Thesis section: Restorative neurology


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Thesis section: Restorative neurology

  1. 1. Restorative neurology Essay In Neuropsychiatry Submitted for partial fulfillment of Master Degree By Samy moussa Seliem M.B.B.CH Supervisors ofProf. Mohammed Yasser Metwally Professor of Neuropsychiatry Faculty of Medicine-Ain Shams University www.yassermetwally.comProf. Naglaa Mohamed Elkhayat Professor of Neuropsychiatry Faculty of Medicine-Ain Shams University Dr. Haitham Hamdy salem Lecturer of Neuropsychiatry Faculty of Medicine-Ain Shams University Faculty of Medicine Ain Shams University 2011 i
  2. 2. ContentsSubject1. Introduction and aim of the work2. Stem cell3. Stem cell therapy in Parkinson disease4. Stem cell therapy in in stroke5. Stem cell therapy in demyelinating disease6. Stem cell therapy in in amyotrophic lateral sclerosis7. Stem cell therapy in muscular dystrophy8. Stem cell therapy Huntington chorea9. Stem cell therapy for Alzheimers Disease10.Stem cell therapy in degenerative diseases in children11.Stem cell therapy in retinal degeneration12.Stem cell therapy in spinal cord injury13.Stem cell therapy in peripheral nerve injurySummaryReferences i
  3. 3. Introduction Stem cells are unspecialized cells in the human body that arecapable of becoming specialized cells, each with newspecialized cell functions. The best example of a stem cell is thebone marrow stem cell that is unspecialized and able tospecialize into blood cells, such as white blood cells and redblood cells, and these new cell types have special functions,such as being able to produce antibodies, act as scavengers tocombat infection and transport gases. Thus one cell type stemsfrom the other and hence the term “stem cell.” Basically, a stemcell remains uncommitted until it receives a signal to developinto a specialized cell. Stem cells have the remarkable propertiesof developing into a variety of cell types in the human body.They serve as a repair system by being able to divide withoutlimit to replenish other cells. When stem cell divides, each newcell has the potential to either remain as a stem cell or becomeanother cell type with new special functions, such as blood cells,brain cells, etc. (Bongso and Lee, 2005). Stem cells also known as progenitor cells which are cellsthat have not undergone differentiation to acquire specificstructure or role. They have the potential to self-renew, divideand differentiate into specialized cell types. They are also,sometimes, termed ‘pluripotent’ or ‘undifferentiated’ cells i
  4. 4. because they can differentiate and develop into various cell lines(Metwally, 2009). Scientists and researchers are interested in stem cells forseveral reasons. Although stem cells do not serve any onefunction, many have the capacity to serve any function afterthey are instructed to specialize. Every cell in the body, forexample, is derived from first few stem cells formed in the earlystages of embryological development. Therefore, stem cellsextracted from embryos can be induced to become any desiredcell type. This property makes stem cells powerful enough toregenerate damaged tissue under the right conditions (Crosta,2010). Perhaps, the most important reason that stem celldevelopment is so appealing to neurologists can be found in thestatement “The adult human brain, in contrast to other organssuch as skin and liver, lacked the capacity for self repair andregeneration” (Lin et al., 2007). The types of stem cells include: Bone marrow-derivedmesenchymal stem cells (BMSCs), embryonic stem cells(ESCs), Adult (somatic) stem cells, and neural stem cells(NSCs). BMSCs also termed bone marrow stromal cells areanother example of a somatic stem cell being studied for itstherapeutic potential in the central nervous system (CNS) and inother tissue (Abdallah and Kassem, 2008). ii
  5. 5. BMSCS generate neurotransmitter-responsive cells withelectro-physiological properties similar to neurons (Diana andGabriel, 2008). ESCs are pluripotent cells isolated from the inner cell massof day 5-8 blastocyte with indefinite self-renewal capabilities aswell as of the ability to differentiate into all cell types derivedfrom the three embryonic germ layers. The primary therapeuticgoal of ESCs research is cell replacement therapy (Aoki et al.,2007). Adult (somatic) stem cells: it has a capacity to differentiateinto tissue-specific types and represent a potential source ofautologus cells for transplantation therapy that eliminateimmunological complications associated with allogenic donorcells as well as bypass ethical concern associated with ESCs,All types are generally characterized by their potency, orpotential to differentiate into different cell types (such as skin,muscle, bone, etc) (Lin et al., 2007). Scientists discovered ways to obtain or derive stem cellsfrom early mouse embryos more than 20 years ago. Many yearsof detailed study of biology of mouse stem cells led to thediscovery, in 1998, of how to isolate stem cells from humanembryos and grow the cells in the laboratory. These are calledhuman embryonic stem cells. The embryos used in these studieswere created for infertility purposes through in vitro fertilization iii
  6. 6. procedures and when they were no longer needed for thatpurpose, they were donated for research with the informedconsent of the donor (Ordrico et al., 2001). The concept that the adult mammalian CNS contains NSCswas first discovered from evidence of neuronal turnover in theolfactory bulb and hippocampus in the adult organism cells withmore restricted neural differentiation capabilities committed tospecific subpopulation lineage, have been generated fromhuman ESCs or directly isolated from neurogenic regions offetal and adult CNS, such as the subventricular zone, whichprovides neuroblasts to replenish inhibitory interneurons in theolfactory bulb (Lin et al., 2007). Stem cell differentiation must be turned on, given direction,and turned off as needed in order to properly supply the basicbuilding blocks of tissues in different organ systems. Thisrequirement for precise regulation applies to an even greaterdegree to the differentiation of neuronal progenitor cells,because effective neural function depends on establishingprecise linkage and interactions between different individualneurons and classes of neurons (Metwally, 2009). Most tissue repair events in mammals are dedifferentiationindependent events brought about by the activation of pre-existing stem cells or progenitor cells. By definition, aprogenitor cell lies in between a stem cell and a terminallydifferentiated cell (Crosta, 2010). iv
  7. 7. With the therapeutic application of NSCs forneurorestoration in mind, a clearer picture is emerging. Both innormal neurodevelopment and stem cell biology, the precursorcells display preprogrammed behavior modified by cues fromthe local environment. The fundamental assumption is thatdifferentiation and predictable behavior of NSCs can beachieved if the appropriate cocktail of soluble/diffusible orcontact-mediated signals is present. In addition, severalcorollary considerations are quickly evident. For example, canwe use NSCs from different sources in an equivalent fashion?The answer to this important question requires that weunderstand the developmental potential of all the types of NSCs(Marquez et al., 2005). Medical researchers believe that stem cell therapy has thepotential to dramatically change the treatment of human disease.A number of adult stem cell therapies already exist, particularlybone marrow transplants that are used to treat leukemia. In thefuture, medical researchers anticipate being able to usetechnologies derived from stem cell research to treat a widervariety of diseases including cancer, Parkinsons disease, spinalcord injuries, Amyotrophic lateral sclerosis, multiple sclerosis,and muscle damage, amongst a number of other impairmentsand conditions (Goldman and Windrem, 2006). v
  8. 8. Aim of the work: The aim of this work is to study and summarize recentprogress in stem cell therapies aimed at neurodegenerativedisorder and illustrate how some of aforementioned methodsand strategies are being utilized to formulate clinically viabletreatments. vi
  9. 9. Stem cellsDefinition: A stem cell is a cell that has the ability to divide(self replicate) for indefinite periods, often throughout the life ofthe organism. Under the right conditions, or given the rightsignals, stem cells can give rise (differentiate) to the manydifferent cell types that make up the organism. That is, stemcells have the potential to develop into mature cells that havecharacteristic shapes and specialized functions, such as heartcells, skin cells, or nerve cells (Charron et al., 2009). The word “stem” actually originated from old botanicalmonographs from the same terminology as the stems of plants,where stem cells were demonstrated in the apical root and shootmeristems that were responsible for the regenerativecompetence of plants. Hence also the use of word “stem” in“meristem” (Kiessling and Anderson, 2003).Historical overview of stem cell therapy: The stem cell is the origin of life. As stated first by the greatpathologist (Rudolph Virchow), “All cells come from cells”.The fertilized egg is formed from fusion of the haploid progenyof germinal stem cells. The fertilized egg is totipotent; from itforms all the tissues of the developing embryo. Duringdevelopment of the embryo, germinal stem cells are formed,which persist in adult to allow the cycle of life to continue. In 1
  10. 10. the adult, tissue is renewed by proliferation of specialized stemcells, which divide to form one cell that remains a stem cell andanother cell that begins the process of differentiation tospecialized function of a mature cell type, normal tissue renewalis accomplished by the differentiating progeny of stem cells, theso-called transit amplifying cells. For example, blood cells aremature cells derived from hematopoietic stem cells in the bonemarrow; the lining cells of the gastrointestinal tract are formedfrom transit amplifying cells, progeny of stem cell in the base ofintestinal glands (Crosta, 2010). Nineteenth century pathologists first hypothesized thepresence of stem cells in the adult as “embryonal rests” toexplain the cellular origin of cancer and the studies indicate thatthe most cancers arise from stem cells or their immediateprogeny, the transit-amplifying cells. Cancer results from animbalance between the rate at which cells are produced and therate at which they terminally differentiate or die. Understandinghow to control the proliferation and differentiation of stem cellsand their progeny is not only the key to controlling and treatingcancer, but also to cell replacement and gene therapy for manymetabolic, degenerative, and immunological diseases (Virchow,1985).Stem cell properties: Stem cells have a capacity for self-renewal giving rise tomore stem cells, and the ability to differentiate into tissues of 2
  11. 11. various lineages under appropriate conditions. They may betotipotent, pluripotent or multipotent, depending on type. Onlythe embryo is totipotent. Embryonic stem cells (ESCs) arepluripotent, as they are capable of differentiating into manytissue types, whereas differentiation of adult stem cells isgenerally restricted to the tissue in which they reside, as withhepatocytes in the liver, and haemopoietic stem cells in blood(figure 1) (Bongso and Lee, 2005). Figure (1): Stem cell self-renewal and differentiation (Bongso andLee, 2005). 3
