Mesenchymal Stem/Progenitor cells for Therapeutic and Tissue engineering applicationsYu-Hui Tsai, Ph.D.( Daniel Tzu-bi Shih)Grad. Inst. Medical SciencesTaipei Medical University
Stem/Progenitor Cells Stem cells • Self Renew (Proliferation) • Muti-differentiation potential (Plasticity) : Trans-differentiation, Transformation, De-differentiation Progenitor cells • Functional Undetermined Tissue-cell Precusors. • 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.
The Niche Determines The Fate of Stem Cells Ecologic/Epigenetical Microenvironment of Stem Cells
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.
Wound Healing 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).
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 Definition: ▪ Cell surface antigens expression : SH2/SH4/CD105 ▪ Mesenchymal Multi-lineage differentiation potential Sites : ▪ Connective tissue / stromal source EX : Bone marrow /Adipose tissue / Skin / Muscle ▪ Umbilical cord blood
Bone Marrow Mesenchymal stem cells ( BM-MSCs ) Multipotent differentiation (mesoderm-derived tissue cell type) : adipocyte / osteoblast / chondrocyte / myoblast / endothelial cell (Ectoderm): Neural cells Interact & Support tissue 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.
Creation of long-lasting blood vessels NATURE|VOL 428 |11 MARCH 2004|
Gene expression profiling of BM-MSCs Multipotent : Undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages. Tremain,N.2001 / Woodbury,D.2002 / eshi,B.2003 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 CD45-GlyA- cells (MPCs) MNC CD34+ cells (HSCs) FACS
Adherent cells -PLA c. Processed Lipoaspirate (PLA)
Cell surface antigens expression Multi-lineage differentiation abilities How to distinguish tissue stem cells ?
Is There Specific Niche for Each Type of Tissue Stem/Progenitor Cells in Human? Objective :To understand the tissue MSC specificity for better stem cell therapy applications Hypothesis: Mesenchymal stem cells in different tissues have their own specific potentials in maintenance of their tissue specific niche
How Specific Are Tissue Niche Need? By Combining Cellular and Molecular Characterizations of MSCs derived from Different Tissues in Human, ….
Strategy : Comparative analysisgene profiling and functional gene characterization Target gene expression Multi-lineage differentiation related genes Early gene expressions Highly differential expression genes Cytokines and growth factors genes Confirm the protein products
Functional genomic characterization Part 1. Multi-lineage differentiation related genes ostogenic / chondrogenic / adipogenic / myogenic / neurongenic / endothelial cell differentiation
Molecular Analytic results suggest that BM-MSCs preferentially expressed functional genes differences from AD-MSCs in: • Secretion ofHemagiogenic 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
2. The tissue cells supporting. Hematopoiesis LIF / SDF-1 / BMP-4 / Jagged-1 / HGF Angiogenesis HGF / PIGF-1 / PIGF-2 / HB-EGF
3. The ECM remodeling properties ECM remodeling protease MMP-1 / MMP-9
Conclusions 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
The Haematopoietic System • Humoral Regulation Multipotent Stem Cell Modulating IL1 IL4 MIP-1 TNF TGF Early Acting SCF FL Tpo G-CSF IL3 IL6 IL7 IL11 Self-renewal ? CLPs CMPs CFU-G, E, M, Mega CFU-GM BFU-E CFU-Mega SCF IL3 GM-CSF SCF IL3 GM-CSF SCF IL3 IL6 Tpo CFU-G CFU-M CFU-Baso CFU-Eo CFU-E SCF IL3 IL6 G-CSF GM-CSF GM-CSF M-CSF IL3 IL4 GM-CSF IL5 Epo IGF-1 Mega- karyocyte Eosinophilic Monoblast Basophilic Monoblast Proerythroblast Myeloblast Monoblast G-CSF GM-CSF Epo IGF-1 IL3 IL4 IL5 GM-CSF M-CSF Tpo Erythrocyte Neutrophil Monocyte Eosinophil Basophil Platelet IL3 IL4 GM-CSF M-CSF Mast Cell Macrophage