Amity Institute of Biotechnology

Faculty: Pankaj Sharma, PhD

 STEM CELLS

 Remarkable potential to develop into different cell types and tissues

 Serve as source of new cells for internal repair

KINDS OF STEM CELLS

 Embryonic Stem Cells
 Adult (Somatic) Stem Cells
 induced Pluripotent Stem Cells
 STEM CELL FEATURES

 Potential for long-term division and renewal

o Factors regulating long-term proliferation and self-renewal?
o Why are ES cells different?

 Unspecialized but have potential to differentiate into specialized cells

o Signals (internal/external) for differentiation?
 Asymmetric Division Is a Unique Property
of the Stem Cell

Stem
Cell
Stem
Cell Asymmetric Division

Differentiated
Cell

• In asymmetric cell division two daughters differ

in their fates.
• This requires genetic, epigenetic and
microenvironmental instructions.
 The Classical View of Stem Cells

• A Totipotent Stem Cell is capable of giving rise to the

whole organism and the placenta.

• A Pluripotent Stem Cell gives rise to the whole organism.

• A Multipotent Stem Cell is capable of producing more than

one type of differentiated cells.

• An Unipotent Stem Cell is a progenitor cell that can

differentiate to a single type of mature cells.
 The Current View of Stem Cells

Stem Cells (SC) as well as Differentiated Cells (DC) are more

plastic than we once thought; SC can trans-differentiate to DC
of unexpected lineages, and DC can de-differentiate to SC.

Differentiation
Stem Differentiated
Cell Cell
De-differentiation

Although differentiated state of the cell is much more stable than

its undifferentiated state, nuclear transfer can override that
stability by nuclear reprogramming.
Embryonic Stem Cells
 ES CELLS
CULTURE
 Isolation
 Culture
- Use of feeder layer (MEFs or laminins)
- Passage (subculture) at low confluence
- Suspension culture
CHARACTERIZATION
 Capacity for long-term renewal in undifferentiated state
 Chromosomal and genomic stability
 Potential for growth after freezing, thawing and re-plating
 Potential for spontaneous and directed differentiation
 Capacity for teratoma formation in mouse models
 ADULT/SOMATIC STEM CELLS
 Undifferentiated cell found among differentiated cells in tissue/organ
with potential to differentiate into all cell types
 Normally quiescent, primary role to maintain and repair tissue
 Located in a “specific stem cell niche” (pericytes?)

Tests for Adult Stem Cells

 Long-term renewal and maintenance
 Ability to differentiate into all cell types of tissue/organ
 In vivo labeling and tracking of differentiation
 Ex vivo labeling and ability to repopulate, in vivo, the tissue of origin
 DIFFERENTIATION OF ADULT STEM CELLS
Direct Differentiation
 Hematopoeitic Stem Cells - All types of blood cells (RBCs, B and T
lymphocytes, neutrophils, basophils, eosinophils, macrophages, monocytes,
mast cells, NK cells, dendritic cells
 Mesenchymal Stem Cells - Bone cells (osteocytes), cartilage cells
(chondrocytes), fat cells (adipocytes), other connective tissue cells (tendons)
 Neural Stem Cells - Neurons, astrocytes and oligodendrocytes
 Gastrointestinal Epithelial Stem Cells - Absorptive cells, goblet cells,
paneth cells, enteroendocrine cells
 Skin Stem Cells - Keratinocytes, hair follicles

Transdifferentiation: Not clearly understood, low efficiency

Dedifferentiation: Change from multipotent to pluripotent
Nuclear Fusion: Fusion of ES cell nucleus with somatic cells
 ADULT STEM CELLS - QUESTIONS
 Kinds
 Evolution (left over ES cells?)
 Maintenance of undifferentiated state
 Characterization of stem cell niche
 Transdifferentiation capacity- Reliability and Efficiency
 Reasons underlying beneficial effects - Tropism, secretion of
specific factors, cell-to-cell contact

