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Shree Ram Singh (ed.), Somatic Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 879,DOI 10.1007/978-1-61779-815-3_1, © Springer Science+Business Media, LLC 2012

Chapter 1

Current Thoughts on the Therapeutic Potential of Stem Cell

Pranela Rameshwar

Abstract

Stem cells are considered as potential therapy for in fl ammatory disorders, tissue repair, and gene delivery, among others. The heterogeneity of a disease and the underlying disorder of a patient bring up the question on the method by which stem cells should be delivered. This summary discusses potential complex interac-tions among mediators at sites to tissue insults with stem cells. The chapter selects mesenchymal stem cells (MSCs) as a model, although the discussion is relevant to all stem cells. The review examines how MSCs and their differentiated cells can develop cross communication with soluble factors and cells within the region of tissue damage. In fl ammatory cytokines, IL-1, TNF a , and TGF b are selected to explain how they can affect the responses of MSCs, while predisposing the stem cells to oncogenic event. By understanding the varied functions of MSCs, one will be able to intervene to form a balance in functions, ultimately to achieve safety and ef fi cient application. Cytokines can affect the expression of pluripotent genes such as REST and Oct - 4 . REST is a critical gene in the decision of a cell to express or repress neural genes. Since cytokines can affect microRNAs, the review incorporates this family of molecules as mediators of cytokine effects. IFN g , although an in fl ammatory mediator, is central to the expression of MHC-II on MSCs. Therefore, it is included to discuss its role in the transplantation of stem cells across allogeneic barrier. In summary, this chapter discusses several potential areas that need to be addressed for safe and ef fi cient delivery of stem cells, and argue for the incorporation of microenvironmental factors in the studies.

Key words: Therapeutic potential , Pluripotency , Stem cell , Stem cell self-renewal , MiRNAs , Mesenchymal stem cells

The area of stem cells is not a new subject. However, during the past century the focus has been on hematopoietic stem cells. This has led to the premise that the bone marrow is a unique organ due to the presence of stem cells. Since the hematopoietic stem cells are the source of immune and blood cells, there was no controversy on the existence of stem cells in bone marrow. As would be expected for any type of research, there were chal-lenges in studies to identify hematopoietic stem cells and lineage

1. Introduction

4 P. Rameshwar

differentiation. Despite the time spent on studies to understand lineage maturation of hematopoietic stem cells, new reagents continue to identify additional pathways in the development of hematopoietic stem cells. If the longest studied stem cells still require investigation, this underscores the complex mechanisms in the maturation of stem cells to mature cells.

There is little to no doubt among investigators that a small subset of bone marrow cells can reconstitute lethally irradiated animals. The very low frequency of these stem cells in an individual can be explained by the self-renewal mechanism reported by Drs Till and McCullock ( 1 ) . Other investigators reported on chromo-somal preservation as a method to reduce the frequency of muta-tion in the stem cells. These fi ndings, mostly determined in studies with hematopoietic stem cells, can be extrapolated to stem cells in other organs and tissues.

The identi fi cation of the major histocompatibility complex (MHC)-Class I and II molecules has been most valuable in causing transplantation of hematopoietic stem cells across allogeneic barrier. This has been met with remarkable success for hematological disorders, solid tumors, and autoimmune disease. The success of these transplants is met with the attitude of improving protocols with the incorporation of the new science. As an example, during the early time of transplantation to repopulate the immune system, relatively young individuals were considered as quali fi ed for the procedure. As the physician gain more experience with transplants, and as the science progress, the ages of individuals are older.

During the past few years, stem cells have been identi fi ed in all organs. Although it is unclear about the physiological functions of these stem cells, studies on their differentiation to specialized cells indicate that the endogenous stem cells in the various organs might be required for daily replacement of damaged cells. This has led to the enormous investigation across the globe on cell replacement and protection by stem cells. In light of the information from different laboratories, it appears that stem cells could be among the new wave of therapy for cell replacement and repair, among other treatments.

The experimental studies are not limited to repair tissues of stem cells from the same organs, but to use stem cells of another organ. In addition, scientists have focused on the immature nature of stem cells to explore their plasticity, which is de fi ned as the ability of stem cells to generate cells of another germ layer. As an example, stem cells of mesodermal origin can form cells of ectodermal origin. Based on the vast number of publications on this subject, it is obvious that adult stem cells are functionally plastic. The current challenge is to utilize the most ef fi cient method to translate the experimental studies. Unlike the experimental studies, stem cells, when placed in patients, could be under stress due to the different microenvironment and can behave differently. This chapter discusses

51 Current Thoughts on the Therapeutic Potential of Stem Cell

some of the challenges and identi fi es some solutions that could be explored to succeed in the translation of stem cells.