  12. 12. A) Stem cell self renewal: The defining feature of a true stem cell is the capacity forself-renewal. Self renewal occurs when a cell that has beenactivated to divide does so asymmetrically. The result producesone cell that is exactly like the mother cell and one cell thattakes on biological functions that are different from those of themother cell. Without self-renewal, each activation event wouldresult in the progressive loss of the originating stem cellpopulation (Andeson et al., 2001).B) The stem cell life cycle: Stem cell activation is generally followed by a clonalexpansion of the daughter cell that is produced. This isassociated with a series of biological processes that includeproliferation, migration, differentiation, and at some point celldeath. Regulation of these downstream events determines thenet effect that, each stem cell activation has on new tissueformation (Song et al., 2007). C) Stem cell plasticity: The term plasticity means that a stem cell from one adulttissue can generate the differentiated cell types of another tissue.At this time, there is no formally accepted name for thisphenomenon in the scientific literature. It is variously referredto as “plastisity” “unorthodox differentition” or“transdifferentiation” (figure 2) (Joanna et al., 2009). 4
  13. 13. To show that the adult stem cells can generate other celltypes requires them to be tracked in their new environment,whether it is in vitro or in vivo. In general, this has beenaccomplished by obtaining the stem cells from a mouse that hasbeen genetically engineered to express a molecular tag in all itscells. It is then necessary to show that the labeled adult stemcells have adopted key structural and biochemicalcharacteristics of the new tissue they are claimed to havegenerated (Gussoni et al., 2002). Also it is necessary to demonstrate that the cells canintegrate into their new tissue environment, survive in the tissue,and function like the mature cells may assume the characteristicof cells that have developed from the same primary germ layeror a different germ layer, for example, much plasticityexperiments involve stem cells derived from bone marrow,which is a mesodermal derivative. The bone marrow stem cellsmay then differentiate into another mesodermally derived tissuesuch as skeletal muscle, cardiac muscle or liver (Kocher et al.,2001). Stem cell lineage differentiation and commitment isconventionally viewed as progressively downstream, unidirectional and irreversible. Thenotion of unidirectional tissue-lineage commitment of stem cellsis being challenged by evidence of plasticity, or lineageconversion, in adult stem cells. Mechanisms allowing for such plasticity include trans- differentiationwhich describes the conversion of a cell of one 5
  14. 14. tissue lineage into a cell of an entirely distinct lineage, withconcomitant loss of the tissue-specific markers and function ofthe original cell type, and acquisition of markers and function ofthe trans-differentiated cell type (Bianco et al., 2005). Alternatively, adult stem cell may differentiate into a tissuethat, during normal embryonic development, would arise from adifferent germ layer. For example, bone marrow derived cellsmay differentiate into neural tissue, which is derived fromembryonic ectoderm and neural stem cell lines cultured fromadult brain tissue may differentiate to form hematopoietic cells, Figure (2): Evidence of plasticity of stem cell (Joanna et al., 2009). 6
  15. 15. or even give rise to many different cell types in embryo. Inboth cases cited above, the cells would be deemed to showplasticity, but in the case of bone-marrow stem cells generatingbrain cells, the finding is less predictable (Song et al., 2007). Alternative mechanisms for explaining apparent stem cellplasticity involve cell-cell fusion between a stem cell and atissue specific cell, the existence of multiple stem cellpopulations in one pool of cells, and the ability of the stem cellsto differentiate to a more primitive, less specialized cell lineage,and then re-differentiate down another lineage (Bongso andLee, 2005).The differentiation potential of stem cells: Many of the terms used to define stem cells depend on thebehavior of the cells in the intact organism (in vivo), underspecific laboratory conditions (in vitro), or after transplantationin vivo, often to a tissue that is different from the one fromwhich the stem cells were derived (Joanna et al., 2009). So they are three classes of stem cells exist: totipotent,pluripotent multipotent and unipotent.1) Totipotent: Totipotency is the ability of a cell to divide and produce allof the undifferentiated cells within an organism, from the Latinword totus, meaning entire; For example, the fertilized egg issaid to be totipotent, because it has the potential to generate allthe cells and tissues that make up an embryo and that support itsdevelopment in uterus. After fertilization, the cell begins to 7
  16. 16. divide and produce other totipotent cells; these totipotent cellsbegin to specialize within a few days after fertilization. Thetotipotent cells specialize into pluripotent cells, which theydevelop into the tissues of the developing body. Pluripotentcells can further divide and specialize into multipotent cells,which produce cells of a particular function (Svendsen andEbert, 2008). Adult mammals, including humans, consist of more than 200kinds of cells. These include nerve cells (neurons), muscle cells(myocytes), skin (epithelial) cells, blood cells (erythrocytes,monocytes, lymphocytes, etc.), bone cells (osteocytes) andcartilage cells (chondrocytes). Other cells, which are essentialfor embryonic development but are not incorporated into thebody of the embryo, include the extraembryonic tissues,placenta, and umbilical cord. All of these cells are generatedfrom a single, totipotent cell, the zygote or fertilized egg(Joanna et al., 2009).2) Pluripotent: Pluripotent stem cells can give rise to any type of cell in thebody except those needed to develop a fetus or adult becausethey lack the potential to support the extraembryonic tissue(e.g., the placenta). Most scientist use the term pluripotent todescribe stem cells that can give rise to cells derived from allthree embryonic germ layers (endoderm, mesoderm, and 8
  17. 17. ectoderm). These three germ layers are the embryonic source ofall cells of the body (figure 3) (Svendsen and Ebert, 2008). Figure (3): Pluripotent stem cells (Svendsen and Ebert, 2008). The term “pluri” is derived from the Latin word plures,means several or many. Thus, pluripotent cells have thepotential to give rise to any type of cell, a property observed inthe natural course of embryonic development and under certainlaboratory conditions. Pluripotent stem cells are isolated fromembryos that are only several days old; cells from these stem 9
  18. 18. cell lines can be cultured in the lab and grown without limit(Sonja et al., 2006).3) Multipotent: Multipotent cells, in contrast, can only give rise to a smallnumber of cell types and they can produce only cells of aclosely related family cell. As haematopiotic stem cells thatdifferentiate to red blood cells, white blood cells and platelets.A hematopoietic cell, or a blood stem cell, can develop intoseveral types of blood cells but cannot develop into liver cells orother types of cells; the differentiation of the cell is limited inscope. A multipotent blood cell can produce red and whiteblood cells (figure 4) (Svendsen and Ebert, 2008). Figure (4): Multipotent stem cell (Svendsen and Ebert, 2008). 10
  19. 19. 4) Unipotent: Unipotent stem cells, a term that is usually applied to a cellin adult organisms, means that the cells in question are capableof differentiating along only one lineage. The term “uni” isderived from the Latin word unus, which means one. Also, itmay be that the adult stem cells in many differentiated,undamaged tissues are typically unipotent and give rise to justone cell type under normal conditions. This process wouldallow for a steady state of self renewal for the tissue. However,if the tissue becomes damaged and the replacement of multiplecell types is required, pluripotent stem cells may becomeactivated to repair the damage (Avasthe et al., 2008). F igu re (5): Differentiation of human stem cells (Bongso and Lee,2005). 11
  20. 20. Classification of stem cells according to their sources: Stem cells can be classified into four broad types based ontheir origin, stem cells from embryos; stem cells from the fetus;stem cells from umbilical cord; and stem cells from the adult.Each of these can be grouped into subtypes (Andeson et al.,2001).1) Embryonic stem cells: In mammals; the fertilized oocyte, zygote, 2-cells, 4-cells, 8-cells and morula resulting from cleavage of the early embryoare examples of totipotent cells (ability to form a completeorganism) (figure 6) (Avasthe et al., 2008). 12
  21. 21. Figure (6): Development and differentiation of human tissues (Avasthe et al., 2008). The inner cell mass (ICM) of the 5 to 6 days old humanblastocyte is the source of pluripotent embryonic stem cells(HESCs) and consisting of 50–150 cells (figure 7) (Bongso andLee, 2005). 13
  22. 22. Figure (7): Human blastocystshowing inner cell mass andtrophectoderm (Bongso and Lee,2005). 14
  23. 23. Figure (8): How human embryonic stem cells are derived? (Bongsoand Lee, 2005).Characteristics of human embryonic stem cells: They can maintain undifferentiated phenotype and thesecells are able to renew themselves continuously through manypassages leading to the claim that they are immortal, also these 15
  24. 24. cells are pluripotent, meaning that they are able to create allthree germ layers of the developing embryo and thus they candevelop into each of the more than 200 cell types of the adultbody (figure 9) (Junying et al., 2006). Figure (9): Characteristics of embryonic stem cells (Junying et al.,2006). 16
  25. 25. Nearly all research to date has taken place using mouseembryonic stem cells (MES) or Human embryonic stem cells(HESCs). Both have the essential stem cell characteristics, yetthey require very different environments in order to maintain anundifferentiated state. Mouse ES cells are grown on a layer ofgelatin and require the presence of Leukemia Inhibitory Factor(LIF) (Bongso and Lee, 2005). HESCs are grown on a feeder layer of mouse embryonicfibroblasts (MEFs) and require the presence of basic FibroblastGrowth Factor (bFGF or FGF-2). Without optimal cultureconditions or genetic manipulation, embryonic stem cells willrapidly differentiate (Avasthe et al., 2008).Identification of the human embryonic stem cells: Laboratories that grow human embryonic stem cell lines useseveral kinds of tests to identify the human embryonic stemcells.These tests include: 1- Growing and sub-culturing the stem cells for many months. This ensures that the cells are capable of long term self-renewal. Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated (Lawrence et al., 2006). 2- Using specific techniques to determine the presence of surface markers that are found only on undifferentiated cells. Another important test is for the presence of a 17
