Stem Cells

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PEPTIDE APTAMER PVGL52 INDUCING MESENCHYMAL STEM CELL LOCALISATION INTO HEART:A CELL THERAPY MODEL STUDY IN CHICK PRESENTED BY R.PRIYADHARSHNI ROLL NO:11706214043 S.VANIDEVI ROLL NO:11706214057

 

INTRODUCTION

 



In 1960S,The existence of stem cells in an irradiated mouse was reported by Canadian scientists Ernest A. A. McCulloch and James E. Till.



Stem cells have the remarkable potential to develop

into many different cell types in the body during early life and growth. 

They are found in most, if not all, multimulticellular organisms.They organisms.They serve as a sort of  internal repair system. eg:wound healing

 

2

important characterstics characterstics of stem cells ce lls areare-



They are unspecialized cells capable of renewing themselvess through cell division and differentiation themselve giving rise to specialised cells.



under certain physiologi physiologicc or experimental conditions, conditions, they can be induced to become tissuetissue- or organorga or gan nspecific cells with special functions. eg:Addition of growth hormones

 

Stem cells are divided or classified based onon1.Source 2.Potency Based on source stem cells can be classified into following: 

 Embryonic stem cells come from a five to sixsix-day -day day-old embryo. They have the ability to form virtually

any type of cell found in the human body. 

 Embryonic germ cells are derived from the part of  a human embryo or foetus that will ultimately

 produce gametes (eggs or sperm).

 



 Adu Adult

stem cells are undifferentiated cells found

among specialised (differentiated) (differentiated) cells in a tissue or organ after birth. Based on current research, adult stem cells appear to have a more restricted ability to produce different cell types and to selfself renew than embryonic stem cells. 

U mbilical mbilical

cor d  d  blood  stem cells are used to treat a

range of blood disorders and immune system conditions.

 

Based on potency Stem cells are classified as as-"Potency" is a term that describes how many types of  cells a stem cell can become. 

Totipotent stem cells are cells that have not begun differentiating differentiati ng at all. They are capable of developing into any other type of body cell.



Pluripotent cells are almost as potent as totipotent stem cells. They have barely started differentiatin differentiating g and can develop into almost any other type of  cell,except placenta.

 





Multipotent stem cells are stem cells that have begun differentiating differentiati ng into a general type of cell. For eg,a  blood cell giving rise to a blood cell only but not  brain cell.eg:MSC Oligopotent stem cells can differentiate into only a few types of cell. For example, a lympoid stem cell can become any of the blood cells found in the lymphatic system system (T cells, B cells, and plasma cells),  but not a different kind of blood cell, such as a red  blood cell or platelet.

 



Unipotent stem cells can only become one type of cell  ² their own.  ²  own. They are considered considered stem cells cells because because they can reproduce indefinitely. An example is skin cells, which can renew themselves indefinitely, but which cannot become any other type of cell.

WHAT ARE MSCs? Mesenchymal stem cells SCscells , or M , or Marrow Stromal Cell are mult multipotent ipotent stem that can differentiate into a variety of cell types which include osteoblasts, chondrocytes and adipocytes.

 

SOURCES OF MSCs Mesenchymal stem cells (MSCs) are adult stem cells  Mesenchymal traditionally found in the bone the bone marrow. marrow . 



Bone marrow is the flexible tissue found in the hollow interior of bones. In adults, marrow in large  boness produce  bone producess new blood cells . There are two types of bone marrow: 1.red marrow 1.red marrow (consisting mainly mainly of hematopoietic tissue) yellow ow ma marrow rrow (consisting mainly of fat cells). 2.yell

 

Bone marrow contains three types of stem cells: 

Hematopoietic stem cells give rise to the three classes Hematopoietic of blood cells that are found in the circulation: white white  blood cells (leukocytes), red blood cells (erythrocytes), (erythrocyte s), and platelets (thrombocytes).



Mesenchymal stem cells are found arrayed around the central sinus in the bone marrow. They have the capability to differentiate into osteoblasts osteoblasts,, chondrocytes, myocytes, and many other types of  chondrocytes, cells.



Endotheliall stem cells Endothelia

 



The other sources sources of bone marrow include cord  blood,perip  blood,peripheral heral blood,fallopian tube,foetalicliver and lung adipose tissue,skeletal tissue,skeleta l muscle,amniot muscle,amniotic fluid,synovium fluid,syno vium and circulatory system.

CHARACTERISTICS



Morpholog Morph ogy y: They are bigger in size having a large, round nucleus with a prominent nucleolus, when compared with normal cells. It also contains Golgi apparatus, rough endoplasmic endoplasm ic reticulum, mitochondria mitochondria,, and a nd  polyribosomes.