The categories of stem cells are broadly divided into embryonic stem cells, fetal stem cells, and adult stem cells. Since the cord blood and placenta contain fetal stem cells, these types are consid-ered as subsets of fetal stem cells. Despite embryonic stem cells being the most primitive cells, if the ethical issues are dissected, the science of these stem cells makes them undesirable for tissue repair. Simply, embryonic stem cells need to be MHC matched. However, there are few reports that showed an immune suppressive role of embryonic stem cells when they are placed as third party cells to two-way mixed lymphocyte reaction ( 2, 3 ) . However, the type of immune response of embryonic stem cells is still unresolved since others have reported on their ability to induce both humoral and cellular responses ( 4 ) . In the best case if embryonic stem cells can be transplanted across allogeneic barrier through their ability to be immune suppressive, this does not address the instability of these stem cells to form tumors ( 5 ) .

Similar to the adult system, the developing hematopoietic stem cells are most studied in the embryo and fetus. In parallel with the renewed interest in stem cells, scientists continue to examine stem cells from fetus, in particular from tissues that are discarded: umbil-ical cord blood and placenta ( 6 ) . Stem cells from other regions of the fetus have been studied. These include, but are not limited to multipotential progenitors, non-hematopoietic stem cells from human fetal livers ( 7 ) . An interesting report involves the isolation of amniotic stem cells from both adult and mice ( 8 ) . The authors were able to expand these cells and showed that they can form different types of all three germ layers. Stem cells from the amniotic fl uid will be important because a large number of women undergo amniocentesis annually. If a small amount of fl uid after diagnosis can be expanded, this will be invaluable to stem cell therapy. Indeed, stem cells from amniotic fl uid continue to be a major subject of research for several clinical disorders ( 9, 10 ) .

Adult stem cells are found in all organs. Each organ has its own unique microenvironment and requires that the stem cells are studies in the context of the milieu within the organ, or at least, to develop experimental models that recapitulate the organ. These types of investigations as well as evolving novel methods will achieve the following: (a) acquire these stem cells in cases where they would be discarded, such as during surgery. If they can be expanded and show plasticity ex vivo, they would be excellent source of stem cells for tissue repair. (b) By studying the stem cells

2. Challenges

6 P. Rameshwar

within their own microenvironment, it will be possible to stimulate them endogenously during tissue insult to replace damaged tissue. As an example, during wounds, it might be possible to induce the endogenous skin stem cells to repair the damaged cells ( 11 ) . (c) If the environment of a tissue is changed by an in fl ammatory process, investigative studies will determine if, instead, of targeting the stem cells, the microenvironment could be regulated by particular drugs. The premise is that changing the microenvironment will cause the endogenous stem cells to mature into a desired cell types. (d) It is necessary to keep in mind that several genes linked to pluripotency are also oncogenic or tumor suppressor. Although still speculative, it is believed that stem cells can transform as the source of tumors ( 12 ) . Therefore, an understanding of the biology of stem cells, and how it interacts with its microenvironment during self-renewal and differentiation would provide insights on malignancies.

This section will not discuss the speci fi c markers on stem cells since this continues to be a subject of investigations. A close examination of the biology of some stem cells, e.g., placenta, adipose tissue, and amniotic fl uid, strongly indicate that these stem cells are function-ally similar to mesenchymal stem cells (MSCs). Therefore, why are these cells discussed as completely different stem cells? To explain why this difference continues to permeate the fi eld of stem cell biol-ogy, one one speculates that scienti fi c meetings are held within nar-row speciality that discusses organs, accounting for the designation of MSCs from different organs. This obstacle should be overcome by broad societies, such as those speci fi c for stem cells. It is expected that time will solve these obstacles as the fi eld is still relatively new. On the other hand, does the difference in name due to intellectual property rights. If so, these are expected to be hindrance going forward in the treatment of various disorders with stem cells.

Stem cell biologists should refer to the literature for published markers. However, the investigator should perform functional assays and used the published literature only as guide. If this approach is made, it is likely that a small group of laboratories will add signi fi cantly to the literature to the fi eld of stem cell biology. Contrary to this approach could result in numerous publications in the wrong directions of the fi eld. This is demonstrated in the fi eld of cancer stem cells where a particular marker such as CD133, fi rst touted as the marker of cancer stem cells, could have subset of cancer cells ( 13 ) . However, published markers of stem cells should not be discounted since they could provide guide to isolate different subsets of lineage-speci fi c cells.