  26. 26. protein called oct-4, which undifferentiated cells typically make. Oct-4 is a transcription factor, meaning that it helps turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development. 3- Examining the chromosomes under a microscope. This is a method to assess whether the chromosomes are damaged or if the number of chromosomes has changed. It does not detect genetic mutations in the cells. 4- Determining whether the cells can be subculture after freezing, thawing, replanting (junying et al., 2006).Differentiation of human embryonic stem cells: In order to start differentiation, the HESCs must be removedfrom the feeder layer and the cell replated and will formembryoid bodies (Ebs), spherical aggregates in which theHESCs undergo mixed spontaneous differentiation towardlineages of all three dermal layers. Another protocol ofdifferentiation directly without formation of embryoid bodiesstage have resulted in more controlled differentiation and betteryield of the required cells (figure 10) (Joanna et al., 2009). 18
  27. 27. Figure (10): Fluorescent markers can be used to identify stem cells hiddenamong ordinary adult cells. Here, human embryonic stem cells arerecognized by the marker proteins they express (green) (Joanna et al.,2009).Ethical considerations: The promise of stem cell therapy has ignited public disputeon the ethics of using aborted embryos for medical purposes.Individual attitudes are usually influenced by religious andliberal views but also by concerns that the practice of embryonictissue transplantation will increase the pressure to performabortions and create a black market in which pregnancy andaborted tissues will be sold to the highest bidder. The regulatedbanking of stem cell lines may solve some of the ethical issues.As in other cases in which medical and scientific advancesfound society without the means to deal with their ethical, legal,and social consequences, it is important to discuss these issuesin public, with the active participation of the medical andscientific community (Christopher, 2008). 19
  28. 28. 2) Fetal stem cells: The identification of human fetal stem cells has raised thepossibility of using autologus cells for in utero treatments. Thehuman fetal stem cells population extracted from fetal bloodcontains adherent cells that divide in culture for 20 to 40passages and can differentiate into mesenchymal lineagesincluding bone and cartilage, but also have the ability to formoligodendrocytes and hematopoiotic cells. These cells, whichcan be found circulating only during the first trimester, aresimilar to hematopoiotic populations in fetal liver and bonemarrow (Avasthe et al., 2008).3) Umbilical cord stem cells: These are cells harvested from the cord blood. Cord blood isrich in the stem cells and after appropriate human leukocyteantigen [HLA] matching may be used to treat a variety ofconditions. Characteristics of these cells are identical to adultstem cells except that they are not derived from adults and thattheir concentration is far more in umbilical blood as comparedto adults. The use of umbilical cord stem cells in orthopedics isstill in a nascent stage and most studies currently focus on theuse of the stem cell (Crosta, 2010).4) Adult stem cell: It is an undifferentiated cell that is found in a differentiatedtissue, it can renew itself and become specialized to yield all the 20
  29. 29. specialized cell types of the tissue from which it originated.Adult stem cells, like all stem cells, share at least twocharacteristics. First, they can make identical copies ofthemselves for long period of time; this ability to proliferate isreferred to as long term self renewal. Second, they can give riseto mature cell types that have characteristic morphologies(shapes) and specialized functions (Charron et al., 2009). Typically, stem cells generate an intermediate cell type ortypes before they achieve their fully differentiated state. Theintermediate cell is called a precursor cells in fetal or adulttissues are partially differentiated cells that divide and give riseto differentiated cells. Such cells are usually regarded as“committed” to differentiating along a particular cellulardevelopment pathway, although this characteristic may not be asdefinitive as once thought (Bianco et al., 2005). Adult stem cells are rare. Their primary functions are tomaintain the steady state functioning of a cell, called(homeostasis) and with limitation to replace cells that die due toinjury or disease. For example, only an estimated 1 in 10,000 to15,000 cells in the bone marrow is a hematopoietic (blood-forming) stem cell (HSC). Furthermore, adult stem cells aredispersed in tissues throughout the nature of animal and behavevery differently, depending on their local environment. Forexample, HSCs are constantly being generated in the bonemarrow where they differentiate into mature types of blood 21
  30. 30. cells. Indeed, the primary role of HSCs is to replace blood cells(Abdallah and Kassem 2008). Unlike embryonic stem cells, which are defined by theirorigin (the inner cell mass of the blastocyte), adult stem cellsshare no such definitive means of characterization. In fact, noone knows the origin of adult stem cells in any mature tissue.Some have proposed that stem cells are somehow set asideduring fetal development and restrained from differentiating.Definition of adult stem cells vary in the scientific literaturerange from a simple description of the cells to a rigorous set ofexperimental criteria that must be met before characterizing aparticular cell as an adult stem cell. Most of the informationabout adult stem cells comes from studies of mice. The list ofadult tissues reported to contain stem cells is growing andincludes bone marrow, peripheral blood, brain, spinal cord,dental pulp, blood vessels, skeletal muscle, epithelia of skin anddigestive system, cornea, retina, liver, and pancreas(Christopher, 2008). Ideally, adult stem cells should also be clonogenic. In otherwords, a single adult stem cell should be able to generate a lineof genetically identical cells, which then gives rise to all theappropriate, differentiated cell types of the tissue in which itresides. Again, this property is difficult to demonstrate in vivo;in practice, scientists show either that a stem cell is clonogenicin vitro, or that a purified population of candidate stem cells canrepopulate the tissue (Avasthe et al., 2008). 22
  31. 31. Sources of adult stem cells:(I) Bone Marrow-Derived Stem/Progenitor Cells: Adult bone marrow-derived stem cells are presently the celltypes most widely used in stem cell therapy. A heterogeneoussubset there of, termed autologous bone marrow-derivedmononuclear cells (ABMMNCs), comprises the following typesof stem cells, (Mesenchymal stem cells, Hematopoietic stemcells and Endothelial progenitor cells), that have potentialtherapeutic uses (figure 11) (Svendsen and Ebert, 2008).Figure (11): Some of the known sources of adult stem cells (Svendsen and Ebert, 2008). 23
  32. 32. (a) Mesenchymal stem cells (MSCs): MSCs are a proper stem cell which can be greatly andefficiently expanded in culture and can differentiate to severalspecific mesenchymal cell lineages. Mesenchymal (Stromal)stem cells (MSCs) are found in various niches of adult tissue.MSCs are rare in bone marrow (<0.01% of nucleated cells, bysome estimates) and 10 times less abundant than hematopoieticprogenitor cells but MSCs can be readily grown in culture.However, more recently, other sources of MSCs have beendescribed including placenta, adipose tissue, cord blood andliver (junying et al., 2006). The human Mesenchymal stem cells (HMSCs) from bonemarrow can be cloned and expanded in vitro more than 1million-fold and retain the ability to differentiate to severalmesenchymal lineages. Researchers have not yet foundconditions that allow continuous, indefinite HMSC growth, yetit is possible to produce billions of MSCs in vitro for cellulartherapy from a modest bone marrow aspirate drawn through theskin. MSCs need to be expanded ex vivo because theyapparently are very contact inhibited, and there is little evidenceof in vivo expansion as MSCs labeled with membrane dyes, thatwould be diluted and undetected from dividing cells after about3 divisions, are found months later even in repairing tissue(Sottile et al., 2002). 24
  33. 33. Advantages of Mesenchymal Stem Cells: Ease of isolation, high expansion potential, genetic stability,reproducible characteristics in widely dispersed laboratories,compatibility with tissue engineering principles and potential toenhance repair in many vital tissues. There they may be thecurrent preferred stem cells model for cellular therapeuticdevelopment (Diana and Gabriel, 2008).Biology of mesenchymal stem cells (MSCs): The anatomical locations of phenotype of MSCs have no yetbeen well defined in vivo. Some have used expression of Stro-1and VCAM-1 to analyses putative MSC in vivo in human. Ageneral consensus among researchers in the field is that MSCcan be successfully defined based on staining with surfacemarkers such as CD44, CD90, CD73, CD105 and CD166.However, none of these antigens are unique to MSC. Usingmarkers such as Stro-1 (human) and Sca-1 (mouse), severalreports indicate that MSC reside adjacent to endothelium in thebone marrow and possibly other tissues (Zannettino et al.,2007).(b) Hematopoietic stem cells (HSCs): HSCs are presented in umbilical cord blood with a frequencyof just under one in 1 million mononuclear cells (one in 3million MNCs) or mobilized peripheral blood (one in 6 millionMNCs). They are capable of unlimited cell proliferation in bonemarrow and must undergo at least 20 to 23 divisions on their 25
  34. 34. way to produce mature blood cells, even assuming no cell deathalong the way (Emerson et al., 2008).Biology of heamtopiotic stem cells: Much effort has been focused on discovering cell surfacemarkers that can identify those cells that have true functionalstem cell properties. Perhaps clinically most familiar is CD34, aglycoprotein present on the cell surface of stem and progenitorcells which is used to enrich stem cells mobilization andcollection for HSCs, but even within the CD34+ population,only a small percentage are HSCs (Emerson et al., 2008). For decade scientists and hematologists have struggled withthe difficulty that HSCs cannot be purified based onphenotypical characteristics and perhaps more importantly,cannot be expanded and cloned ex vivo. Recent evidence hasemerged suggesting that HSCs can be expanded ex vivo. Butthere is still no evidence to support the idea of clonality. Forthese reasons HSCs are not ideally suited for in vitroexperiments designed to test plasticity. In this regard HSCsdiffer dramatically from MSCs in bone marrow and neural stemcells (NSCs) in the central nervous system, both of which canbe clonally derived and tested for multiple differentiationpathways (figure 12) (Joanna et., 2009). 26
  35. 35. Figure (12): Hematopoietic and stromal stem cell differentiation (Joanna et al., 2009). (c) Endothelial progenitor cells (EPCs): A subset of bone marrow-derived hematopoietic progenitor cells: endothelial progenitor cells (EPCs). These cells can give rise to endothelial recovery and new capillary formation after ischemia (Einstein and Ben-Hur, 2008). (II) Neural stem cells: The concept that adult mammalian CNS contains NSCs was first inferred from evidence of neuronal turnover in olfactory 27
