Tissue Engineering. Mesenchymal Stem Cells for. Tissue Engineering

Transcription

1 Tissue Engineering Mesenchymal Stem Cells for Tissue Engineering Reference: Culture of Cells for Tissue Engineering (Culture of Specialized Cells), Chapter 2 Shu-Ping Lin, Ph.D. Date: Institute of Biomedical Engineering splin@dragon.nchu.edu.tw Website:

2 Tissue engineering: Reconstructing biological tissues includes a) cells that fabricate the lost or damaged tissue, b) materials intended to serve as delivery vehicles and scaffolds for the cells, c) cytokines and other bioactive factors consistent with appropriate cell proliferation and differentiation Except differentiated cells, autologous stem cells as the source of donor cells in tissue engineering low donor site morbidity, diminished or absent immune response, high proliferative potential, and relative ease of access to the cell repository Stem cells, capacity to self-renew and to generate differentiated progeny, naturally exist in essentially all tissues (especially those that rapidly proliferate or remodel) and are present in the circulation. There are two predominant lineages of stem cells: Mesenchymal (Bone marrow stromal) give rise to connective tissues (bone, cartilage, etc.) although found in some tissues, typically isolated from bone marrow Hematopoietic Stem Cells give rise to blood cells and lymphocytes isolated from bone marrow, blood (umbilical cord) Stem cells are rare; bone marrow typically has: a single mesenchymal stem cell for every 1,000,000 myeloid cells a single hematopoietic stem cell for every 100,000 myeloid cells

3 History of Stem Cells and What are Mesenchymal Stem Cells (MSC) The presence of nonhematopoietic stem cells in bone marrow was first suggested by the observations of the German pathologist Cohnheim 130 years ago. (First isolated by Friedenstein et al in 1974 Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method Fibroblastoid cells - spindle-shaped) 1976 Stem cells are present in bone marrow was suggested by Friedenstein, based on characterization of clonal populations of marrow stromal cells; and proposed a model of differentiation for progenitor cells in the stromal system analogous to that of the hematopoietic system; multipotential and could differentiate into osteoblasts, chondrocytes, adipocytes, and even myoblasts. Adherent to tissue culture glass or plastic High growth potential

4 Why are Stem Cell Interesting? Rat MSC genetically tagged are microinjected into a mouse blastocyte Blastocyte reimplanted into another rat Blastocyte allowed to develop into baby rat -> sacrificed Gene tags are found in many of the baby rat tissues tested ->Embryonic stem cells? Identification of adult stem cell: (1) label the cells in a living tissue with molecular markers and then determine the specialized cell types they generate; (2) remove the cells from a living animal, label them in cell culture, and transplant them back into another animal to determine whether the cells replace (or "repopulate") their tissue of origin. Demonstrated that a single adult stem cell can generate a line of genetically identical cells that then gives rise to all the appropriate differentiated cell types of the tissue. To confirm experimentally that a putative adult stem cell is indeed a stem cell, scientists tend to show either that the cell can give rise to these genetically identical cells in culture, and/or that a purified population of these candidate stem cells can repopulate or reform the tissue after transplant into an animal.

5 What is Known about Adult Stem Cell Differentiation? Differentiation pathways of adult stem cells: Hematopoietic stem cells: give rise to all the types of blood cells: red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, and macrophages. Mesenchymal stem cells: give rise to a variety of cell types: bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of connective tissue cells such as those in tendons. Neural stem cells: in the brain give rise to its three major cell types: nerve cells (neurons) and two categories of non-neuronal cells astrocytes and oligodendrocytes. Epithelial stem cells: in the lining of the digestive tract occur in deep crypts and give rise to several cell types: absorptive cells, goblet cells, paneth cells, and enteroendocrine cells. Skin stem cells: occur in the basal layer of the epidermis and at the base of hair follicles. The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. The follicular stem cells can give rise to both the hair follicle and to the epidermis.

6 Differentiation of MSC Marrow stem cells Committed progenitors of fibroblastic, reticular, adipogenic, osteogenic, and possibly other cells. Our vision of the lineage pathways available to mesenchymal stem cells was originally depicted in the mesengenic process diagram.

7 How to Identify MSC? In vivo ceramic cube assay, described in Section 6, is the standard for identifying MSCs. Human MSC-ceramic composites implanted subcutaneously in immunocompromised host animals almost always produce bone, whereas bone and cartilage are never present when dermal fibroblasts are used in the same manner marrow-derived MSCs from different species produce different proportions of bone and cartilage. Cartilage, almost never seen in human MSC ceramic composites, is frequently observed with rat MSCs, but much less so than in vitro assays, each of which is specific for different differentiated cells. Morphology Adherent to glass or plastic What cell markers are relevant to identifying MSCs? Surface markers: SH2, SH3, SH4, STRO-1, ICAM-2, NCAM, integrins, PDGF, IL-IR Capable to be induced in vitro to differentiate into osteoblasts, chondrocytes, and adipocytes