Common functions in pluripotency include self-renewal of stem cells, multiple lineage differentiation, and the expression of

3. Markers of Stem Cells

71 Current Thoughts on the Therapeutic Potential of Stem Cell

genes linked to stem cells such as Oct4 , Nanog , and Sox2 ( 14 ) . Ideally, the stem cells should reconstitute an organ. As an example, stem cells from the mammary gland should reconstitute an organ to generate cells of all lineages ( 15 ) .

Based on the scienti fi c issues with embryonic stem cells, the desir-able stem cells should be of adult or fetal source. Since placenta and amniotic stem cells seem to exhibit functions, similar to MSCs, this section will discuss the desirability of MSCs. The information would be extrapolated to other stem cells with similar properties.

MSCs are generally considered as heterogeneous and can be found in several adult and fetal tissues ( 16– 18 ) . The adult bone marrow is the major organ of MSCs. In bone marrow, MSCs sur-round blood vessels and are in contact with the trabeculae ( 17, 19 ) . The frequency of MSCs varies, depending on the source. For example, the frequency is low in umbilical cord blood, but high in adult bone marrow and adipose tissues ( 20, 21 ) . In contrast to the blood of the cord, the perivascular region and Wharton Jelly of the cord have a higher frequency of MSCs ( 22, 23 ) . Similar to other stem cells, MSCs differentiate along multiple lineages to generate fi broblasts, adipocytes, chondrocytes, osteogenic cells, and carti-lage ( 24 ) .

It appears that subsets of cultured MSCs express genes to indicate their ability to generate specialized cells ( 25 ) . For example a population of CD146-expressing cells have been shown to gen-erate osteogenic cells and fi broblasts, suggesting that CD146(+) MSCs could be osteoprogenitors ( 26 ) . In other functions, the CD146-expressing cells can be transplanted to form a supporting hematopoietic microenvironment ( 26 ) . This information is signi fi cant for speci fi c application of MSCs. If speci fi c markers can be identi fi ed to select a subset of stem cells, it will be possible to select a particular subset to treat a speci fi c disease. MSCs have been reported to express neural-associated markers, such as neural gan-glioside, GD2 ( 27– 29 ) . At present, it is unclear if this marker is limited to a particular subset of MSCs that can form neural cells, such as neurons ( 30– 33 ) .

Regarding techniques to characterize MSCs, these stem cells are morphologically symmetrical with fi broblastoid appearance ( 34 ) . However, phenotypic analyses indicated that they express CD44, CD29, CD105, CD73, and CD166 and lack markers of hematopoietic lineage, in particular CD45 ( 35, 36 ) . The markers seem to be expanding with CD200 added as a marker with immu-nomodulatory property ( 37 ) . Human MSCs have been suggested to be perivascular, also referred to as pericytes ( 38, 39 ) . Pericytes

4. Mesenchymal Stem Cells: Overview

8 P. Rameshwar

have been isolated from different human organs, express CD146, NG2, and PDGF receptor type 1, and form myogenic cells ( 38 ) .

A discussion regarding the origin of embryonic MSCs is relevant to an understanding of their ability to generate different cell types. The origin of MSCs have been reported to be mesodermal and neuroepithelial ( 24, 40 ) . They show smooth muscle type structures that make them stem cells of mesodermal origin. The neuroepithe-lial origin of MSCs would explain the ease by which they form functional neurons and other neuronal cells ( 30– 33, 41 ) . As meso-dermal stem cells, the generation of functional neurons indicates that the MSCs have crossed germ layer by forming cells of ecto-dermal origin, validating the plasticity of MSCs. On the other hand, if they are neuroepithelial stem cells, they should be able to generate ectodermal cells and this might deter their ability to be designated as plastic cells in their generation of functional neurons ( 30, 32, 42, 43 ) .

To understand the safety of MSCs, including their transplantation across allogeneic barrier, requires investigation into their involve-ment in immune responses. MSCs could suppress the functions of dendritic, natural killer, and T- and B-cells ( 44– 47 ) .

The safety of MSCs has been determined in several clinical trials, including trials in which MSCs are used as third party cells for graft vs. host disease ( 48, 49 ) . As far as we are aware, there is no report on adverse effect, including the formation of tumors. It is almost accepted that MSCs can be transplanted across allogeneic barrier. This indication is based on early reports as well as more recent studies that show immune suppressor role of MSCs ( 34, 36 ) . Based on the experimental studies, MSCs show promise as immune sup-pressor cells for bone marrow transplantation as well as for organ transplant. MSCs could also be applied to patients with other in fl ammatory disorders such as asthma ( 50, 51 ) , in fl ammatory bowel disorders ( 52 ) , and skeletal disorders ( 53 ) . This list of disor-ders contained only few disorders that can be treated with MSCs, indicating that the potential application for MSCs could be exhaus-tive and therefore warrant in-depth discussion.