  36. 36. bulb and hippocampus in the adult organism. The multipotencyof NSCs was demonstrated in vitro in 1990 by their ability todifferentiate into neurons, astrocytes, and oligodendrocytes aswell as various forms of neural precursors. In addition, in vivodelivery of these cells to animal models of neurodegenerativediseases was associated with varying degrees of functionalrecovery. Currently, there is no set of markers or proteinexpression profiles that precisely define and fully characterizeundifferentiated NSCs. Neural stem cells (NSCs) and neuralprecursor cells (NPCs) can be isolated from the developing oradult CNS and can be safely expanded in chemically definedculture media for an extended (Song et al., 2007).(a) Adult neural precursor cells (NPCs): New neurons are derived in adulthood from a population ofadult NPCs, which are primarily found in the subependymallayer of the ventricular zone and the dentate gyrus of thehippocampus, although they are also probably found in othersites. However, the behavior of the neural precursor cells(NPCs) found in all these sites is different, and may relate asmuch to the environment in which they find themselves as totheir intrinsic properties, eg; nigral NPCs appear to onlydifferentiate into astrocytes in situ or when grafted to the adultnigra, but when they are cultured in vitro or transplanted into thehippocampus they can form neurons (Gronthos et al., 2003). 28
  37. 37. Properties of neural stem cells:(1) Immunosupressive effect of NSCs: Although NSCs may exert their therapeutic effects bydirectly replacing missing cells, transplantation rarely results insignificant numbers of transplant-derived terminallydifferentiated neurons. The beneficial effect of NSCs in diseasemodels may be attributable to alternative biologic properties.The first indication of an anti-inflammatory effect of NPCscame from transplantation experiments in rats with experimentalautoimmune encephalomyelitis (EAE). It was showntransplantation of NPCs reduced brain inflammation and clinicaldisease severity, it was suggested that the benefit of NPCtransplantation was mediated by an anti-inflammatory effect(Raisman and Li, 2007). The exact mechanisms by which transplanted NPCsattenuate brain inflammation are unclear. Some suggests animmunomodulatory effect by which NPCs promote apoptosis oftype 1 T-helper cells, shifting the inflammatory process in thebrain toward a more favorable climate of dominant type 2 T-helper cells. Alternatively, a nonspecific bystanderimmunosuppressive effect of NPCs on T-cell activation andproliferation has been suggested. The suppressive effect ofNPCs on T cells was accompanied by a significant suppressionof pro-inflammatory cytokines. This nonspecific anti-inflammatory mechanism may be of major importance in theapplication of transplantation therapy in immune-mediated 29
  38. 38. diseases because it can protect the host CNS and graft fromadditional immune attacks (Einstein and Ben-Hur, 2008).(2) Neuroprotictive effects of transplanted NSCs: Neuroprotective effect was observed in other nonautoimmune experimental disease models. Neural stem cellsrescued dopaminergic neurons of the mesostriatal system in aParkinson disease (PD) model in rodents. These findings led tothe concept that NSCs are endowed with inherent mechanismsfor rescuing dysfunctional neurons. This effect was found to beimportant in other neurologic diseases. Neural stem cells seededon a synthetic biodegradable scaffold and grafted into the hemi-sectioned adult rat spinal cord induced significant improvementin animal movement by reduction of necrosis in the surroundingparenchyma and by prevention of inflammation, glial scarformation, and extensive secondary cell loss (Einstein and Ben-Hur, 2008).(3) Neurotrophic effects of transplanted NSCs: After sectioning of the adult spinal cord, NSCtransplantation induced a permissive environment for axonalregeneration. Similarly, in a model of retinal degeneration, NPCtransplantation promoted neural growth in the optic nerve. Inboth cases, this effect was mediated by induction of matrixmetalloproteinases that degrade the impeding extracellularmatrix and cell surface molecules, enabling axons to extendthrough the glial scar. Transplantation of olfactory-ensheathingcells into the sectioned spinal cord also promoted axonal 30
  39. 39. regeneration in long fiber tracts, with a return of lost function.This was explained by the creation of proper realignment,enabling axonal growth through a permissive tract. In addition,the cells increased axonal sprouting, remyelination, andvascularization of the injured spinal cord (Raisman and Li,2007).Isolation of human NSCs: To date, they are primary isolated and propagated in vitro ascells that form free-floating neurospheres when cultured inserum-free medium on non adherent surfaces in the presence ofmitogenic factors such as basic FGF or FGF-2 and epidermalgrowth factors, although there have also been reports ofmonolayer cultures (McBride et al., 2004).(III) Pancreatic stem/progenitor cells: There is strong evidence that new pancreatic islets canderive from progenitor cells present within the ducts and islets,in a process called “neogenesis”. Furthermore, when thesepseudo-islets were transplanted into non-obese diabetic (NOD)mice, diabetes reversal was observed. Candidate pancreaticstem/progenitor cells have also been described within acini, butcontamination with endocrine and ductal cells in cultures couldnot be excluded in these experiments (Limmbert et al., 2008). The isolation of a distinct stem/progenitor cell within theendocrine pancreas depends on the identification of a specificprogenitor marker. The exciting observation that nestin positiveislets cells display endocrine differentiating capacity led to the 31
  40. 40. hypothesis that this intracytoplasmic filament protein mightcorrespond to a pancreatic stem/progenitor cell marker. Morerecently, in two important studies a population of cells in thedeveloping and adult mouse pancreas was identified, whichunder differentiation conditions, released insulin in a glucose-dependant manner. After differentiation, these cells expressedspecific developmental pancreatic endocrine genes (e.g. Ngn3,Pax-4, Pax-6 and PDX-1) and contamination with mature betacells was ruled out (Limbert et al., 2008). While mature beta cell replication appears to be majorphysiological beta-cell regenerative process, identification ofpancreatic cells with progenitor features might open animportant and promising strategy for cell replacement andregeneration therapy. Anyhow, to be clinically relevant, in vitroproliferation of progenitor cells from human pancreas mustproduce large amounts of cells, in order to allow cells isolatedfrom one single donor to be sufficient to treat a given diabeticpatient. It would be even better to have one single donor forseveral diabetics. For these reasons, acinar isolatedstem/progenitor cells might be of interest, considering thatexocrine tissue constitutes 90% of pancreatic tissue and isdiscarded during islet isolation (Kushner et al., 2005).(IV) Other sites: First identified in human bone marrow, a population ofmesenchymal progenitor/stem cells (MSC) with wellcharacterized immunophenotype and distinct from 32
  41. 41. hematopoietic stem cells, was shown to possess a high proliferation rate and great plasticity. Under specific culture conditions these cells differentiate into mesenchymal tissues, such as bone, cartilage, muscle, tendon, adipose and stroma, as well as neuronectodermal tissues (Limbert et al., 2008). Adult tissues and organs known to have stem cellsSource DescriptionBrian Stem cells of the brain can differentiate into the three kinds of nervous tissue-astrocytes, oligodendrocytes, and neurons-and in some cases, blood-cell precursor.Bone marrow These occur as hematopoietic stem cells, which give rise to all blood cells, and as stroma cells, which differentiate into cartilage and bone.Endothelium These stem cells are called hemangioblasts and are known to differentiate into blood vessels and cardiomyocytes. They may originate in bone marrow, but this is uncertain.Skeletal muscle These stem cells may be isolated from muscle or bone marrow. They mediate muscle growth and may proliferate in response to injury or exercise.Skin Stem cells of the skin are associated with the epithelial cells, hair follicle cells, and the basal layer of the epidermis. These stem cells are involved in repair and replacement of all types of skin cells.Digestive Located in intestinal crypts, or invaginations. These stem cells aresystem responsible for renewing the epithelial lining of the gut. Many types are believed to exist, but examples have yet to bePancreas isolated. Some neural cells are known to generate pancreatic β cells. The identity of liver stem cells is still unclear. Stem cells from boneLiver marrow may repair some liver damage, but most repairs seems to be carried out by the hepatocytes (liver cells) themselves. Table (1): Sources of adult stem cells (Limbert et al., 2008). 33
  42. 42. Identification of the adult stem cells: The scientists often use one or more of the following threemethods to identify and test adult stem cells:1- Labeling the cells in a living tissue with molecular markers and then determining the specialized cell types they generate. Then2- Removing the cells from living animals, labeling them in cell culture, and transplanting them back into another animal to determine whether the cells repopulate their tissue of origin. Then3- Isolating the cells, growing them in cell culture, and manipulating them, often by adding growth factors or introducing new genes, to determine what differentiated cells types they can become (Raisman and Li, 2007).The similarities and differences between embryonic and adultstem cells: The adult and embryonic stem cells differ in the number andtypes of differentiated cells types they can become. Embryonicstem cells can become all cell types of the body because arepluripotent. Adult stem cells are generally limited todifferentiating into different cell types of their tissue of origin.However, some evidence suggests that adult stem cell plasticitymay exist; increasing the number of cell types a given adultstem cell can become (figure 13). Large numbers of embryonicstem cells can be relatively easily grown in culture, while adultstem cells are rare in mature tissues and methods for expanding 34
  43. 43. their numbers in cell culture have not yet been worked out. Thisis an important distinction, as a large number of cells are neededfor stem cell replacement therapies (Limbert et al., 2008). A potential advantage of using stem cells from an adult isthat the patient’s own cells could be expanded in culture andthen reintroduced into the patient. The use of patient’s ownadult stem cells would means that the cell would not be rejectedby the immune system. This represents a significant advantageas immune rejection is a difficult problem that can only becircumvented with immunosuppressive drugs. Embryonic stemcells from a donor introduced into a patient could causetransplant rejection, however, whether the recipient would rejectdonor embryonic stem cells has not been determined in humanexperiments (Sonja et al., 2006).Figure (13): Sources of stem cells (Limbert et al., 2008). 35