8 Assays for Phenotypic Potencies What assays are relevant to identifying MSCs? Elevated alkaline phosphatase activity, although not unique to osteogenic cells, is a useful early marker for osteogenic differentiation. Accordingly, biochemical and cytochemical assays for alkaline phosphatase are used early in the culture of MSCs in osteoinductive conditions. Calcium biochemistry and von Kossa staining to detect mineralization are used later in the culture period. These assays and the conditions that promote osteogenic differentiation are described in Section 7. Aggregate or pellet cultures can be established in a defined medium to promote chondrogenic differentiation of MSCs [Johnstone et al., 1998]. Such cultures will not be covered in this section as they are treated thoroughly in Chapter 4 of this book. Differentiation of MSCs to adipocytes can be induced through the use of a unique medium in cultures seeded at a slightly higher density than that used for osteogenic differentiation. Flow cytometric analysis after Nile Red staining is used to assess expression of the adipocytic phenotype after induction. This methodology is described in Section 8

9 Assays for Phenotypic Potencies What cell markers are relevant to identifying MSCs? Stem cells are identified by the expression of specific antigens on their surface, for example: Hematopoietic stem cells express CD45, CD34 and CD14 Mesenchymal stem cells do not express these markers (i.e. CD34 -, CD45 -, CD14 - ) Selective separation of positive marker cells (in a mixed cell population) can be done by several techniques (e.g. immunomagnetic methods). Characterization and Commitment: The most common approach to characterize multi-lineage- or single lineage-committed stem cells is through colony-forming assays: Cells grown under culture conditions that promote their proliferation and differentiation Clonal progeny of a single progenitor cell stay together to form a new colony of mature cells Colony-forming assays are used to: characterize stem cells from different sources (e.g. BM, umbilical cord blood) investigate responses to growth factors, cytokines and other drugs expansion, commitment, etc. quality control for collection, processing and cryopreservation

10 Key Technical Details Selection of the proper lot of fetal bovine serum (FBS) for use in culturing MSCs is perhaps the most important parameter in MSC technology and is described briefly in Section 9. It has been our experience that it is Not good practice to purchase serum off the shelf (that is, without testing). We have also found that a batch of serum optimal for MSCs from one species will probably not support proliferation of MSCs from another. Although the selection of the proper batch of FBS is extremely important, all of the technical details involved in the isolation, expansion, and analysis of MSCs are also important.

11 Isolate MSC Sources: BM, periosteum, synovium, fat, cord blood, peripheral blood, foetal liver and lung Adherent to culture bottle; haemopoietic stem cells do not Easily subcultured

12 Preparation of Media and Reagents

13 Preparation of Solutions -1

14 Preparation of Solutions -2

15 Isolation of Human Marrow-Derived Mesenchymal Stem Cells (HMSCS) Aspiration of Human Bone Marrow: Aspiration of bone marrow from the posterior superior iliac crest receive the bone marrow sample in a 20-ml syringe and proceed to the steps listed in Protocol v=_eysgvbcxz8&nr=1 v=waycgpmfuow&playnext=1&l ist=plf33aeb6529ee64f9

16 Enrichment of Mesenchymal Stem Cells from Human Marrow Cell fraction enriched for mesenchymal stem cells is isolated by density gradient centrifugation.

17 Primary Culture of MSC from Bone Marrow High density ( nucleated cells per 10-cm dish), the total cell inoculum A small fraction of erythrocytes, many nucleated cells, probably from the hematopoietic lineage, that are not capable of attaching to the culture substrate. change medium smaller subset of cells becomes anchored to the substrate (attanegatively charged culture dish) primarily fibroblast-like cells begin to proliferate and form colonies - fibroblastoid cells, which can first be seen around days 4 6 of culture (ability of these cells to attach to the culture substrate represents the first and most important step in the selection of MSCs from the total nucleated cell population. Because of variability among individual marrow donors, the number of colonies per dish may vary; in our experience a typical 10-cm dish may contain 100 to 200 colonies. Colonies increase in size next 7 days and should be subcultured before the cells become dense and multilayered (proliferation of the cells is not contact inhibited). Colony of these cells arises from a single attached cell, we would conclude that these cells are rare indeed (1 2 per 10 5 nucleated cells from the marrow of young donors).

18 My Sister's Keeper Anna Fitzgerald, (a genetic match for her older sister, Kate, who suffers from acute promyelocytic leukemia) was born in order to keep her alive, looks to earn medical emancipation from her parents who until now have relied on their youngest child to help their leukemia-stricken daughter Kate remain alive. Anna and Kate's older brother, Jesse reveal that Anna's actions are actually under Kate's instruction; Kate, not wanting to live any longer, and knowing Sara refuses to hear any option other than her desired one, had gently persuaded Anna to refuse to donate her kidney.

19 In vitro MSC growth patterndoubling time 33hr (Conget et al 1999)