 Factors controlling proliferation and differentiation

 Factors controlling tissue/organ-specific homing

 STEM CELLS - EMBRYONIC vs. ADULT

 Ease of Isolation
 Numbers
 Ease of Culture/Maintenance

 Transplantation Rejection
 Homing to target tissue/organ
Currently Available Methods to Generate Pluripotent Stem Cells
from Adult Somatic or Germ Cells

Yamanaka S, 2007
Only Four Master Genes Can Reprogram
the Nucleus of a Differentiated Cell
States of Cells Sets of Gene Expressed
HG SG DG
Stem On On Off

Differentiated On Off On

Cell Nov 30, 2007

Putative Roles of the Factor Quadruplets in
the Induction of iPS Cells

Yamanaka S, 2007
 Nuclear Reprogramming

States of Cells Sets of Gene Expressed

HG SG DG
Stem On On Off

Differentiated On Off On

HG = House Keeping Genes

SG = Genes for Stemness
DG = Genes for Differentiation

The Nuclear Reprogramming is mediated by

Chromatin Remodeling.
Chromatin Structure

Core Histones
Nucleosome H2A, H2A
H2B, H2B
10 nm
H3, H3
H4, H4
Linker
The First Mammal Cloned by Nuclear Transfer
Using an Adult Differentiated Cell

Dolly, the Cloned Sheep Prof. Ian Wilmut, Ph.D.

(Jul 5, 1996 – Feb 14, 2003) (1944- )

Nature 385:810-813, 1997

 Cat and Pigs Cloned by Nuclear Transfer
Using Adult Differentiated Cells

Nature 415: 859, 2002 Nature 415: 1035, 2002

Nature 415:859, 2002
CRITERIA FOR STEM CELL-BASED THERAPIES

• Extensive Proliferation

• Efficient Differentiation into desired cell types(s)

• Survival in Receipient after transplantation

• Integration into surrounding tissue

• Appropriate function for the duration of receipient’s life span

• No undesired side-effects
CURRENT STEM CELL-BASED THERAPIES

 Hematopoietic Stem Cell Transplant

Human HSC : CD34+, CD59+, Thy1/CD90+, CD38low/-, C-kit/CD117+, lin-

Mouse HSC : CD34low/-, SCA-1+ , Thy1.1+/low, CD38+, C-kit+, lin-

Sources
 Bone Marrow- Hematopoietic and mesenchymal
 Umbilical Cord Blood
 Peripheral Blood
 Amniotic Fluid

 Embryonic Stem Cells - For terminally ill, incurable patients

- Spinal Cord Injury, Parkinson’s, Alzheimer’s
- Ethical Issues?
BONE MARROW AS A SOURCE OF STEM CELLS
HEMATOPOIETIC STEM CELLS
MESENCHYMAL STEM CELLS
 UMBILICAL CORD BLOOD AS SOURCE OF
STEM CELLS
 Rich source of hematpoeitic stem cells
 Neonatal stem cells- Less mature than bone marrow cells

Advantages:
 Non-invasive
 Large number of cells may be obtained
 No graft-vs.-host disease
Disdvantages:
 Limited number of stem cells in a single umblical cord
 Risk of graft failure
 Still at experimental stage
 POTENTIAL BENEFITS OF
STEM CELLS
 Brain damage, Parkinson’s disease, Alzheimer’s disease
 Amyotrophic lateral sclerosis (ALS), muscular dystrophy and spinal
cord injuries
 Cardiovascular diseases and heart damage
 Aging - Baldness, Graying hair, Eye changes, Memory loss, hearing loss
 Dentistry- Regeneration of damaged coronal dentin and pulp,
peridontal regeneration, whole tooth regeneration
 Congenital birth defects- Neural and behavorial
 Diabetes- type II and type I
 Orthopaedic conditions
 Wound healing and tissue repair
 Infertility

 Cancer- Hematopoietic malignancies and solid tumors