  44. 44. Types of Stem Cell transplantation: Stem cell transplantation can be classified according to thegenetic relation between the donor and recipient into 4 classes: 1- Autograft: In which the donor and recipient is the same individual. 2- Isograft or syngenic graft: In which the donor and recipient are genetically identical (e.g., monozygotic twins). 3- Allograft or homograft: In which the donor and recipient are genetically unrelated but belong to the same species. 4- Xenograft or heterograft: In which the donor and recipient belong to different species (David, 2009).Application of stem cells: 1) Basic science application:Stem cells are ideally suited to allow for the study of complexprocesses that direct early unspecialized cells to differentiateand develop into the more than two hundred cell types in thehuman body (Bianco et al., 2005). 2) Medical research applications:Stem cell studies may allow researchers to follow the processesby which diseases and impairments caused by geneticabnormalities first manifest themselves biochemical orstructurally in cells and tissues. Using stem cells to producelarge numbers of genetically uniform cultures of organ tissuesfor example, liver, muscle, or neural would allow controlledcomparison of the effects of drugs or chemical on these tissues. 36
  45. 45. Alternatively, testing drugs against stem cell tissues varyinggenetic makeup could allow tissue specific stem cell mayprovide a constant in vitro source of such cellular material(Bianco et al., 2005).The site of stem cell implantation: The transplantation can be described as orthotropic orheterotropic: 1- Neurologic transplantation: Refers to donor tissue implantation in the anatomically correct position in the recipient. 2- Heterotropic transplantation: Refers to the relocation of the implant in the recipient at a site different from the normal anatomy (David, 2009).Route of stem cell delivery: Reports have indicated that after stereotacticintraparenchymal, intracerebro-ventricular, intravenous andintraarterial transplantation, stem cells can home to sites ofinjury in the CNS and induce functional recovery. Of thesevarious transplantation techniques, those that depend onintravascular delivery of stem cells for stroke are particularlyattractive.Intravascular delivery: In addition to its minimal invasive nature, intravasculardelivery may allow stem cells to have a superior interactionwith injured tissue. A comparative study revealed that directintracerebral transplantations resulted in the largest number of 37
  46. 46. cells at the lesion site, followed by intracerebro-ventricular andintravenous transplantations (Guzman et al., 2008). However, researchers in that study only assessed theabsolute number of cells in the perilesional area and took noaccount of whether these cells were therapeutically distributingto all injured areas of brain parenchyma on a microscopic level.Many believe that intravascular delivery of stem cells may leadto a wider distribution of cells around the lesion as comparedwith focal perilesional transplants, thereby leading to superiorstem cell–injured tissue interactions (Xiao et al., 2007).Mechanism of wide distribution: The cells travel in the blood stream and follow a chemoattractant gradient generated by inflammation in the injuredbrain. Unfortunately, intravenously delivered cells pass throughthe systemic and pulmonary circulation systems and home toother organs as well, which significantly reduces cell homing tothe injured brain. Intravenous injection of human MSCs intorats 24 hours after stroke showed that only 4% of the cellsentered the brain, the number of cells entering the brainincreased over time and peaked at Day 21 post-stroke. At Day56, 60% of these surviving cells differentiated into glia, and20% into neurons. Despite the fact that the number of cellsentering the brain was limited, functional recovery wasenhanced by intravenous delivery (Pluchino et al., 2005). 38
  47. 47. Intracarotid injection: Another route of intravascular delivery is intra arterial,which would circumvent body circulation. The first pass of stemcells injected into the carotid artery would be the brain, thisroute of delivery have demonstrated functional recovery afterstroke and traumatic brain injury. In 2006 Shen and colleaguesinjected donor rat BMSCs into the internal carotid artery of rats24 hours post-stroke and successfully induced functionalrecovery. In another study, the same group injected donor ratBMSCs into rats 24 hours after stroke and observed thatinjected cells localized around the infarction area in the brainand very few were found in the heart, lungs, liver, spleen, andkidney (figure 14) ( Guzman et al., 2008).Figure (14): Confocal laser scanning microscopy images revealingnumerous cells in the stroke border zone and the hippocampus ipsilateral tothe stroke (A). Inset shows doublecortin bromodeoxyuridine labeled cells.The VCAM-1 (arrows) is highly expressed in the stroke affectedhemisphere 48 hours after stroke (B). DCX = doublecortin; BrdU =bromodeoxyuridine; DAPI = 46-diamidino-2-phenylindole (Guzman et al.,2008). 39
  48. 48. The debate over the best delivery route is furthercomplicated by the fact that there is still a great deal ofcontroversy concerning the mechanism by which stem cells leadto enhanced functional recovery in patients who haveexperienced stroke. The 2 most discussed mechanisms are asfollows: 1) cellular replacement, by way of the functionalintegration of stem cells; and 2) secretion of neurotrophic andangiogenic factors. If the mechanism of recovery is cellularreplacement, then transendothelial migration is necessary andthe methods that allow the highest concentrations of stem cellsin the injured brain areas ought to be pursued; however, there issignificant evidence that stem cells may provide their benefitsby secreting various neuroprotective factors (Guzman et al.,2008). In summary, the best route of human stem cell delivery hasnot been determined, but the intravascular route is particularlyattractive because of its ease of administration, minimalinvasiveness, and potential for widespread cell distributiontogether with widespread secretion of neuroprotective,proangiogenic, and immunomodulatory factors. Intuitively, theintraarterial route of delivery seems better than the intravenous,given that injected cells first pass the target organ that is, thebrain prior to being redistributed in the systemic circulation(Pluchino et al., 2005). 40
  49. 49. Timing of transplantation Undoubtedly the fate and function of transplanted cells willdepend on any or all of these alterations, and the optimal time oftransplantation is unknown. The timing of transplantationdepends mainly on the goal of treatment, for example,neuroprotection, which should happen early after the insult, orneuroregeneration/cell replacement, which can be done once alesion has stabilized. We can envision a future in which we willrely on multimodal stem cell treatment, depending on acombination of early and late administrations of different celltypes (Guzman et al., 2008).Early intravascular cell delivery In animal models with a neuro inflammatory componentsuch as stroke, traumatic brain injury, spinal cord injury, andmultiple sclerosis, therapeutic somatic stem cells (for example,BMSCs, umbilical cord blood stem cells, MSCs, and NPCs)target inflamed CNS areas where they persist for months andpromote recovery through neuroprotective mechanisms. It isthought that the process of transendothelial migration of somaticstem cells may be regulated in a manner similar to that ofinflammatory cells. As early as 30 minutes after stroke, theinfiltration of leukocytes, both polymorphonuclear leukocytesand monocytes/macrophages, can be observed (Goldman andWindrem, 2006). Chemoattraction, adhesion, and transendothelial migrationof inflammatory cells is regulated by specific inflammatory 41
  50. 50. mediators, which have been identified in experimental andhuman stroke. The temporal expression profile of adhesionmolecules, cytokines, and chemokines after stroke has been welldescribed. Vascular cell adhesion molecule–1 (VCAM-1) hasbeen shown to reach a peak level 24 hours after experimentalstroke. At the bedside, soluble VCAM-1 concentration inplasma is increased in patients with acute stroke. Intercellularadhesion molecule–1 levels have been elevated as early as 4hours after stroke with sustained levels for up to 1 week (Zhangand Lodish, 2005). Monocyte chemo-attractant protein–1, a key chemoattractantfactor for the recruitment of circulating peripheral cells to thestroke area and an important factor for stem cell migration, isupregulated 3 days after stroke and then returns to baseline after1 week. Similarly, stromal-derived factor–1(SDF-1) is known tobe a potent chemoattractant for inflammatory as well as stemcells (including BMSCs and NSCs) and is expressed early afterstroke. Anatomically, adhesion molecule upregulation as well aschemokine expression has been shown to be highest in thestroke-affected penumbral region. Blocking the differentpathways of chemoattraction and cell adhesion in stroke-affected rodents reduced the number of infiltratinginflammatory cells (Belmadani et al., 2006). In mice lacking intercellular adhesion molecule–1 (ICAM-1), a significant reduction in inflammatory cellular infiltrate anda reduction in lesion size were noted. Treatment with anti– 42
  51. 51. ICAM-1 antibodies was a successful neuroprotective means ofreducing lesion size and apoptosis in experimental stroke.However, a clinical trial exploring the feasibility of using anICAM-1 blocking antibody failed to demonstrate any beneficialeffects in the patients. There is some evidence thatintravascularly administered stem cells undergo the sameprocess as inflammatory cells, including chemoattraction,adhesion, and transendo-thelial migration after stroke,potentially making this route an ideal way of cell delivery (Hillet al., 2004).Late intraparenchymal cell transplantation In contrast to the acute intravascular cell treatment, theintraparenchymal approach has been hindered by poor outcomesif the stem cells are transplanted too early after stroke.Excitotoxicity, oxidative stress, and inflammation post ischemiamake the ischemic brain a hostile environment forintracerebrally transplanted cells. In fact, we have found anegative correlation between graft survival and inflammation.Additionally, we demonstrated that human NSCs transplantedtoo close or into the stroke area have very limited survival atdays after stroke (Belmadani et al., 2006). Transplanting cells 3 weeks after stroke, when there is asignificant decrease in inflammation, led to greater graftsurvival than transplanting 5–7 days after stroke. Takentogether, early intravascular cell therapy might benefit from theprocesses tied to post-stroke inflammation but might be 43
  52. 52. detrimental to cells directly transplanted intra-parenchymally.Therefore, intra-parenchymal cell replacement therapy might beuseful as a second line or delayed stem cell treatment strategy(Grabowski, 2010). 44
