at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
STEM CELLS (2)
Dr. Judit Pongrácz
Three dimensional tissue cultures and tissue engineering – Lecture 3
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Cord blood stem cells
• Approx. 130 million babies born yearly – the
umbilical cord blood is the largest potential source of stem cells for regenerative medicine
• In the past 36 yrs 10000 patients were treated for
over 80 different diseases
Cord blood stem cells and fetal stem cells
Cord blood collection from umbilical vein (after birth)
The cord blood cells are frozen in bag or cryovials
Liquid nitrogen storage tank (-150°)
Analysis of blood Cell separation
Mix and settle Concentrate cells Freeze cells
35 min 10 min
Express
Express Add cord
blood
1 2 3
Cryopreservation
• Cryopreservation of primary cells is possible for long term (so far 20 yrs).
• The low-temperature is maintained at -150-196 o C in
liquid nitrogen.
Cord blood processing
1. Red cell depletion (using Ficoll, Hetastarch, Lymphoprep, Prepacyte)
2. Depletion of plasma for smaller storage size 3. Testing of the final cell pool (infection, volume,
cellularity, stem cell content, CD34 + )
cryopreservation
• Cord blood is primarily useful in hematological disorders
• Cord blood is collected at birth
• Either processed or just simply frozen in DMSO
Cord blood banking
• Cord blood banks should be set up in every
metropolitan city with HLY specification and linked to an international computer network
• Keeping cord blood for a considerable length of
time is costly
Pluripotenciy of cord blood stem cells
Cord Blood
Stem cells
CBE MSC
Purification
Endodermal Mesodermal Ectodermal
Hepato-Biliary Blood Neural
Stem cell population in cord blood
Adherent Pre-MSC MSC
Non-adherent
CBE
Lin - CD133 + CD34 +
Cord Blood
Bone marrow
Peripheral Blood
Disorders treatable with cord blood I
Oncologic disorders
Acute lymphoblastic leukemia Acute myeloid leukemia
Autoimmune lymphoproliferative disorders Burkitt lymphoma
Chronic myeloid leukemia
Cytopenia related to monosomy Familial hystocytosis
Hodgkin’s disease
Juvenile myelomonocytic leukemia Langerhans cell hystocytosis
Myelodysplastic syndromes Non-Hodgkin’s lymphoma
Immune deficiencies
Ataxia telangiectasia Cartilage-hair hypoplasia
Chronic granulomatous disease DiGeorge syndrome
Hypogammaglobulinaemia IKK gamma deficiency
Immune dysregulation polyendocrinophaty Mucolipidosis type II
Myelokathesis
Severe combined immunodeficiency Wiscott-Aldrich syndrome
X-linked agammaglobulinaemia,
immunodeficiency, lymphoproliferative
syndrome
Disorders treatable with cord blood II
Hematological disorders
Autoimmune neutropenia Cyclic neutropenia
Diamond Blackfran anemia Evan’s syndrome
Red cell aplasia Refractory anemia Severe aplastic anemia Sickle cell disease Thalassaemia Fanconi’s anemia Galnzmann’s disease
Congenital sideroblastic anemia
Juvenile dermatomyositis and xanthogranulomas
Metabolic disorders
Adrenoleukodystrophy Alpha mannosidosis Type I diabetes Gaucher’s disease Gunther disease
Hermansky-Pudlak syndrome Hurler syndrome
Hurler-Scheie syndrome Krabbe’s disease
Maroteau-lamy syndrome Metachromatic leukodystrophy Mucolipidosis Types II, III
Neimann Pick syndrome, Types A and B Sandoff syndrome
Sanfilippo syndrome
Tay Sachs disease
Fat stem cells (ASC)
Fat or adipose tissue stem cells (ASC):
• Easily obtainable
• Consistent immunophenotype
• Similar to BMSC
• Multipotent
• Manipulation by genetic engineering
Types of adipose tissues
TYPE FUNCTION
1 Bone marrow Fills in space no longer used for hematopoiesis 2 Brown adipose tissue (BAT) Protects vital organs in the newborn
3 Ectopic adipose tissue Abnormal fat accumulation in the liver, skeletal or cardiac muscle (e.g. in metabolic syndrome)
4 Mammary adipose tissue Lactation – energy and nutrient source 5 Mechanical adipose tissue Protection from mechanical trauma
6 White adipose tissue (WAT) Insulation, energy storage, reservoir, endocrine
organ
Isolation procedures
Digestion with collagenase at 37 o C, 1hr
Stromal vascular fraction (SVF) Wash in PBS
Aspiration of lipocytes
300g, 5 min
