MESENCHYMAL STEM CELLS���� �

ZURAIDAH YUSOFF

DIVISION OF CORD BLOOD

NATIONAL BLOOD CENTRE

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Introduction

What is stem cells (SC)?

• Undifferentiated cells capable of self renewal through cell division.
• ability to differentiate into specialised cells.

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Types of stem cells

1. Embryonic Stem Cells (ESC): Obtained from the inner cell mass of

the blastocyst formed after egg fertilization or zygote (except extra-

embryonic layers).

2. Adult Stem Cells: Multipotent SC with limited capability to

differentiate along specific lineage.

3. Induced Pluripotent Stem Cells (iPS): Cells that have been engineered to behave like embryonic stem cells.

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Definition of MSC

Classical definition

• capable of self-renewal and lineage specific differentiation potential.
• give rise to cells of mesodermal lineages such as bone, adipose, cartilage, tendon and skeletal muscle

(Reyes et al. 2001; Pittenger et al. 1999; Fridenstein et al. 1974a).

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As a matter of fact…..

• Trans-differentiate into non-mesodermal lineage tissues including pancreatic islet cell (Dang et al. 2015), cardiac muscle (Potdar and Prasannan 2013), hepatocyte (Kazemnejad et al. 2009) and neural cell (Kopen et al. 1999).

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Trans-differentiation Potential of MSC into �Non-mesodermal lineages

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Basic history of MSC…… �

• Discovered in 1970’s by Alexander Friedenstein in BM.

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• Characterised as non-phagocytic adherent cells that form fibroblast like colonies .

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• First to address as non-haematopoietic stem cells.

(in BM, MSC co-exist with HSC).

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• Could differentiate into bone and adipose tissue.

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• Limited number of these cells within tissues could be replicated by using a culture medium.

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Bone marrow derived MSC (BM-MSC)

Not always acceptable due to:

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• Viral exposure during aspiration.
• Reduction of proliferation and differentiation capacity with increasing age of donor.
• Inadequate cellular fractions in bone marrow.
• Invasive procedure and may cause pain and discomfort to donors which may lead to infection, excessive bleeding and other complications.

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Sources of MSC

• Canine (Seo et al. 2009)
• Ovine (Gao et al. 2013)
• Murine (Li et al. 2013)
• Bovine (Cortes et al. 2013)
• Equine (Barberini et al. 2014)
• Swine (Yueying et al. 2014)

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Human Sources of MSC – alternative to BM

Bone marrow 0.001 - 0.01% of the nucleated

cells are constituted by MSC (Caplan, 1991).

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• Adipose tissue (Locke et al. 2009)
• Menstrual blood (Mou et al. 2013)
• Tonsil (Park et al. 2014)
• Dental pulp (Navabazam et al. 2013)
• Cardiac tissue (Williams et al. 2013)
• Cartilage (Sandrasaigaran et al. 2014)

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MSC from birth-associated waste tissues

• Umbilical cord (Sarugaser et al. 2009).
• Placenta (Vellasamy et al. 2012)
• Amniotic membrane (Pirjali et al. 2013)
• Umbilical cord blood (Jin et al. 2013; Divya et al. 2012)

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Methods for generation of MSC

• Buffy coat isolation
• Enzymatic digestion (Romanov et al. 2003)
• Enzymatic-mechanical dissociation (Tong et al. 2011)
• Explants method (Cardoso et al. 2012).

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Enrichment Methods of MSC

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• + ve selection using monoclonal -antibody labelling such as CD105 (Roura et al. 2006) and CD133.
• - ve selection through depletion of unwanted haematopoietic and endothelial cells (Tondreu et al. 2005).
• modified protocols and culture media including serum pre-selection and additional of growth supplement

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�Generation of Human MSC from WJ�- Explant Method�

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Sample collected in transportation media.

Disinfected in 70% ethanol for 30 seconds.

Soaked in 1XPBS

Blood vessels removed

UC tissues minced into paste-like consistency.

Paste-like tissue seeded into T25 flask and cultured.

Minimal Characterisation of MSC

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Morphological Analysis of UC-MSC - TEM

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FE-UCMSC

Phase Contrast Microscopic

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Spindle-shaped and fibroblast-like cells

Transmission electron microscopy:

MSC contain a small cell body with cell processes that are long and thin. The cell body contains a large euchromatic nucleus with prominent nucleolus. The remainder of the cell body contains small amount of Golgi apparatus, endoplasmic reticulum, mitochondria and polyribosome. (Asuman et al. 2013)

Basic Laboratory Testings

• Colony Forming Unit-Fibroblast
• Growth Kinetics
• Population Doubling Time
• Immunophenotyping
• Mesodermal Differentiation

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So why MSC?