  53. 53. Stem cell therapy in Parkinsonism Parkinson’s disease (PD) otherwise known as ‘’paralysisagitans’’ or ‘’shaking palsy’’ was classically described by JamesParkinson in 1817. His description of “Involuntary tremulousmotion with lessened muscular power, in parts not in action andeven when supported, with a propensity to bend the trunkforward and to pass from a walking to a running pace, thesenses and intellect being uninjured” has stood the test of time.PD is also defined as a debilitating neurodegenerative disorderof insidious onset in middle or late age characterized by theselective loss of nigrostriatal dopaminergic neurons and loss ofdopamine in the striatum (Abayomi, 2002). Parkinson’s disease is second only to Alzheimer’s diseasewith a prevalence of 1 in 10,000. Although it is uncommon inpeople under age 40 years, the incidence of PD greatly increaseswith age, affecting approximately 1% of individuals older than60 years (Lane et al., 2008).Pathology: The basic pathology is cell degeneration and loss ofpigmented neurons in the pars compacta of the substantia nigraand locus ceruleus with atrophy and glial scarring. Thedegenerated pigmented neurons contain Lewy bodies which areintracytoplasmic eosinophilic hyaline inclusions composed ofprotein filaments (ubiquitin & synuclein), and do not have theelectronic microscropic appearance of any known viral or 45
  54. 54. infective agent (fig. 15). Lewy bodies are characteristic ofParkinson’s disease except in post-encephalitic Parkinson’s andparkengene mutants. However, they could be found in 4% ofbrain without parkinsonian features and these are likely cases ofsubclinical Parkinson’s as 80% of the zona compacta cells mustdegenerate before clinical symptoms become apparent(Abayomi, 2002). The pars compacta contains 450,000 dopaminergic neurons.With the loss of dopaminergic neurons at those sites, there isdeficiency of dopamine in the basal ganglia, chiefly the striatum(caudate nucleus and putamen). Furthermore, the enzymesrequired for dopamine synthesis, DOPA decarboxylase and therate limiting enzyme tyrosine hydroxylase are reduced. Inaddition, there is deficiency of neurotropic factors such as glialand brain derived neurotrophic factors. However, neurons in thestriatum with dopamine receptors remain intact and areresponsible for the therapeutic effects of levodopa. In theParkinsonism unresponsive to levodopa, striatal neurons aredegenerated. Genetic and environmental factors are important inthe mechanism of neuronal deaths due to neuronal necrosis orapoptosis. In neuronal necrosis there is disintegration of cell andorganelles and subsequent removal by phagocytic andinflammatory response with increased cellular permeability. Inapoptosis on the other hand, there is rapid programmed celldeath in response to a toxic stimuli. There is chromatincondensation, DNA fragmentation and cell shrinkage, with 46
  55. 55. relative sparing of organelles without inflammatory changes orincreased cellular permeability (Golbe, 2003). Among the factors that have been implicated in neuronaldegeneration in Parkinson’s disease are mitochondrialdysfunction, oxidative stress, and the actions of excitotoxins,deficient neurotrophic support and immune mechanisms. HLA-DR positive reactive microglial cells and cytokines such asinterleukin 1 (IL-1) and tumor necrosis factor-a play significantrole in the pathogenesis of Parkinson’s disease. Oxidative stresswith excess reactive oxygen species and free radical damageinvolving one or more unpaired electrons react with nucleicacids, proteins and lipids, this metabolic derangement results ingeneration of toxic byproducts and increased oxidative stresswith resultant cellular damage (figure 15) (Lane et al., 2008). 47
  56. 56. Figure (15): Neuronal Pathways that degenerate in Parkinsons disease.Signals that control body movements travel along neurons that project fromthe substantia nigra to the caudate nucleus and putamen (collectively calledthe striatum) (Lane et al., 2008).Cell replacement therapy: The idea of growing dopamine cells in the laboratory to treatParkinson’s is the most recent step in the long history of cell ortissue transplantation to reverse this devastating disease. Theconcept was, and still is, straightforward: implant cells into thebrain that can replace the lost dopamine releasing neurons.Although conceptually straight forward, this is not an easy task.Fully developed and differentiated dopamine neurons do notsurvive transplantation, so direct transplantation of fullydeveloped brain tissue from cadavers, for example, is not an 48
  57. 57. option. Moreover, full functional recovery depends on morethan cell survival and dopamine release; transplanted cells mustalso make appropriate connections with their normal targetneurons in the striatum (Lindvall and Kokaia, 2010). Under the basic principle of restoring dopamine producingneurons via neural grafts, extensive studies have been done tobring this to fruition. One of the first attempts at using celltransplantation in humans was tried in the 1980s. This surgicalapproach involved the transplantation of dopamine producingcells found in the adrenal glands, which sit atop the kidneys inthe abdomen dramatic improvement in Parkinson’s patients bytransplanting dopamine producing chromaffin cells from severalpatients’ own adrenal glands to the nigrostriatal area of theirbrains; it showed dramatic improvement in Parkinson’s patients.Another strategy was previously attempted in the 1970s, inwhich cells derived from fetal tissue from the mouse substantianigra was transplanted into the adult rat eye and found todevelop into mature dopamine neurons (Panchision, 2006). The functional integration of dopamine neuron grafts provethe efficacy of the cell replacement principle, but in reality, thisclinical outcome is extremely inconsistent with respect to thepercentage of cells that survive the grafting procedure and theamount of dopamine produced by the new neurons. In fact,average functional improvement of patients in the experimentsonly rises about 20%. Across the board, subjects achievefunctioning levels less than or equal to that of patients 49
  58. 58. undergoing deep brain stimulation, which carries a lowermorbidity risk (Lane et al., 2008).Cell transplantation: Transplantation of primary ventral mesencephalic tissue intothe striatum aims to restore brain circuitry and function lost as aresult of PD. The main objective of primary tissuetransplantation has been to provide proof of principle thatgrafted dopaminergic neurons can i) survive and restoreregulated dopamine release, ii) integrate with the host brain toreinstate frontal cortical connections and activation, and iii) leadto measurable clinical benefits together with improved qualityof life. Preclinical work in animal models of PD has shown thatgrafted dopaminergic neurons, extracted from the developingventral mesencephalon (VM) can survive, reinnervate thelesioned striatum, and improve motor function (Winkler et al2005). Over the past two decades, a series of open label clinicaltrials have provided convincing evidence to show that humanembryonic nigral neurons taken at a stage of development whenthey are committed to a dopaminergic phenotype can survive,integrate and function over a long time in the human brain.There is good evidence of graft survival, with grafted neuronsdeveloping afferent and efferent projections with the hostneurons. Long term survival of dopaminergic grafts is possibleup to 10 years after transplantation, and there have been no 50
  59. 59. reported cases of overt immunorejection even after several yearsof withdrawal from immuno-suppression (Olanow et al 2003). Evidence from Positron Emission Tomography (PET)scanning has revealed significant increases in activation in theareas reinnervated by the grafted cells, and longitudinal clinicalassessments indicate significant functional recovery for motorcontrol, in some cases for more than 10 years, in the mostsuccessful cases, patients have either reduced dependency for orcompletely withdrawn from L-dopa treatment. Post mortemstudies similarly show good survival of transplanted neuronsand well integrated grafts (figure 16) (Winkler et al., 2005).Figure (16): Dopamine Neuron Transplantation: PET images from aParkinson’s patient before and after fetal tissue transplantation, the imagetaken before surgery (left) shows uptake of a radioactive form of dopamine(red) only in the caudate nucleus, indicating that dopamine neurons havedegenerated. Twelve months after surgery, an image from the same patient(right) reveals increased dopamine function, especially in the putamen(Winkler et al., 2005). 51
  60. 60. The precise mechanism responsible for these dyskinesiaremains unknown but it does not appear to be related to graftovergrowth resulting in excessive dopamine release. Onepossibility surrounds the quality of dissected tissue. Successfultrials have used either freshly dissected tissue or tissue that hasbeen stored in culture for only a few days. One of the trialsreporting cases of severe dyskinesias used tissue stored inculture for up to four weeks and it may be that holding tissue inthis way reduces its dopaminergic composition (Olanow et al.,2003). A further issue concerns the identification of densehyperdopaminergic areas within the graft of some patients withgraft induced dyskinesias. This may have caused uneven striatalinnervation and excessive dopamine release into nonreinnervated areas. It is also possible that variable side effects ofgraft induced dyskinesias are related to patient selection.Greater functional improvement is associated with youngerpatients, and in patients with less advanced disease. This is mostlikely because the neuropathology is relatively confined to thenigrostriatal pathway and may have better trophic supportcompared to patients with more advanced disease (Winkler etal., 2005).Stem cells: Stem cells could provide one such source and wouldovercome the issue of limited availability of fresh primary fetalcells. A wide range of stem cells are being investigated as 52
  61. 61. potential sources of dopaminergic neurons for transplantation,stem cells can be obtained from various sources (Morizane etal., 2009). The majority of research thus far with respect to theformation of dopaminergic neurons for the treatment of PD is inembryonic stem cells and neural stem cells. Dopaminergicneurons are more easily obtained from neural stem cells in thedeveloping ventral mesencephalon (VM) than other parts of thedeveloping central nervous system but the number ofdopaminergic cells produced is still very low. Despite geneticmanipulation and the addition of various growth anddifferentiation factors, generating large numbers ofdopaminergic cells from this cell type has had mixed results(Panchision, 2006). However, greater success has been achieved with the morecomplex ES cells. Derived from blastocysts donated followingin vitro fertilization these cells are truly pluripotent. Promisingdata have been obtained with dopaminergic neurons derivedfrom mouse ES cells, significantly improving motor function ina rat model of PD. However, directing the differentiation ofhuman ES cells has proved complex and while 50% of cellsspontaneously differentiate into neurons upon leukemiainhibitory factor (LIF) withdrawal, few are dopaminergic. Thus,there is the need to develop protocols to ‘direct’ differentiation.The most successful published protocols describe multipleculture stages in which different transcription and growth 53
