Presentation on theme: "1 STEM CELL ISOLATION, CULTURE AND APPLICATION By P.B.TIRUPATHI PICHIAH II M.Sc., Animal Biotechnology, Dept Of Animal Science. BHARATHIDASAN UNIVERSITY."— Presentation transcript:
1 STEM CELL ISOLATION, CULTURE AND APPLICATION By P.B.TIRUPATHI PICHIAH II M.Sc., Animal Biotechnology, Dept Of Animal Science. BHARATHIDASAN UNIVERSITY
2 SYNOPSIS What is stem cells ? How Stem Cells studies started Classification of stem cell A. Based on potency. - Totipotent, Pluripotent, Multipotent B. Based on source - Embryonic Stem Cell and Adult stem cell. Properties of Stem Cells Embryonic Stem Cell Vs Embryonic germ cell. Maintenance of Stem Cells. Transcription Factors Operating in Early Mouse Development and ES Cells
3 Can divide and renew them selves for long periods of time. Are unspecialized. Can divide and become specific specialized cell types of the body. What are Stem Cells ?
4 How Stem Cells studies started? In 1998, James Thomson (University of Wisconsin-Madison) isolated cells from the inner cell mass of the early embryo, and developed the first human embryonic stem cell lines. In 1998, John Gearhart (Johns Hopkins University) derived human embryonic germ cells from cells in fetal gonadal tissue (primordial germ cells). Pluripotent stem cell “lines” were developed from both sources
5 How Stem Cells studies started? Stem cells were discovered in the semineferous tubules, where male germ cells, arise, by Leblond and co-workers. However, it has been extensively studied in the haematopoietic system. Prof. Lazlo Lajtha and co-workers, extensively studied haematopoietic stem cells and laid the foundation of Stem cells Biology Clinical uses of stem cells to treat diseases of the haematopoietic system such as leukemia and genetic anaemia, which are due to diseased or transformed stem cells followed animal experiments by Till Mc Culloch in Prof Donnel Thomas, carried out the first human bone marrow transplantation to successfully treat a thalassaemic patient.
6 Classification of stem cell Based on potency. Based on potency Based on source. Based on source
8 Based on potency Totipotent: Cells can develop into a new individual. (Source: Cells from early (1-3 days) embryos ). Pluripotent : Cells can form any (over 200) cell types. (Some cells of blastocyst (5 to 14 days). Multipotent :Cells differentiated, but can form a number of other tissues. (Fetal tissue, cord blood, and adult stem cells)
9 Properties of Embryonic Stem Cells * Derived from the inner cell mass/epiblast of the blastocyst. * Capable of undergoing an unlimited number of symmetrical divisions without differentiating (long-term self-renewal). Exhibit and maintain a stable, full (diploid), normal complement of chromosomes (karyotype). can give rise to differentiated cell types that are derived from all three primary germ layers of the embryo. [* Not shown in human EG cells. # Not shown in human ES cells. All of the criteria have been met by mouse ES cells.]
10 Properties of Embryonic Stem Cells * # Capable of integrating into all fetal tissues during development. * # Capable of colonizing the germ line and giving rise to egg or sperm cells. * Clonogenic. Expresses the transcription factor Oct-4,. Can be induced to continue proliferating or to differentiate [* Not shown in human EG cells. # Not shown in human ES cells. All of the criteria have been met by mouse ES cells.]
11 Properties of Embryonic Stem Cells Lacks the G1 checkpoint in the cell cycle. ES cells spend most of their time in the S phase of the cell cycle. ES cells do not require any external stimulus to initiate DNA replication. [* Not shown in human EG cells. # Not shown in human ES cells. All of the criteria have been met by mouse ES cells.]
12 ES Cells Vs EG Cells are derived from the inner cell mass of the preimplantation blastocyst (4-5 day old blastocyst). Can be propagated for several hundred population doublings. Embryonic - like stem cells, embryonic germ cells are derived from primordial germ cells, which were obtained from the gonadal ridge and mesenchyma (5-10 week old fetus). Can be maintained for only 70 to 80 population doublings.
