Mesenchymal Stem/Progenitor cells for Therapeutic and Tissue engineering applications Yu-Hui Tsai,...

Mesenchymal Stem/Progenitor cells for Therapeutic and Tissue engineer

ing applications

Yu-Hui Tsai, Ph.D. ( Daniel Tzu-bi Shih)

Grad. Inst. Medical SciencesTaipei Medical University

Stem/Progenitor CellsStem cells• Self Renew (Proliferation)• Muti-differentiation potential (Plasticity) :

Trans-differentiation, Transformation, De-differentiation

Progenitor cells• Functional Undetermined Tissue-cell Precu

sors.• Muti-differentiation potential (Plasticity)

Types of Stem Cells

– ES Cells (ESCs) [Derived from Embryo inner mass]

– EG Cells (EGCs) [Derived from Fetus gonad]

– Somatic Tissue Stem /Progenitor Cells [ Isolated from Fetus, Neo-natal and Adult Tissues] (tSC, tS/PC): e.g. Neural SCs, Mesenchymal Stem/Progenitor Cells (MSC, MSCs/MPCs), Hemapoietic Stem/Progenitor Cells (HSC, HSCs/HPCs), and Karotinocytes etc.

Development of the Human Preimplantation Blastocyst

Development of Human Embryonic Tissues

The Niche The Niche Determines The Fate Determines The Fate

of Stem Cellsof Stem CellsEcologic/Epigenetical Microenvironment of Stem Ce

lls

Tissue Stem Cells (tSCs)

Develop & Maintain within Specific Niche

A. Epidermal---Basal Layer

Hair Folocale & Epidermis--- The bulge (Tcf3)

B. Small Intestine--- The crypt Cells ( Tcf4)

C. Neural Precursors Cells--- Ependymal or the Sub Ventricular Zone Cells (AP)

D. Hematopoietic SCs--- Bone Marrow

Tissue Maintenance and Repair

• I) Tissue Stem/Progenitor Cells

• II) Environmental Cellular Metrix (ECM)

• III) Associate Stroma Cells

• IV) Cytokines

Basic Requirements for Tissue Regeneration

*Tissue Stem/Progenitor Cells

*Stromal Cells for MicroEnvironmental Support of the Tissue Survival and Normal Functioning.

*Cytokines (Autokine & Parakines) & Chemokines

*Oxygen Supply: Red Blood Cells and Neovascularization

*Sensory Nuron: nurogenesis.

Adapted from speech of Dr. Bruce A. Mast, M.D.: The Regulation of Wound Healing. Division of Plastic and Reconstructive Surgery Department of Surgery Univeristy of Florida. (http://www.medinfo.ufl.edu/cme/grounds/mast/index.html).

Wound Healing

CD34+ HS/PCs Functions:

Hematopoietic Reconstitution

Immunotherapy

Neovasculogenesis, Neurogenesis, &Tissue Regenerations etc.

Comparative analysis of the similar stem cell populations from various origins

The Niche of HSCs

Marrow(MSC & ECM)

& Soluble Factors

Tissue Mesenchymal Stem Cells

▪ Connective tissue / stromal source

EX : Bone marrow /Adipose tissue / Skin / Muscle

▪ Umbilical cord blood

Definition:

Sites :

▪Mesenchymal Multi-lineage differentiation potential▪Cell surface antigens expression : SH2/SH4/CD105

Bone Marrow Mesenchymal stem cells ( BM-MSCs )

Multipotent differentiation

Interact & Support tissue cells

(mesoderm-derived tissue cell type) : adipocyte / osteoblast / chondrocyte / myoblast / endothelial cell

(Ectoderm) : Neural cells

Hematopoiesis and angiogenesis

Hypotheses to explain the generation of nonhematopoietic tissues from HSC-enriched populations. Totipotential cells derived from the embryo could persist into adult life and be sequestered in the bone marrow, where they could form a reservoir of precursors from which definitive HSCs could develop. Protocols for the enrichment of HSCs based on their phenotypic and physical properties might also result in the isolation of these totipotent cells, which in turn contribute to skeletal or cardiac muscle or cells of the gastrointestinal tract after various transplantation protocols. Alternatively, precursors whose developmental potential is more limited might also be present in the bone marrow. Finally, a pluripotent HSC (PHSC) may be able to alter its genome and transdifferentiate.

