24 hrs
15–20 rpm
Single ES cells
oxygenator membrane
17. 17(Nir et al., 2003)
Fig. 8. RT-PCR analysis demonstrated the generation of tissues
derived from the three germ layers in the two system cultures in the
human model.
Germ layer markers expression
18. 18
Germ layer markers expression
Fig. 9. RT-PCR analysis ectoderm layer markers expression in different
size by the entrapment. (A)RT-PCR analysis. (B) The quantification of
relative gene expression. (Park et al., 2007)
0
(A)
(B)
(B)
19. 19
α-fetoprotein (AFP)
Germ layer markers expression
Fig. 10. RT-PCR analysis demonstrated mouse EB endoderm mark (α-
fetoprotein, AFP) expression for day 4 and day 8 in vitro. (A)RT-PCR
analysis. (B) The quantification of relative gene expression. ** indicate
p < 0.01 as compared to 200 μm EBs.
(Choi et al., 2010)
(A) (B)
20. 20
Efficiency of differentiating
Fig. 11. Illustration of the cumulative percentage of EBs containing
contracting area derived from STLV, hanging drop and suspension
culture. Scale bar=500 μm
D
(Rungarunlert et al., 2009)
A B C
21. 21
Efficiency of differentiating
(Choi et al., 2010)
Fig. 12. The beating frequency of EBs. EB beating frequency refers to
the number of concave microwells containing beating EBs divided by
the total number of concave microwells. Error bars are standard
deviation and Scale bars are 500 μ m.
22. Efficiency of differentiating
22
(Choi et al., 2010)
Fig. 13. Quantitative analysis of neurite numbers and lengths from
EBs retrieved from the entrapment, showing that larger EBs had a
greater number of neurites than smaller EBs. Error bars are
standard deviation; ** indicate p < 0.01 as compared to 200 μm EBs.
23. Summary
• Small (100 μm) aggregates showed increased
expression of the ectodermal marker
compared to that in large (500 μm)
aggregates, which had an increased
expression of mesodermal and endodermal
markers.
• Large EBs (500/1000 μm) had batter
differenting efficiency than small EBs (200
μm).
23
25. Conclusion
• It's better to chose the best protocol which
consider experiment design and lab
condition.
• It appears that the size of EBs exerts a
stronger influence on their differentiation
potential than the method by which EBs are
derived. 25
27. 27
Diagram depicting important steps for the conversion of hESCs to
cardiomyocytes. Early mesoderm differentiates via pre-cardiac mesoderm
and committed cardiac progenitors further to functional cardiomyocytes
(CM). Early and late cardiomyocytes are mainly discriminated based on
their electrophysiological properties and structural organization. Typical
markers for each step are indicated as well as some functional features of
the differentiated cells.
(Vidarsson et al., 2010)
28. 28
(Choi et al., 2010)
Differentiation of GOlig-mESC into OPCs. (A) Scheme showing the protocol of the
embryoid body (EB)-based and small molecule-driven differentiation. At D8, the
EBs were disaggregated and plated. The cells were passaged once per week when
they became confluent. (B) Morphology of day 4 EBs.
29. 29
Cost Unity Quantity Technicality
SC Low Low Random Low
HD Middle Middle Limited High
Ent. Middle High Limited Middle
Bioreactor High Middle to
High
Random Low
Table 1. Comparison of current in vitro cell culture systems for production of EBs
and other cell types. Suspension culture (SC). Hanging drop (HD). Entrapment
(Ent.).
(Dang et al., 2001; Rungarunlert et al., 2009; Xu et al., 2001)
30. 30
•Ngn2=Neurogenin 2
•HES1=Transcription factor HES-1 is a protein
that in humans is encoded by the HES1 gene.
•ASCL1=Achaete-scute homolog 1 is a protein
that in humans is encoded by the ASCL1 gene.
Ectoderm markers
32. 32
Nestin
It is expressed in stem cells of the central
nervous system (CNS) but not in mature CNS
cells.
A class VI intermediate filament protein.
Intermediate filaments within cells; characteristic
of primitive neuroectoderm formation.
(R&D systems web)
33. AFP(α-fetoprotin)
A major plasma protein produced by the yolk
sac and the liver during fetal life.
A marker of the visceral endoderm. (Hogan et
al., 1981)
33
(R&D systems web)
34. Brachyury
Member of the T-box family of transcription
factors.
Brachyury is required in the early determination
and differentiation of mesoderm.
Brachyury is essential for the formation of the
posterior body in all vertebrates.
34
(R&D systems web)
35. 35
Germ layer markers expression
Phase DAPI AFP
Fig. 11. Immunofluorescence assay of primate EB endoderm mark (α-fetoprotein,
AFP) expression in vitro. Scale bar = 150 μm.
(Park et al., 2007)
36. Germ layer markers expression
36
SSC
HD
D1 D2 D3 D4 D5 D6 D7
Brachyury
Fig. 9. RT-PCR analysis mesoderm layer marker Brachyury expression in the two
system cultures in the mouse model. There had no diameter data.
(Mogi et al., 2009)
37. 37
Quantitative analysis of EB diameter distribution. EBs retrieved from concave
microwells after culturing for 4 days in vitro were more homogeneous in size
and their sizes were significantly regulated by microwell widths (200, 500, and
1000 mm).
(Choi et al., 2010)