13 Maintenance of ES cells Grow on feeder layer of MEF cells or add LIF to the growth medium. Inhibit feeder layer proliferation by addition of Mitomycin C. LIF binds to receptor complex of LIF receptor and gp 130 receptor. Binding of LIF triggers activation of latent transcription factor STAT3, a necessary event in vitro for proliferation of mSC cells. STAT3 and Oct-4 may interact and affect the function of common set of target genes.
14 Maintenance of ES cells In vivo, signaling through the gp130 receptor is not necessary for normal early embryonic development but is required to maintain epiblast during diapause. Self renewal is inhibited by SHP-2 (tyrosine phosphatase) and ERK (extra cellular regulated kinase). Both ERK and SHP-2 counteract the proliferative effects of STAT3 and promote differentiation
15 Maintenance of ES cells Nanog, a homeodomain protein act to restrict the differentiation inducing potential of Oct-3/4. Both Nanog and Oct-3/4 are essential to maintain ES cell identity, but STAT3, following LIF activation plays an accessory role.
16 Maintenance of ES cells In combination with BMP (bone morphogenetic protein), LIF sustains self-renewal. BMP is to induce expression of Id (“inhibitor of differentiation) genes via the SMAD pathway. Wnt activation sustains expression of the pluripotent stage-specific transcription factors Oct-3/4 and Nanog. Other markers are Rex-1, Sox2, Genesis, GBX2, UTF1, Pem and L17.
17 Transcription Factors Operating in Early Mouse Development and ES Cells System Oct4 is crucial for the first embryonic lineage specification, and Nanog is crucial for the second. Maintenance of the pluripotent epiblast of postimplantation embryos requires Oct4, Sox2, and FoxD3. In the ES cell Oct4, Sox2, Stat3, and Nanog are essential for self renewal: the pools of target genes controlled by each transcription factor or combination of factors are shown in color.
18 Properties of Adult Stem Cells Rare. Difficult to identify and their origins are not known. long-term self-renewal. Multipotent. Trans differentiation/ Plasticity. derived from brain, bone marrow, peripheral blood, dental pulp, spinal cord, blood vessels, skeletal muscle, epithelia of the skin and digestive system, cornea, retina, liver, and pancreas.
19 Localization of Adult Stem Cells
21 Cell-cycle related pathways PathwaysComments Intrinsic and extrinsic signaling Growth factors, cytokines, etc. can regulate stem cell number and trigger expansion of the stem cell pool/ production of progenitors. DNA repair and apoptosis pathways Stem cells resist DNA damage due to up regulation of DNA repair pathways. When DNA damage is detected, e.g., UV radiation, ES cells undergo apoptosis rather than delay at the G1/S check point. Regulating cell cycle ES cells are regulated at the S/G2 check point, somatic cells and differentiating cells are regulated at G1/S check point. Symmetric/ asymmetric division The controls of differentiation vs cell growth (symmetric division) are not well known. Factors such as piwi, Pten, Bmi-1, Notch, etc have been identified as playing a role
22 Pathways regulating senescence PathwaysComments Maintaining telomere lengthTelomerase activity tends to be high in stem cell populations and it is likely that specific components of this pathway will be high in stem cell populations. DNA repair and apoptosis pathways Stem cells are radiation sensitive but often resistant to other apoptotic signals. Regulating cell cycleThe ability to undergo prolonged periods of quiescence and then reenter the cell cycle suggests that cell-cycle regulation will differ from most other cell populations Mitochondrial and oxidative stress pathways Prolonged lifespan requires special mechanisms to regulate mitochondrial stability and response to oxidative stress Immortality genesGenes that prolong lifespan or bypass senescence have been identified. These include Mortalins, and MORF’s. Igf/akt/PTENThe igf/akt/PTEN pathway has been shown to be important in regulating cell size and proliferation as well as lifespan. It is likely that components of this pathway will be shared by stem cell populations
23 Similarities and differences Between embryonic and adult stem cells ES cells are pluripotent. Adult stem cells are multipotent, generally limited to differentiating into different cell types of their tissue of origin. Large numbers of ES cells can be relatively easily grown in culture, while adult stem cells are rare in mature tissues and methods for expanding their numbers in cell culture have not yet been worked out.