  62. 62. factors are added at controlled time points (Goldman andWindrem, 2006). However, despite good yield of dopaminergic neurons invitro, clinically relevant long term survival and behavioralrecovery in animal models rivaling that of primary tissue has yetto be convincingly demonstrated. Neuronal stem cells unlikeembryonic stem cells, which are only derived from theembryonic blastocyst, neural stem cells can be found both inembryonic neural tissue and also in specific neurogenic regionsof the adult brain. If the in vivo survival of neural stem cells canbe improved they hold the potential to provide autologoustransplantation as patients provide the cells for their ownrecovery (Morizane et al., 2009). Interestingly, stem cells may not just be useful as dopaminefactories in the striatum. Some studies in both rodent andprimate models have shown significant behavioral recoveryfollowing transplantation with neural stem cells. In addition tothe generation of a small population of dopaminergic neuronsother cells within the graft were found to be releasing growthfactors which are purportedto exert neuro-protective orneuroregenerative influences. While more evidence needs to beaccumulated on the longevity of this effect, it broadens thepotential of neural stem cells from simple dopaminereplacement to preserving and enhancing remainingdopaminergic neurons (Svendsen and Langston, 2004). 54
  63. 63. Stem cell based approaches could be used to providetherapeutic benefits in two ways: first, by implanting stem cellsmodified to release growth factors, which would protect existingneurons and/or neurons derived from other stem cell treatments;and second, by transplanting stem cell derived DA neuronprecursors/neuroblasts into the putamen, where they wouldgenerate new neurons to ameliorate disease-induced motorimpairments (figure 17) (Lindvall and Kokaia, 2010).Figure (17): Stem cell based therapies for PD. PD leads to the progressivedeath of DA neurons in the substantia nigra and decreased DA innervationof the striatum, primarily the putamen (Lindvall and Kokaia, 2010).Neurogenesis: As mentioned above endogenous stem cells are present inspecific regions of the brain. While the occurrence ofneurogenesis in the striatum and substantia nigra is debated, one 55
  64. 64. indisputable neurogenic region is the subventricular zone (SVZ)lying adjacent to the striatum. The cells in the region are anassortment of stem and progenitor cells that have the potential tobe mobilized and induced to differentiate by the presence ofgrowth factors or other small molecules. In the normal condition75%–99% or the cells differentiate into granular GABAergicneurons, with the rest forming periglomular neurons expressingeither tyrosine hydroxylase or GABA. The control ofproliferation and mobilisation of these cells may bedopaminergic as both MPTP and 6-OHDA mediated dopaminedepletion reportedly decreases proliferation in this zone (Zhaoet al., 2008). An additional source of endogenous source of newdopaminergic neurons may be described presence of tyrosinehydroxylase positive cell bodies in the striatum, which increasein quantity with dopaminergic denervation. As yet there are noimminent therapeutic strategies heading towards the clinic thatmanipulate these endogenous systems but their potential iswaiting to be harnessed. Therapeutic strategies to increasestriatal dopamine could involve recruiting newly producedneurons in the SVZ and encouraging them to migrate into thestriatum and differentiate into dopaminergic neurones or tostimulate cells resident in the striatum. In order for this to beachieved understanding more about these two processes ofneurogenesis and phenotypic switching in the striatum isnecessary, determining the intrinsic or extrinsic factors 56
  65. 65. responsible may provide an alternative set of mechanisms thatcould be utilized to treat PD (Lane et al., 2008).Graft standardization: Grafting methods for the past 20 years have differed ineverything from procurement process to tissue composition toimplantation technique. To add even more variability, multipledonors are needed to create a graft large enough to carry somepromise of efficacy. This, undoubtedly, plays an important rolein determining survival, growth, and integration of thetransplant (Morizane et al., 2009). As stem cells can theoretically provide an endless source ofquality consistent neurons, standardization of the transplanttissue will enhance the reliability of the procedure and itsresults. One promising study has shown that implantation ofundifferentiated human neural stem cells (hNSCs) inParkinsonian primate brains can restore functionality.Furthermore, the repair process not only reestablishes the grossanatomical structure of the organ but does so with appropriateproportions of neuron types. Guided by signals of themicroenvironment of the damaged brain, uncommitted hNSCsare induced to differentiate into dopaminergic neurons, as wellas other cells that mediate neuroprotection (Lane et al., 2008).Patient standardization: With standardization of transplant material, patients mustlikewise be evaluated for variables in their presentation of thedisease. Specifically, the distribution of the damaged neurons 57
  66. 66. should be taken into account before and after graft implantation.In earlier studies, patient selections overlooked the preoperativemagnitude of the lesions, making it difficult to evaluate theextent of the graft incorporation. Similarly, it was also unknownwhether continued postoperative degeneration of non graftedregions would affect clinical response. Conversely, patients withlittle or no postoperative damage showed the best functionaloutcome. Because the decline of dopaminergic cells in areasoutside the nigrostriatal region seems to arise as PD progressesto a more severe state, it may be that implantation during earlierstages will exhibit a higher rate of success. This is mirrored inthe survival of transplanted tissue, which survives and integratesbetter in younger patients. The reasons why this occurs have yetto be fully determined, but it is known that neural growth factorsare expressed more in younger brains (Morizane et al., 2009). 58
  67. 67. Stem cell therapy in stroke Stroke is defined as an abrupt focal loss of brain functionresulting from interference with the blood supply to part of thecentral nervous system. It is one of the major causes of deathand disability among the adult population in the world. In spiteof the extensive research in the field of stroke biology, there islittle effective treatment for a completed stroke. Most strokesfall into two main categories: ischemic (80%) or hemorrhagic(20%) (Caplan, 2011).The biology of cellular transplantation: The transplantation of human neuronal cells is an approachto reducing the functional deficits caused by CNS disease orinjury. Several investigators have evaluated the effects oftransplanted fetal tissue, rat striatum, or cellular implants intosmall animal stroke models for the most part, clinical trialdesigns using primary human fetal tissue into patients withneurodegenerative diseases have lessened. The widespreadclinical use of primary human tissue is likely to be limited dueto the ethical and technical difficulties in obtaining largequantities of fetal neurons at the same time; much effort hasbeen devoted to developing alternate sources of human neuronsfor use in transplantation (Kondziolka and Wechsler, 2008). When transplanted, these neuronal cells survived, extendedprocesses, expressed neurotransmitters, formed functionalsynapses, and integrated with the host. During the retinoic acid 59
  68. 68. induction process, significant changes were seen in the neuronprecursor cells that resulted in the loss of neuroepithelialmarkers and the appearance of neuronal markers. The final cellproduct was a ≥ 95% pure population of human neuronal cellsthat appeared virtually indistin-guishable from terminallydifferentiated, post mitotic neurons. The cells were capable ofdifferentiation to express a variety of neuronal markerscharacteristic of mature neurons, including all 3 neurofilamentproteins (L, M, and H); microtubule associated protein 2, thesomatic/dendritic protein, the axonal protein. Thus, the neuronalphenotype made these cells a promising candidate forreplacement in patients with CNS disorders, as a virtuallyunlimited supply of pure, post mitotic, and differentiated humanneuronal cells (Lindvall and Kokaia, 2010). The concept of restoring function after a stroke bytransplanting human neuronal cells into the brain was conceivedin the mid 1990s. Research conducted in a rat model of transientfocal cerebral ischemia demonstrated that transplantation offetal tissue restored both cognitive and motor functions.Ischemic stroke leads to the death of multiple neuronal typesand astrocytes, oligodendrocytes, and endothelial cells in thecortex and subcortical regions. Stem cell based therapy could beused to restore damaged neural circuitry by transplanting stemcell derived neuron precursors/neuroblasts. Also, compoundscould be infused that would promote neurogenesis fromendogenous SVZ stem/progenitor cells, or stem cells could be 60
  69. 69. injected systemically for neuroprotection and modulation ofinflammation (Kondziolka and Wechsler, 2008). Behavioral testing was conducted using a passive avoidancelearning and retention task and a motor asymmetry measure.Animals that received transplants of neurons and treatment withcyclosporine showed amelioration of ischemia inducedbehavioral deficits throughout the 6 month observation period.They demonstrated complete recovery in the passive avoidancetest, as well as normalization of motor function in the elevatedbody swing test. In comparison, control groups receivingtransplants of rat fetal cerebellar cells, medium alone, orcyclosporine failed to show significant behavioral improvement.Subsequent studies have shown that these cells released glialderived neurotrophic factor after transplantation into ischemicrats (figure 18) (Lindvall and Kokaia, 2010).Figure (18): Stem cell based therapies for stroke (Lindvall and Kokaia,2010). 61
  70. 70. Embryonic stem cells: Animal models have demonstrated that ESCs, whentransplanted into adult hosts, differentiate and develop into cellsand tissues and thus may be applicable for treating a variety ofconditions, including Parkinson’s disease, multiple sclerosis,spinal injuries, stroke, and cancer. Transplanted ESCs areexposed to immune reactions similar to those acting on organtransplants; hence, immunosuppression of the recipient isgenerally required. It is possible, however, to obtain ESCs thatare genetically identical to the patient’s own cells usingtherapeutic cloning techniques (Kalluri and Dempsey, 2008). Several studies showed that these cells are able to migrate inresponse to damage, using MRI, that ESCs that were implantedinto the healthy hemisphere of rat brains 2 weeks after focalcerebral ischemia (FCI), migrated along the corpus callosum tothe ventricular walls, and populated en masse at the border zoneof the damaged brain tissue (i.e., the hemisphere opposite to theimplantation sites). Another study showed that undifferentiatedESCs xenotransplanted into the rat brain at the hemisphereopposite to the ischemic injury migrated along the corpuscallosum toward the damaged tissue and differentiated intoneurons at the border zone of the lesion. In the homologousmouse brain, the same murine ESCs did not migrate, butproduced highly malignant teratocarcinomas at the site ofimplantation, independent of whether they werepredifferentiated in vitro to NPCs. These results imply that 62