 



Diff erentiation

cap ca pacity ty::

MSCs have a large capacity for selfself -renewal -renewal while maintaining their multipotency. The standard test to confirm multipotency is differentiation of the cells into osteoblasts, adipocytes, adipocytes, and chondrocyte chondrocytess as well as myocytes and possibly neuronneuron-like -like cells. 

Immun mmuno omodulat dulator ory y

eff ects: ects:

 Numerous studies have demonstrated that human MSC avoid allorecognition, interfere with dendritic cell and T-cell function and generate a local immunosuppressive microenvironment by secreting cytokines.(Ryan JM et al 2005)

 

The minimal requirement for a population of cells to I

i

nterrnat onal qualify MSCs as suggested by nte by Societyas orMSCs, Cyto Cyt o,the ther rapy is threefold: f or

1.They must be plastic adherent under standard culture conditions. 2.They

should express CD105,CD73 and CD90 and lack the expression of CD45,CD34,CD14,CD19 and HLA-DR surface molecules.

3.They should possess tripotential mesodermal capacity into osteoblasts,chondrocytes and adipocytes.

 

DIFFERENTIATION OF MSC 

Under defined conditions, MSCs can differentiate into chondrocytes, osteoblasts, and adipocytes, and they also serve as hematopoies hematopoiesis hematopoiesisis-supporting is-supporting stromal cells.



MSCs have also been reported, to differentiate into myocytes and cardiomyocytes cardiomyocytes and even into cells of  non mesodermal origin, including hepatocytes hepatocytes and neurons.

 

Mesenchymal stem cells

Ecto Ec tode derm rmal al ce cell lls s

Nerve cells

Meso Me sode derm rmal al st stem em ce cell lls s

endo en dode derm rmal al ce cell lls s

Adiposecells bone cartilage cardiac skeletal muscle muscle

liver cells

pancreatic cells

 

REGULATION OF MSC 

Growth factors such as members of the transf orm orming grow growth th f actoractor a ctoror-beta -beta (TGF(TGF-) -) su sup perf am amily, the insulin-lik e grow growth th f actor actors, s, the fibrob broblast last grow growth th f actor actors, s, the platelet latelet--de derrived th f actor actor,, and Wnts. growth grow



Several Hormonal factors like phosphatonins phosphatonins FGF 23 and sFRP4, Receptors for PTH/PTHrP ((Franz Franz Jakob et al, 2006)



Peptides derived from growth factors(Jae Sam Lee et al,2009) al,2009)

 

REGENERATION BY MSC 



MSCs are a promising cell type for regenerative medicine  because of their ease of isolation and expansion, their  multipotency and their low immunogenicity. The MSCs has the following applications as a regenerative medice: regeneration of cardiac muscle.(Pittenger, muscle.(Pittenger, Mark F et al,2002) regeneration of musculoskeletal tissues.(Edward tissues.(Edward J. Caterson et al,2001). regeneration of bone marrow(K  marrow(K Fukuda, Fuk ukud uda, a,2003) etc.

 

MODEL STUDY IN CHICK 

 



Model org Model organ anism is a nonnon-human -human species that is extensively extensivel y studied to explore potential causes and treatments treatm ents for human human disease diseasee . diseas



These can be classed as: genetic models (with short generation times, such as the fruitfly and nematode worm)

1.

2.

3.

experimental models, and genomic models, with a pivotal position in the evolutionary evolutiona ry tree.

 



The chick en domesticus)) is a domesticated en (Gallus gallus domesticus fowl.



It has descended primarily from the Red Junglefowl (Gallus  gallus)) and is scientificall  gallus scienti scientifically fically y classified classified as as the same species . it is an amniote and excellent for micromanipulation (e.g. tissue grafting) and overover -expression -expression of gene products.



TAXONOMIC CLASSIFICATION: CLASSIFICATION: Kingdom: Animalia Phylum: Chordata Class: Aves Order: Family:Galliformes Phasianidae Genus: Gallus Species: G. gallus Binomial name: Gallus gallus

 

OBJECTIVES

 



To culture stem cells and isolate the MSCs.



To subculture the cells.



Induction of growth and differentiation.



Cell therapy



Staining techniques



RT--PCR  RT -PCR 



Agrose gel ele Agrose electr electrophoresis ctropho ophores resis is

 

METHODS

 

MEDIUM USED: 1.L--15 1.L -15 medium(Leibovitz) 2.DMEM(Dulbecco's Modified Eagle's Medium Modified ) PREPARATION PREPARA TION OF LL-15 -15 and DMEM MEDIUM: 1.4 grams of LL-15 -15 (in powder form) was added to 100ml of autoclaved 1XPBS. 1XPBS. 10grams of DMEM powder in 1000ml of HBS(Hanks  buffered--saline).  buffered -saline).