Stem cells find their niche

NATURE|VOL 428 |11 MARCH 2004|

Creation of long-lasting blood vessels

Gene expression profiling of BM-MSCs

Undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages. Tremain,N.2001 / Woodbury,D.2002 / eshi,B.2003

Multipotent :

Growth factors / Matrix proteins / Cell surface molecules :

BM-MSCs expressed various kinds of growth factors,matrix proteins, and cell surface molecules. Silva,W.A.,Jr2003

Adipose tissue Mesenchymal stem cells (AD-MSCs)

• Similar to Bone marrow mesenchymal stem cells

CD marker antigens

Multipotent differentiation ▪▪

A Study on Human Tissue Stem/progenitor cells for Medical Application and Drug Discovery

• Bone Marrow• Placental and

Peripheral Blood• Fat Tissue• Scalp• Amnoitic tissue• Fore Skin• Placenta • Teeth

Stem Cell Isolation and Processing

a. Hematopoietic Stem Cells (HSCs) and Mesodermal Progenitor Cells (MPCs)

MACS

FACS CD34+ cells (HSCs)MNC CD45-GlyA- cells (MPCs)

c. Processed Lipoaspirate (PLA)

Adherent cells -PLA

Cell surface antigens expression

Multi-lineage differentiation abilities

How to distinguish tissue stem cells ?

Hypothesis: Mesenchymal stem cells in different tissues have their own specific potentials in maintenance of their tissue specific niche

Objective : To understand the tissue MSC specificity for better stem cell therapy applications

Is There Specific Niche for Each Type of Tissue Stem/Progenitor Cells in Human?

By Combining Cellular and Molecular Characterizations of MSCs derived from

Different Tissues in Human, ….

How Specific Are Tissue Niche Need?

Strategy :

Comparative analysis gene profiling and functional gene characterization

Highly differential expression genes

Cytokines and growth factors genes

Multi-lineage differentiation related genes

Target gene expression

Early gene expressions

Confirm the protein products

Functional genomic characterization

ostogenic / chondrogenic / adipogenic /myogenic / neurongenic /

endothelial cell differentiation

Part 1. Multi-lineage differentiation related genes

Molecular Analytic results suggest that BM-MSCs preferentially expressed functional genes differences from AD-MSCs in:

•Secretion of Hemagiogenic factors (LIF / Jagged-1/ BMP-4 / HGF / PIGF-1 / PIGF-2

/ HB-EGF ),

•Supply Homing/ mobilzing elements (:CD106(VCAM-1)/ jagged-1/ SDF-1), and

•Generation of ECM remodeling factors (mmp9/mmp1) for HS/PCs (CD34+, CD38-, and CXCR-4+)

Highly differential expression of genes is primarily involved in

Growth factors /Extra cellular matrix molecules /Cell surface molecules /Receptors /

Suggest that the major difference between BM-MSCs and AD-MSCs : cell-cell interactions regulate or support tissue cells

▪

1. The tissue cells homing and mobilization.

CD106 (VCAM-1) / SDF-1 / MMP-9/HGF

Journal of Cellular biochem S 38:29-38 (2002)

BM-MSCs exppressed more key factors involved in Homing and mobilization of hematopoietic stem cells :CD106 / SDF-1 / MMP-9

Hematopoiesis LIF / SDF-1 / BMP-4 / Jagged-1 / HGF

AngiogenesisHGF / PIGF-1 / PIGF-2 / HB-EGF

2. The tissue cells supporting.

3. The ECM remodeling properties

ECM remodeling protease MMP-1 / MMP-9

Our present study indicated the tissue specificity of mesenchymal stem cells exhists as analyzed by molecular characterization of their gene and protein expressions.

In addition to their cell surface phenotypic descriptions and multipotency examinations in vitro, this approach may be indispensable in further realizing the nature of tissue stem cell populations

Conclusions

Thanks for Your Attensions

Proerythroblast

MultipotentStem Cell

Self-renewal ?

Early ActingSCF FL Tpo

G-CSFIL3IL6IL7

IL11

Erythrocyte Platelet Neutrophil Monocyte Eosinophil Basophil

Macrophage Mast Cell

Mega-karyocyte

Myeloblast MonoblastEosinophilicMonoblast

BasophilicMonoblast

CFU-ECFU-G CFU-M CFU-Eo CFU-Baso

BFU-E CFU-Mega CFU-GM

CFU-G, E, M, Mega

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G-CSFGM-CSF

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GM-CSFM-CSF

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The Haematopoietic System

• Humoral Regulation

ModulatingIL1IL4

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CLPsCMPs