The results of early clinical trials with MSCs are mixed. The con-tradictory outcome of these trials cannot be explained. Jones and McTaggart ( 46 ) discuss the results of several trials, explaining the discord between the experimental studies and the clinical trials. The clinical trial fails to sustain long-term immune suppression. The question is whether long-term immune suppression is desired and if so, how this will be achieved? Insights into this question require a closer look at the ability of MSCs to establish functional cross talk with mediators within tissue microenvironment.

5. MSCs: Safety and Early Trials

91 Current Thoughts on the Therapeutic Potential of Stem Cell

MSCs express speci fi c receptors for chemokines and cytokines that are expected at sites of tissue injury ( 54– 56 ) . These studies underscore the ability of MSCs to establish functional cross talk with cytokines when they are placed within a milieu of in fl ammatory mediators. Although MSCs have been linked to immune suppres-sor functions, they can also exert immune-enhancing functions such as antigen presentation and autoimmune responses ( 57– 60 ) . Therefore, when MSCs are placed within a milieu of in fl ammatory responses, it might be dif fi cult to predict the outcome. The dual immune responses of MSCs are consistent with the lackluster results of clinical trials ( 46 ) .

The ability of MSCs to establish functional cross talk with immune mediators within a microenvironment of cytokines is not limited to undifferentiated stem cells. MSC-derived neurons also express cytokine receptors such as IL-1, IL-2, IL-6, and TNF a ( 55 ) . More importantly, the question that lingers is whether cells differentiated from MSCs or the host can reject any other stem cells? To explain this further, MSCs are considered desirable for transplantation as off-the-shelf stem cells, indicating their delivery across allogeneic barrier. The initial delivery can be safe due to the immune suppressive properties of MSCs ( 61 ) . However, if future application shows that MSCs can replace endogenous tissues, the new cells will express class I MHC of the donor. The question is whether the new cells will be rejected or if tolerance will be developed. Regardless, this should be important investigations, in parallel to current work with MSCs. Also, the new cells, through the expression of cytokine receptor, would be able to establish cross talk with the microenvironment of tissue injury. The mixed trials with adult stem cells for cardiac disease have provided valu-able information on the complex network of activated cells and soluble factors that stem cells have to accommodate to repair damaged tissue ( 62 ) . Together, this section describes the complex biology that is not mutually exclusive in the ef fi ciency of MSCs, and other stem cells to protect and repair damaged tissues.

This section discusses the immune responses of MSCs in detail since these responses are fundamental to the future clinical trials of these stem cells. MSCs show functional plasticity with regard to their immune properties by exerting both immune suppressor and enhancer functions ( 63 ) . In addition, MSCs might be instructive cells to macrophages as a mechanism of tissue repair ( 64 ) . MSCs produce varied cytokines that can mediate autocrine and/or paracrine stimulation ( 17 ) . Dominci et al. ( 18, 65 ) suggest that MHC-II expression should be included among the minimal

6. Immune Biology of MSCs

10 P. Rameshwar

requirements for cells to be designated as MSCs. Several reports indicated the isolation of pluripotent cells with properties con-sistent for MSCs, but undetectable MHC-II. These cells exhibit plastic adherence and can be differentiated along multiple lineages. It is possible that not all subsets of MSCs express the MHC-II and that its expression does not require prior stimulation with in fl ammatory mediators ( 34, 59 ) . However, the expression of MHC-II on MSCs is a major consideration for stem cell therapy. MHC-II expression provides the cells with the ability to act as antigen presenting cells (APCs) ( 34, 57, 66– 69 ) . Although MHC-II might not be expressed in unstimulated MSCs, its expres-sion can be induced by interferon gamma (IFN g ) ( 59 ) . The signi fi cance of MHC-II expression, whether constitutive or induced, is signi fi cant to transplantation studies because if MSCs begin to serve as APCs, this might create confounds to the treat-ment. Therefore, the analyses of MHC-II and the consequence to cell therapy should be carefully examined, going forward to achieve ef fi cient cell therapy. Another relevant property of MSCs is their ability to endocytose particles ( 57 ) . Again, this is highly signi fi cant for tissue repair where vast amounts of necrotic cells are likely to be present. Therefore, by placing MSCs in these regions of tissue injury, the cells could begin to engulf necrotic tissues to initiate an immune response.