 

MATRIX PREPARATION 

A solid matrix was prepared by adding a dding 2.5grams agar  agar and 1gram gelatin in 100ml 1XPBS.



5 culture bottles were taken and 1ml of this matrix was poured at the bottom of each culture bottle.



Above this 5ml of freshly prepared LL-15 -15 medium was added which contains 1000ul of pencillin and 100ul of amphotericinB.

 

ISOL ATION AND CUL ISOLA CULTURE TURE OF MSC FROM CHICK BONE MARROW To isolate marrow marrow,, kill chick by cervical dislocation Rinse the animal skeleton freely in 70% ethanol Make an incision around the perimeter of the hind limbs Dissect the hind limbs from the trunk of the body by cutting along the spinal cord Bisect each hind limb by cutting through the knee joint and remove the muscle and connective conn ective tissue .

 

After cleaning, store the bones in LL-15 -15 supplemented with  penicillin/streptomycin.  Now the stem cells ce lls are taken by Aspiration of  Bonemarrow(small amount of bone marrow fluid and cells are removed through a needle put into a bone ). Centrifuge the BM cells taken at 6000rpm for 5 min and pellet it

Culture the cells in 5 culture bottles with 1ml of matrix and an d 5 ml of LL-15 -15 media . Incubate the bottles at 37 °°C C for 4 days without without disturbing them.

 

SUBCULTURING After 4 days incubation

Add collagenase into the culture tube Once the media becomes cloudy, cloudy, the cells are detached and starts floating Resuspend the cells in a small volume of fresh serumserum-containing -containing medium to inactivate the collagenase Transfer the required number of cells ce lls to 5 new labeled bottles containing matrix and prepre-warmed -warmed medium.

 

The same procedure for subculturing is repeated twice, in 2 days interval.  MSC  Differentiation Differentiation:: 

The differentiation of the Mesenchymal stem cells was induced induce d  by the addition of synthetic synthetic peptides. 10ul of 5 different test peptides was added to the 5 culture bottles

To avoid contamination 100microlitre of penicillin was added add ed incubated overnight at 37C.

 



After inducing differentiation the culture bottles were checked for  more number of cells using these steps: All the culture bottles were shaken well

1ml of supernatant was removed and transferred to fresh eppendorf tubes.

OD taken at 450nm.

 

 Now the cultured cells were checked for its metabolic activity using PHOS metabolic assay . 1ml supernatant was removed from the culture after shaking well. Centrifuged at 6000rpm for 10 min, supernatant discarded. 1ml of PBS was added to the pellet.

100ul of PHOS solution was added. Incubated at 37ºC for 15 min .

OD measured at 450nm.

 

Following F ollowing the met etaabol etab abolic oliic ol ic aas assa sssay ay, the cul ulture with gr ult greeate grea eater ter  r metabolic mettab me abol olic ic acti ac activity tivi vity ty is is taken an and fu and further furt rthe rt her  her r  su subc bcul ultu ult tture ured ur red ed d  b  by by y di dilu luttion luti tio ion on n method. met etho hod d. The The met method etho hod d is is ca carr rrie ied do out ou ut as as follows: foll fo llow owss: Take the culture with greater activity a ctivity and shake well.

Centrifuge at 6000rpm for 10 min and remove the supernatant. sup ernatant.

To the pellet 1ml of DMEM medium was added and suspended.

 



Take 5 plastic culture flasks ,add 20ml DMEM medium.Now the suspended cells are added as given below(dilution method): 1st flask flask  ± ± 1µl of ce cell cells llss 2nd flask flask - 5µl 5µl of cells cell ce llss 3rd flask flask - 20µl of cells 4th flask flask - 100µl 100µl of cells cells cel ls 5th flask flask  ± ± remaining. rema re main inin ing. g.

 



The flasks are then incubated overnight at 37ºC. OD was measured at 450nm to find which dilution flask has  better number of MSC. 18ml of the medium is removed from that flask and replaced with fresh medium, incubated overnight at 37ºC.

 NOTE: Further studies were carried out with the dilution flask  containing maximum no of cells.

 

CELL THERAPY 

The dilution having the least OD reading was taken as appropriate for further invivo studies in chick.



2ml



Centrifuged at 6000rpm for 10 min, 4ºC and supernatant removed.



To the pellet 100ul of 1XPBS solution was added ad ded and kept aside for injecting into chick.

 



of this culture was taken from the plastic flask 

4 chicks were taken.Out of which 1 was kept kep t as control. 100µl of isoproterenol and 100ul cell sample was given to 3 chicks. 1 chick taken as a control was given 100µl isoproterenol alone. The chicks were left for 3 days.