Immunophenotype of ASCs Positive markers
Marker Name Marker Name
CD9 Tetraspan CD55 Decay accelerating factor
CD10 Common acute lymphocytic
leukemia antigen CD59 Protectin
CD13 Aminopeptidase CD71 Transferrin
CD29 b1-integrin CD73 5’-ectonucleotidase
CD34 Sialomucin CD90 Thy1
CD44 Hyaluronate receptor CD105 Endoglin
CD49d a4-integrin CD146 Muc-18
CD54 Intracellular adhesion
molecule CD166 Activated leukocyte cell
adhesion molecule HLA-ABC Histocompatibility locus
antigen-ABC a-SMA a-smooth muscle actin
Negative markers
Marker Name Marker Name
CD11b ab-integrin CD50 Intracellular adhesion
molecule-3
CD14 LPS receptor CD56 Neural cell adhesion molecule
CD16 Fc receptor CD62 E-selection
CD18 b2-integrin CD104 B4-integrin
CD45 Common leukocyte antigen HLA-DR Histocompatibility locus
antigen-DR
Cytokine profile of ASCs
CYOTOKINE FUNCTION GROUP
• Adiponectin
• Leptin
• Plasminogen activator inhibitor-1
Adipokines
• Hepatocyte growth factor
• Pigment epithelial derived factor
• Vascular endothelial growth factor
Angiogenic
• Flt-3 ligand
• GCSF
• Leukemia inhibitory factor
• IL-7
• MCSF
Hematopoietic
• IL-6, IL-8, IL-11
• Leukemia inhibitory factor
• TNFa Pro-inflammatory
Immunogenecity of ASCs
• Lack of immunogenicity is linked to the absence of the major histocompatibility class II antigens (HLA- DR) on their surface.
• Their immunosuppressive properties are linked to
prostaglandin E2 production.
Differentiation potential of ASCs
• Adipocyte
• Cardiac myocytes
• Chondrocyte
• Endodermal and ectodermal lineages
• Endothelial and smooth muscle cells
• Hematopoietic support
• Neuronal lineage
• Osteoblast
• Skeletal myocytes
Differentiation into adipocytes
ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION WAT etc. Forskolin (AMP agonist) Neutral lipids Functional fat pads
Methylisobutylxanthine
(AMP agonist) Adiponectin Plastic surgery
Glucocorticoid receptor ligands (dexamethasone)
CAAT/enhancer binding protein-a fatty acid binding protein (aP2)
Cosmetic and
reconstructive surgery PPAR-g2 ligands
(thiazolidinediones) Leptin
Insulin Lipoprotein lipase
bFGF PPAR-g2
Differentiation into cardiac myocytes
ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION
BAT 5-azacytadine Sarcomeric actinin Repair injured cardiac
tissue after ischemic injury
WAT Cardiomyocyte extract Connexin-43
Desmin
troponin-I
Differentiation into chondrocytes
ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION Bone marrow adipose
tissue Ascorbate Aggrecan Knee, hip chondroid tissue
BAT Dexamethasone Chondroitin sulfate
WAT TGF-b Collagen type II
3D structure Collagen type IV
BMP-6 Chondrocyte specific
proteoglycans
FGF
Differentiation into osteocytes
ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION
WAT Ascorbate Osteocalcin Bone implantation
Bone marrow adipose
tissue Dexamethasone DMP-1 Bone fracture repair
1,25-dihydroxy vitamin D3 Osteoadherin B-glycerophosphate
BMP-2
BMP-7
Runx2
Differentiation into skeletal myocytes
ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION Low concentration FBS myoD
Horse serum myogenin
Myosin light chain kinase
Differentiation into neuronal cells
ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION Indomethacin Glial fibrillary acidic
protein (GFAP) Central nervous system injury
Insulin Nestin
methylisobutylxanthine Intermediate filament Glutamate receptor subunits
S-100
B-III tubulin
ectodermal lineages
ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION Bone marrow adipoid
tissue Hepatocyte growth factor Albumin Liver
Oncostatin M A-fetoprotein DMSO together with HGF,
bFGF, nicotinamide Urea
ATRA Cytokeratin-18 Epithelial tissue repair
(Crohn’s disease)
Differentiation into endothelial and smooth muscle cells
ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION
WAT CD31 Vascular trauma
calponin Urogenital trauma
A-smooth muscle actin
Hematopoietic support
ORIGIN OF ACS INDUCING AGENT MARKERS INDUCED APPLICATION ASCs secrete:
IL6,IL7, IL8,IL11 SCF, TNFa, MCSF, GMCSF
CD34+ into T, NK, B markers
For patients requireng
hematopoietic stem cells
reconstruction following
high-dose chemotherapy
STEM CELLS (3)
Dr. Judit Pongrácz