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Magic Healing Cells….���remarkable regenerative, reparative and immunosuppressive properties

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Therapeutic Potentials of MSC

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• Regenerative and tissue reparative

- communicate with the injured tissue/cells

- differentiation as well as trans-differentiation ability.

- new tissues and finally treat the damage or degenerative condition.

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• Support Haematopoiesis and development of HSC

- manufacture the mature blood cells

- construction of a unique microenvironment

- Eg:- Graft versus host disease (GvHD)

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• MSC and Gene Therapy

- cancer gene therapy

- vehicles for cell and gene therapies

- easily susceptible for genetic manipulation at in vitro

- ability of tumour-homing

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� Important measures�- technically �

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• Relatively easy to procure from a variety of tissues
• Expand rapidly in cell culture at in vivo or ex vivo.
• Minor spontaneous differentiation during ex vivo

expansion

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Clinical Trials and Applications of MSC

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�����Biological properties of MSC��

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Wang et al., Journal of Hematology & Oncology 2012, 5:19

Immuno-modulatory Effect of MSC

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in 2000, Liechty et al. noticed a unique immunologic characteristics of MSC that enable their persistence in a xenogeneic environment .

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interaction of MSC with a wide range of immune cells including:

• Dendritic cells (Zhang et al. 2009)
• B lymphocytes (Corcione et al. 2006)
• Natural killer cells (Sotiropoulou et al. 2006)
• T lymphocytes (Di Nicola et al. 2002).

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Interestingly....

• inhibit most of the non-specific inflammatory cells which act as the first line

defence to infection.

• suppress the differentiation of immature monocytes into DC.

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Poorly Immunogenic - refrain from the host immune recognition�

• Positive expression for MHC class 1

-escape from Natural killer cells cytotoxicity

(Rasmusson et al. 2007)

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• Negative expression for MHC class II

-avoid alloreactive CD4⁺ T-cell recognition

(Di Nicola et al. 2002).

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• Lacking of co-stimulatory molecules expression such as CD80, CD86 or CD40

-necessary for the second signal for T cell activation

(Le Blanc et al. 2003; Pittenger et al. 1999).

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Important bio-active molecules released by MSC and their functions

Important bio-active molecules released by MSC and their functions

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• 69 of 174 assayed proteins molecules are growth factors, cytokines, and chemokines, able to exert profound effects of anti-apoptotic and regeneration-stimulating.

(Chen et al. 2008)

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• These plethora of growth factors, successively coordinate all relevant cells to foster regeneration and tissue repair through maintenance of endothelial integrity and angiogenesis augmentation (Lee et al. 2011).

Migration and engraftment of MSC

• The inflammatory signals released from site of injury could potentially activate MSC at bone marrow pool which subsequently induce proliferation, differentiation, migration and engraftment of MSC at the injury sites (Spaeth et al. 2008; Krysko et al. 2006).

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• Manufacturing in GMP-Facility
• Starting material or source of population
• Seeding density
• Growth kinetics -number of passages
• Devices for cell expansion
• Type of culture media & supplements –serum and growth factors)
• Clonogenicity
• Storage capacity for cryopreserved cells
• Differentiation potential
• Quality tests – viability, genomic stability, differentiation potential

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http://stemcellassays.com/2014/05/msc-pdl/

Critical issues for clinical-grade ex vivo expansion of MSC for

clinical scale production

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http://stemcellassays.com/2014/05/msc-pdl/

Safety Issues of Clinical Grade Expanded MSC

• In 2005 Rubio first time reported spontaneous human MSC transformation after 6-8 weeks ex vivo expansion .

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• More published reports confirming spontaneous adult stem cell transformation in long-term cell culture in mouse and human.

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• Intriguing reports showing expanded MSC can not overcome “senescence crisis”.

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• Potential risk of carcinogenesis during MSC due to chromosomal instability

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Guidelines on clinical exploitation of MSC formulated by International Society for cellular therapy

• Commercial GMP licensed MSC product
• Generally accepted assays of the potency of MSC.
• Optimal route of MSC delivery.
• The best MSC source.
• Optimal dose for MSC administration.
• Purity of expanded MSC – identification of contaminating cell

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Conclusion

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Safety Issue?

Biological activities

of MSC at in vivo?

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Thank You Very Much