7. REGENERATIVE MEDICINE1 AND CELL REPLACEMENT THERAPY2 1 Therapy that enables the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. 2 The prevention, treatment, cure or mitigation of disease or injuries in humans by the administration of autologous, allogeneic or xenogeneic cells that have been manipulated or altered ex vivo. Why do we need it? Aging population Obesity The source#1: EMBRYONIC STEM CELLS Science. 1998 Nov 6;282(5391):1145-7 The source#2: ADULT STEM CELLS Neural stem cell validation single cell cloning NESTIN differentiation oligodendroglia neuron Hematopoietic stem cell validation in irradiated mice trasnplanted with b-gal-labeled NSC (in vitro BM clonogenic assay) NON-TRANSPLANTED TRANSPLANTED original HSC granulocyte/macrophage macrophage The source#3: INDUCIBLE PLURIPOTENT CELLS (iPS) 8h embryo ICM 2d embryo circulation 2006- INDUCIBLE PLURIPOTENT CELLS (iPS) Oct3/4 Sox2 c-Myc Klf4 HOW TO REPAIR BROKEN HEART? infarcted area necrotic muscle (red) extensive fibrosis (pink) v - blood vessel Fish heart regenerates from undifferentiated (de-differentiated?) progenitor cells epicardial tissue (tbx18 and raldh2 – markers of embryonic epicardium) pre-cardiac markers nuclear-dsRed reporter – differentiated high expression, differentiating – low expression. dpa - days after amputation epicardial invasion via EMT Adult rat heart contains resident cardiac stem cells that can be isolated and expanded in vitro…… Lin (none) – blood lineage c-kit+ (green) - stem cells Nkx2.5 (white) – early cardiac sarcomeric actin (red)- cardiac MEF2C (yellow dots)- early cardiac GATA4 (magenta) – early cardiac cardiac myosin (orange) …and used to repair infarcted heart red – cardiac myosin green -PI yellow (D) – connexin 43 yellow (E)- N-cadherin (F) - non-treated tissue (blue – collagen) cell injections Hematopoietic stem cells can regenerate endothelium and cardiac muscle in ischemic heart LacZ-BM Flt-1 (red) ICAM-1 (green) LacZ (blue) transplanted/infarcted control transplanted transplanted/infarcted a-actinin Mammalian heart can regenerate! (at least during its physiological renewal) But does it happen in disease? SKELETAL MYOBLASTS - remain committed to skeletal muscle fate - do not form gap junctions to couple with host myocardium, do not beat in synchrony with the rest of the heart ADULT HEMATOPOIETIC STEM CELLS + differentiate well into endothelial and smooth muscle compartments of the heart veins - differentiate poor into the myocardium - fuse with myocardial cells ENDOTHELIAL PROGENITORS + excellent in infarct revascularisation - poor contribution to myocardium MESENCHYMAL STEM CELLS (BM-derived) - do not fully transdifferentiate to myocardium - do not form connections or contract RESIDENT MYOCARDIAL PROGENITORS + differentiate into cardiomyocytes (partially), smooth muscle cells and endothelia - fuse with cardiomyocytes MOVIE – hESC-derived cardiomyocytes in gelatin cell culture (Histone 2BeGFP) HUMAN EMBRYONIC STEM CELLS + excellent cardiac potential, full functional differentiation into cardiomyocytes + specific differentiation into ventricular, atrial and nodal/pacemaker cells possible - inefficient cardiogenesis - stem cells often carried-over in transplant MDA-MB-231, 9 days of growth Alamar blue conversion for cell proliferation (red line- seeded cells) LLC – Lewis lung carcinoma cells CD45 – hematopoietic CD31- endothelial ConA-rhodamine injection - capillary REGENERATION IN PLANARIANS: Being the simplest animals with bilateral symmetry, planarians are in a constant cell turnover. Their bodies contain up to 20% of so called neoblasts, characterized by the expression of ATP-dependent RNA helicase similar to Drosophila vasa protein. Neoblasts divide and contain the population of totipotent cells that can form all 15 cell types of the planarian tissues. Following transection, there is a muscular contraction limiting the area of the cut followed by the formation of the wound epithelia that makes up regeneration blastema. The blastema enlarges and redifferentiates to form missing structures. The mechanism of a polarity decision, whether to be a head or tail, is poorly understood and does not likely involve the Hox genes. Regeneration of body parts VERTEBRATE LIMB REGENERATION: Among the vertebrates only certain amphibians can regenerate limbs after surgical removal. These include anuran tadpoles that can regenerate limbs before they reach the metamorphosis as well as many urodele species that regenerate limbs during both larval and adult life. After limb amputation, a wound epithelia forms via migration of epidermal cells over the cut surface followed by dedifferentiation of an underlying tissue. The blastema consists of loose-packed mesenchymal cells surrounded by thick epidermal jacket. The blastema proliferates and then the limb structures redifferentiate in the proximal-distal sequence. Can mammals regenerate body parts? (C) Stages of pedicle development 1 - intramembranous ossification 2 - transitional ossification 3 - endochondral ossification 4 - endochondral ossification and skin formation Antler growth from transplanted perichondrium into the metacarpal bone v – velvet skin p –perichondrium m- mesenchyme cp – chondroprogenitor region c- cartilage bo – bone p – periosteum (B) Velvet skin e - epidermis d - dermis h - hair follicle s – gland (C) Fibrous perichondrium arrow – blood vessel (D) Mesenchymal growth zone (E) Chondroprogenitor region (F) non-mineralized cartilage v - blood vessel (G) mineralized cartilage (H) spongy bone