Three dimensional tissue cultures and tissue engineering – Lecture 4
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Application of ESCs and ASCs
Vascular lumen
Vasculogenic zone Vascular disorders
Gene therapies
Genetically modified stem cell-based delivery Intravenous
injection
Mesoderm
Cardiac diseases CSCs
Atria niche Apex niche
HSC
EPC MSC
Osteoblasts Chondrocytes
Adipocytes Myoblasts Common lymphoid
precursor NK-cells T-lymphocytes Dendritic cells B-lymphocytes Common myeloid
precursor
Macrophages Platelets
Erythrocytes Monocytes Granulocytes HSCs
Endosteum surface niche Microvasculature niche
MSCs
Perivascular surface niche
Bone
Hemopoietic and Immune system disorders
Endoderm
BASCs
Bronchioalveolar duct junction niche Lung disorders
PSCs
Pancreatic duct putative niche
Pancreas
Insulin-secreting b-cells
Endocrine islets of Langerhans Exocrine
acini
Diabetes
Liver
Hepatocytes
HDCs Bile duct (canal of Hering) Hepatic disorders
Lung
Ectoderm
NSCs Subventricular zone niche Hippocampus niche (dentate gyrus region) Neuron Astrocyte Oligodendrocyte
Brain and spinal cord disorders
Eye disorders
RSCs
Ciliary epithelium niche
CESCs Limbus niche Retina
Cornea
KSCs
Basal layer niche bESCs
eNCSCs Bulge niche
SKPs Dermal papilla Skin disorders
Heart
Cardiomyocytes
Embryonic development
Inner cell mass ESCs Blastocyst
Pluripotent ESC
Mesodermal stem cell Endodermal
stem cell
Ectodermal stem cell Hemangioblast
HSC EPC
Vascular wall-resident stem cells EPCs and MSCs New endothelial cell
Bloodstream Macrophage
Platelets
Erythrocyte Monocyte
Eosinophil
NK-cell T-lymphocyte Dendritic cell B-lymphocyte Basophil
Neutrophil
Media
Brain
Eye
Skin
Genetic engineering and gene delivery using ASCs
• Lentiviral vectors can transduce ASCs
• Other recombinant viral vectors
• Nucleofection
stem cell growth and differentiation
Bioreactors come in many sizes and designs and include stirred, rotary and perfused systems. All serve to improve exchange efficiency of nutrients and waste products and delivery of growth factors to enable longer term culture, helping to scale-up cell numbers or to grow larger pieces of tissue.
Scaffold can provide physical (e.g.
surface roughness, porosity, etc.) and also biochemical (e.g. controlled release of doped growth factors) cues to promote attachment, recruitment, differentiation and delivery of cells.
Co-culture with the cells or tissues of interest (i.e. the target for tissue repair) can help to encourage differentiation.
This can include direct physical contact and/or indirect biochemical signaling Biochem factors added to culture medium (including serum) stimulate differentiation. Requires knowledge of factors likely to induce differentiation but is rarely, if ever, 100% effective.
Differential adhesion assays using specific ECM proteins or receptor ligands can help in encouraging selection of specific cell types based on affinity and kinetics of cell- substratum interactions. Related to this is the colony forming unit (CFU) assay.
Cell sorting techniques like MACS or FACS can positively select (or negatively deplete unwanted cell types) using cell surface antibodies or fluorescent transduced markers like GFP.
FACS MACS
Transduction with lineage specific genes can help to drive differentiation.
Reporter tags like GFP aid selection (i.e. FACS) and reveal when and where genes are activated
Gene
Reporter (e.g. GFP)
Reprogramming
Virus carries
reprogramming factors into somatic cell’s nucleus
Pluripotent iPSC line
Culture as per hESCs Somatic cell is
reprogrammed
Differentiation of Cells I
Precursor cell
Regulatory protein 2
Regulatory protein 3
Regulatory protein 1
Cell A Cell B Cell C Cell D Cell E Cell F Cell G Cell H
Regulatory protein 3
Regulatory protein 3 Regulatory
protein 3
Regulatory protein 2
Cell division
Differentiation of Cells II
Blastocyst Zygote
Gastrula
Germ cells
Sperm Egg
Mesoderm (Middle layer)
Smooth muscle Cardiac muscle Skeletal muscle
cells Red blood cells Tubule cell
of the kidney
Endoderm (Internal layer)
Lung cell
(Alveolar cell) Thyroid cell Pancreatic cell
Ectoderm (External layer)
Skin cells of
epidermis Neuron of brain Pigment cell