24 Similarities and differences Between embryonic and adult stem cells Use of the patient's own adult stem cells would not be rejected by the immune system. Es cells from a donor introduced into a patient could cause transplant rejection. However, whether the recipient would reject donor embryonic stem cells has not been determined in human experiments. Up until week 12, fetal/ embryonic stem cells are hypoallergenic as they have little to none of a certain type of protein on their surface (Class II HLA). After the 12th week, fetal stem cells acquire these immune- triggering protein.
25 ES cells Vs Multipotent adult progenitor cells PropertyES cellsMAPCs Growth potentialindefinite Contributes to germ cells?yes? Common growth factorsyes (mouse) Differentiates to most cell typesyesyes (lower efficiency) Expression of telomeraseyes? Expression of Oct-4yeslow levels Expression of Rex-1yes Expression of SSEA-1yesyes (subset) MAPCs lineage markersabsentlow levels Flk-1, Flt-1, AC133, CD44, CDw90, KDR, B2- microglobulin, higher levels CD13, CD49b ES cell lineage markersAldehyde dehydrogenase, Forward/side scatter/SP, Hoescht/rhodamine, alkaline phosphatase, CXCR-4, ABCG-2 transporter, SOX-2, UTF-1, FoxD3, FGFR-4, connexins, glucose transporter GLUT1, Mash2/Hash2, eomesodermin, TRA-1- 81/TRA-1-60 absent
26 ISOLATION OF STEM CELLS
27 ISOLATION AND IDENTIFICATION OF S.C The stem cells are isolated mainly from two sources Bone marrow Umblical cord blood
29 CORD BLOOD SAMPLE Cord blood is collected during pregnancy The collected sample is then processed furtherly for the isolation of stem cells
31 Protocol for s.c sepration from UCB Mix UCB with Hydroxy ethyl starch in 1:2 ratio & mix for 10 min Allow to settle for 30min Pool out the supernatant ¢rifuge at 1500 rpm for 20min Wash the pellet twice with sterile saline Resuspend the pellet in 10ml saline
32 Take 3ml of “ficol- hypague in 15ml centrifuge tube On top of 3ml-layer 10ml of sample with out mixing Centrifuge at 1500rpm for 20min Discard the supernatant Remove the MNC layer into a c.tube
33 Resuspend the layer with 5ml of saline & centrifuge at 1500 Then remove the supernatant & wash the pellet with sterile saline Resuspend the cell pellet in DMEM Check out the cell count – check viability
34 Plate the cells in culture plate with 3ml media in each plate
35 Incubate in co2 incubator at 35˚C for 5-6 days
36 MICROSCOPIC VIEW OF GROWN CELLS
37 Analysis of stem cells by FLOW CYTOMETER Flourescence dye’s for analysis of stem cells FITC-Flouro iso thio cyanate PE -Phyco erythrin MNC’S – Leucocyte –CD45 Stem cells - CD34 CD34 + PE→ Flourescence In green colour CD45 + FITC→ Flourescence In red colour
38 Sample + CD34 + PE→ Flourescence In green colour→confirms presence of stem cells
42 MEDIA FORMULATION FOR STEM CELLS.
44 Differentiation of ES cells in to neuron, pancreatic islets
45 Bone Marrow Stem Cells May Cure Eye Disease Science DailyScience Daily — Adult bone marrow stem cells may help cure certain genetic eye diseases, according to UC researchers. The green in this diagram shows bone marrow stem cells that have been injected into the eye and have taken on the properties of corneal cells. After being injected, the stem cells begin expressing cornea-specific proteins. This technology could one day help cure genetic eye diseases. (Credit: Image courtesy of University of Cincinnati) Scientists have completed a study using mice which showed that bone marrow stem cells can switch roles and produce keratocan, a natural protein involved in the growth of the cornea— the transparent, outer layer of the eyeball. This ability of marrow cells to “differentiate” into keratocan-producing cells might provide a means for treating abnormal corneal cell growth in people. S The green in this diagram shows bone marrow stem cells that have been injected into the eye and have taken on the properties of corneal cells. After being injected, the stem cells begin expressing cornea-specific proteins. This technology could one day help cure genetic eye diseases. (Credit: Image courtesy of University of Cincinnati)