 

RESULTS AND DISCUSSIONS

 

OD FOR SUBCULTURED CELLS SAMPLE

OD(450nm) CONCENTRATION (mg/ml)) (mg/ml

1

0.8176

0.08176

2 3

0.1223 0.1760

0.01223 0.01760

4

0.6230

0.06230

5

0.6530

0.06530

 

OD FOR METABOLIC ASSAY SAMPLE

OD(450nm)

1

0.5795

2

1.3176

3

1.0383

4

0.5745

5

0.4999

 

OD FOR CELLS IN PLASTIC CULTURE FLASKS SAMPLE

OD(450nm)

CONCENTRATION( mg/ml)) mg/ml

1ul flask

0.2455

0.02455

5ul flask

0.2306

0.02306

20ul flask

0.1807

0.01807

100ul flask

0.1501

0.01501

Remaining

0.2308

0.02308

 

FUTURE WORK 

 



Cell viability assay



Cell morphology using PAP stain



Metabolic assays

 

Rna isolation RT--PCR  RT -PCR 



Agarose gel electrophoresis

 

REFERENCE 

Becker AJ, McCulloch EA, Till JE (1963). "Cytological demonstration of the clonal nature of spleen spleen colonies colonies derived derived from from transplanted mouse marrow cells".  N ature ature 197: 452 ±   ±4. 4. doi::10.1038/197452a0 doi a0.. PMID PMID 13970094 139700 139 13970094. 70094 94. Siminovitc Simi Siminovitch novitch h L, McCulloch McCulloch EA, Till JE (1963). "The distribution distribution distributi on of colonycolony-forming -forming cells among spleen colonies".  J ournal ournal of  Cellular and Comparative Physiology 62: 327±  7 ±36. 36. doi::10.1002/jcp.1030620313 doi 0313.. PMID PMID 14086156 14086 140 14086156. 86156 156.



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Friede Fri Friedenstein edenste nstein in AJ, Go Gors Gorskaja rskaj kajaa JF, JF, Kulagin Kulaginaa NN NN (1976). (1976). "Fibroblast "Fibroblast

Frieden Fri Friedenstein edenste stein in AJ, Der Derigl Deriglasova iglasov asovaa UF, Kul Kulagi Kulagina agina na NN, NN,, Pa NN Pana Panasuk  nasu suk k AF, AF, Rudakow Rud Rudakowa akowaa SF, SF, Luria EA, EA, Ruadkow IA (1974). (1974). "Precursors "Precursors "Precur sors for  fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method".  Ex p Hematol  Hematol 2 (2): 83±  83  ±9 92. PMID PMID 445551 4455 44 5551 512  precursors in normal and irradiated mouse hematopoietic organs".

Hematol 4  Ex p Hematol 

(5): 267   74. PMID PMID 976387 9763 97 6387 87

 







 Netter, Frank H. (1987),  M usculoskeletal usculoskeletal system: anatomy, Ciba physiology, and metabolic disorders. disorders . Summit, New Jersey: CibaGeigy Corporation ISBN 0914168886, 0914168886, p.134 ^ Brighton, Carl T. and Robert M. Hunt Hu nt (1991), "Early histologic and ultrastructural ultrastructu ultras tructural ral changes in medullary medullary fracture fracture callus callus", ",  J ournal  ournal  of Bone and  J oint oint Surgery, Surgery, 73 73--A -A (6): 832-847 -847 Engl En Engler  gler er AJ, AJ, Sen S, Sweeny Sweeny HL, Discher  Disc Di sche herr DE (2006). "Matrix Elasticity Directs Stem Cell Lineage Specification". Cell 126 (4): 677±  677  ±689. 689. doi doi::10.1016/j.cell.2006.06.044 006.06.044.. PMID PMID 169 16923388 3388.. ^

Ryan JM, Barry FP, Murphy JM, Mahon BP (2005). "Mesenc "Mesenchymal Mesenchym Mesenchymal hymal al stem cells avoid all allogen allogeneic ogeneic eic rejection". rejecti reje ction" on"..  J  Inflamm  Inflamm (Lond  (   Lond)  ) 2: 8. doi doi::10.1186/1476 10.1186/1476--9 -925555-2-8 -8. PMID PMID 16045800 160458 160 16045800. 45800 00.



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Ryan JM, Barry F, Murphy JM, Mahon BP (2007). "Interferon"Interferongamma does not break, but promotes the immunosuppressive capacity of adult human mesenchymal mesenchym mesenc hymal al stem cells". cells". Clin Clin..  Ex p.  Immunol.. 149 (2): 353±   Immunol  353 ±63 63.

 

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