  71. 71. ESCs might migrate to the damaged site. However, theproduction of teratocarcinoma raises concerns about the safetyof ESC transplantation in patients with stroke (Battler and Leor,2006).Adult neural stem cells: During the last century, the dogma existed that the adultCNS was incapable of generating new neurons (neurogenesis).Over the past decades, convincing evidence emerged thatneurogenesis in the adult CNS is a continuous physiologicalprocess. Neurogenesis is present in two regions: thesubventricular zone (SVZ) and the subgranular zone of thedentate gyrus (figure 19) (Kalluri and Dempsey, 2008).Figure (19): Schematic drawings showing ischemia induced damage in thecortex and neural stem cell proliferation in the subventricular zone anddentate gyrus; these hypothetical sections of brain illustrate the ischemictissue and the neurogenic regions. A: Drawing showing the subventricularzone of the lateral ventricles. B: Drawing showing the dentate gyrus in thehippocampus. Note the migration of cells from the subventricular zonetoward the infarcted area. Red dots represent proliferating and/or migratingneural stem and/or progenitor cells (Kalluri and Dempsey, 2008). 63
  72. 72. Additionally, other studies also indicated the existence ofNSCs in other regions of the CNS, namely the striatum, spinalcord and neocortex. SVZ and dentate gyrus derived NSCs arecharacterized by long term, self renewal capacity andmultipotency. Adult SVZ and dentate gyrus derived NSCspersist throughout the life span of mammals including humans.It is important to note that neurogenesis occurs in aphysiological mode or is exogenously modulated by externalsignals or pathophysiological processes. External globalstimulants such as enriched environment, physical activity andstress or application of defined molecules such as fibroblastgrowth factor-2, vascular endothelial growth factor (VEGF),brain derived neurotrophic factor (BDNF) and erythropoietindifferentially modulate adult neurogenesis. Finally, CNS diseaseconditions such as seizures and traumatic brain injuryrepresented by respective animal models induce neurogenesis(Panchision, 2006).Role of neural stem cells:Proliferation: Neural stem cell proliferation involves the sequentialactivation of several cell cycle dependent enzymes and proteinsto initiate either symmetrical (2 stem cells) or asymmetrical celldivision (1 stem and 1 progenitor cell). Erroneous activation ofcell cycle enzymes in differentiated cells like neurons, however,can lead to either apoptotic death of the neurons or to theformation of cancer cells. A plethora of growth factors are 64
  73. 73. expressed by the ischemic tissue, which may be responsible forthe ischemia induced neurogenesis. The increase in ischemiainduced neurogenesis could therefore be due to the upregulationof growth factor content or their receptor expression (Hass etal., 2005). In addition, not all growth factors are stimulatory infunction. Although a variety of growth and trophic factors areupregulated following ischemic injury, some are stimulators ofneural progenitor proliferation, whereas others block the selfrenewal of cells. It is important to note that ischemia inducedmigration of neuroblasts has been shown to be due to theexpression of monocyte chemoattractant protein (MCP-1),which can attract the progenitor cells away from the neurogenicniche, blocking their proliferation. Likewise, inhibition ofgrowth factor activity by its binding proteins may also interferewith proliferation of cells. Interestingly, insulin like growthfactor–I (IGF-I) can stimulate the proliferation of progenitorcells only in the presence of mitogens like fibroblast growthfactor–2 (FGF-2) and promotes differentiation followingmitogen (FGF-2) withdrawal (figure 20) (Kalluri and Dempsey,2008). 65
  74. 74. Figure (20): Schematic drawing showing proliferation and differentiationof neural stem cells. These cells proliferate in response to mitogens anddifferentiate into neurons, astrocytes, and oligodendrocytes on exposure tovarious growth factors (Kalluri and Dempsey, 2008).Migration: Several studies have shown that ischemia induces themigration of neuroblasts into the striatum and cerebral cortex.An enriched environment, however, increased the strokeinduced neurogenesis in the hippocampus, but decreased the cellgenesis and migration of neuroblasts into striatum. In addition,erythropoietin has also been shown to be involved in theproliferation of progenitor cells in the subventricular zone.Hence, it appears that proliferation and migration of cells maybe interrelated, in the sense that they are elements of the sameprocess (Son et al., 2006). 66
  75. 75. It is thought that chemokines proteins released at the site ofinjury by the inflammatory cells induce migration of the cells.The expression of chemokine receptors on neural stem cells isimportant for an efficient response to chemokines. Theexpression of chemokine receptors is regulated by retinoic acid,and that animals fed on a diet lacking retinoic acid havedecreased numbers of double cortin positive cells. Retinoic acidhas been shown to induce the differentiation of neurons in vitro.Hence, it appears that lack of retinoic acid may have decreasedthe neuronal differentiation and/ or the expression of chemokinereceptors, which results in decreased numbers of neuroblasts(immature neuronal cells expressing double cortin) migratingout of the neurogenic niche (figure 21) (Kondziolka andWechsler, 2008). 67
  76. 76. Figure (21): Schematic drawing showing ischemia induced neurogenesis.Ischemia increases the expression of IGF-I, FGF- 2, TGFb1, and MCP-1.The IGF-I and FGF-2 enhance the proliferation of cells, whereas MCP-1increases the migration of cells away from neurogenic regions toward theischemic tissue. The stem cells leaving the neurogenic regions exit the cellcycle, during which time TGFb1 promotes the differentiation of stem cellsinto neuroblasts (migrating immature neurons). Alternately, if proliferatingcells in the neurogenic region are exposed to TGFb1, it will inhibit theprocess, thus slowing down the proliferation of cells (Kondziolka andWechsler, 2008).Differentiation: The process of generating specialized cells from neural stemcells is called differentiation. Neural stem cells can bedifferentiated into neurons, astrocytes, and oligodendrocytes;this involves interplay between intrinsic cellular programs andextrinsic cues like growth factors provided by the surroundingenvironment. Although neural progenitor cells proliferate inresponse to ischemia, several immature neurons (double cortinpositive cells) were shown to be migrating toward striatum andcortex (Battler and Leor, 2006). 68
  77. 77. Although IGF-I induces the proliferation and differentiationof progenitor cells after ischemia, most of the cells that aremigrating toward the injury site are neuroblasts (immatureneurons) but not oligodendrocytes. Although speculative, aneuronal differentiation factor like transforming growth factorb1 (TGFb1), which is upregulated after ischemia, may beresponsible for the formation of neuroblasts during the postischemic neurogenesis. The differential response of IGF-Itoward proliferation and differentiation was shown to beregulated by a mitogen activated protein kinase pathway.Because matrix metalloproteinase (MMPs) play an importantrole in both extracellular matrix (ECM) digestion and IGFregulation, it is crucial to understand their direct (ECMdigestion) and indirect (IGF metabolism) effects on progenitorcell differentiation. Hence it is possible that after stroke, cellsproliferate in response to mitogens and growth factors, some ofwhich exit the cell cycle due to the chemokine mediatedmigration out of the neurogenic area, and differentiate intoneuroblasts in response to differentiation promoting factors suchas TGFb1. Thus, the final outcome depends on the spatial andtemporal expression of these factors following ischemia (Son etal., 2006).Potential mechanism involved in stem cell mediatedrecovery after stroke: Initial transplantation studies were focused on the potentialof NSCs to replace lost circuitry. Transplanted neural progenitor 69
  78. 78. cell (NPCs) in a rat model of global ischemia have beenreported to express synaptic proteins post transplantation.However, only limited evidence demonstrates that transplantedcells are able to sustain CNS repair through massive cellreplacement, especially to the extent that might be required afterstroke. Regardless of the characteristics of the experimentaldisease, functional recovery achieved through NPCtransplantation does not correlate with absolute numbers oftransplant derived, newly generated, and terminallydifferentiated neural cells (Emerson et al., 2008).Stem cell induced neuroprotection: Transplanted stem cells can provide neuroprotection andreduce host cell death in the post stroke brain. Most authorshave reported functional recovery and a reduction in lesion sizewhen cells are transplanted within the first 24-48 hours afterstroke. The short timeframe in which NPCs affect recoverycannot be explained by the regeneration of new neurons andsynapses, suggesting an important role for neuroprotection inenhancing recovery. In fact, NPCs are known to exert directneuroprotection through the neutralization of free radicals,inflammatory cytokines, excitotoxins, lipases, peroxidases, andother toxic metabolites that are released following an ischemicevent (Ourednik et al., 2005). The neuroprotective effects of transplanted NPCs are usuallyaccompanied by increased in vivo bioavailability of mainneurotrophins such as nerve growth factor, brain derived 70
  79. 79. neurotrophic factor (BDNF), ciliary neurotrophic factor,vascular endothelial growth factor (VEGF), fibroblast growthfactor, and glial derived neurotrophic factor (GDNF).Alternatively, one could argue that the cells did adhere to theendothelium in the affected brain area and exerted their effectthrough the secretion of GDNF but did not eventually engraft. Adifferent aspect of neuroprotection would be an effect onendogenous neurogenesis after stroke. Endogenousneurogenesis is increased after a stroke. Its exact function hasyet to be determined, but it may signify a natural repairmechanism of the brain, which could be enhanced bytransplanted cells (Guzman et al., 2008).Stem cell induced angiogenesis: Transplanted NPCs can also enhance endogenousangiogenesis. Increased vascularization in the penumbra withina few days after stroke is associated with spontaneous functionalrecovery. As early as 3 days after ischemic injury, new bloodvessels are observed in the stroke affected penumbra, andgrowth continues to increase for at least 21 days. Transplantedcell induced blood vessel formation has been reported withBMSCs, NSCs, and cells from human cord blood or peripheralblood. The ability of transplanted cells to increase endogenouslevels of angiogenic factors and chemoattractant factors (forexample, SDF-1) induces the proliferation of existing vascularendothelial cells (angiogenesis) and mobilization and homing of 71