Although MSCs express MHC-II and act as APCs, MHC-II is regulated differently as compared to other APCs such as mac-rophage ( 57, 70 ) . IFN g , which is a major pro-in fl ammatory inducer of MHC-II, shows a bimodal effect on MHC-II expression on MSCs ( 57 ) . MSCs produce baseline IFN g that maintain MHC-II expression ( 57 ) . However, when MSCs are exposed to high level of IFN g , its expression is decreased. If this observation is extrapo-lated to in vivo transplantation for acute in fl ammation, the MHC-II will be decreased and the stem cells will exert immune suppressive function. This will be desirable until the IFN g level is decreased and MHC-II will be re-expressed.

The bimodal expression of MHC-II on MSCs has been attrib-uted to the differential effects of IFN g on the master regulator of MHC-II, CIITA ( 71 ) . At high IFN g levels, the CIITA is retained in the cytosol, thereby preventing MHC-II transcription ( 71 ) . This is a highly relevant fi nding since the retention of CIITA in the cyto-sol could be explored for effective treatment to prevent the re-expression of MHC-II on MSCs.

The effect of CIITA is not only relevant for the stem cells. The mechanism appears to be similar for neurons derived from MSCs that are exposed to IFN g ( 72 ) . This further underscores that signi fi cance of CIITA as future targets for the ef fi ciency of trans-planting MSCs as off-the-shelf stem cells. The expression of MHC-II in cells that were derived from allogeneic MSCs is likely to occur long after their delivery to replace damaged tissue.

111 Current Thoughts on the Therapeutic Potential of Stem Cell

The immune system could develop tolerance for the Class I MHC, which is normally considered as a weak antigen for allogeneic response. However, if the host is subjected to an infection, there will be high levels of IFN g that could cause MHC-II to be expressed. This could result in late rejection of the replaced cells.

A thorough understanding on the roles of cytokines as communi-cators of stem cells would require a separate, but lengthy review due to the existence of numerous cytokines and chemokines as well as other immune mediators such as extracellular matrices. At any sites of tissue injury, the microenvironment is expected to encom-pass a complex network of multiple soluble and insoluble medi-ators as well as several immune cell subsets. Another level of complexity is the timeline changes of the factors as well as the changes at sites close to the area of tissue insult. For example, there could be timeline changes in cytokine levels, following tissue injury. In addition, at a speci fi c time, the cytokines could show a gradient concentration from the site of injury. These changes make it dif fi cult to predict how MSCs, or any other stem cells, should be implanted.

This review brie fl y discusses two cytokines with broad, but opposing functions: interleukin-1 a (IL-1 a ) and transforming growth factor-beta (TGF- b ). IL-1 is selected because it could regulate other cytokines with positive and negative effects. TGF- b is discussed due to its role as a pro- and anti-in fl ammatory mediator. Another reason to discuss TGF- b is due to its association with oncogenesis. The placement of any stem cell within a milieu of in fl ammatory mediators could predispose the cell to transformation. At the time of designing any trial with stem cells, one needs to consider that the genes associated with pluripotency is also linked to oncogenesis.

IL-1 a belongs to the family of cytokines that are central to in fl ammation and host defense ( 73 ) . IL-1 a and IL-1 b appear to exhibit similar effects through the type I IL-1 receptor. The type II receptor subtype lacks an intracellular signaling domain. IL-1 could be signi fi cant in understanding how stem cells respond to tissue factors. For example, IL-1 induces the expression of other in fl ammatory mediators in the stem cells and also in neighboring cells. Since it is most likely that IL-1 will be present at an area of tissue injury, it is expected that this cytokine could initiate a net-work of other cytokines that will cause cross communication between stem cells and other immune cells ( 73 ) . In this regard, IL-1 will be a direct as well as an indirect mediator of stem cell responses ( 74, 75 ) . IL-1 would be able to affect cells derived from

7. Cytokines in Stem Cell Responses

12 P. Rameshwar

MSCs. As an example, IL-1 has been shown to stimulate the type 1 receptor in MSC-derived neurons ( 73 ) . In the case of neurons, IL-1 can induce the production of neurotransmitters, which could cause a cascade reaction to stimulate immune cells at the site of tissue injury, and perhaps indirectly expand to distant organs through the movement of activated immune cells ( 74, 76– 78 ) .

TGF- b 1 belongs to a superfamily of proteins including the activins, inhibins, and bone morphogenic proteins ( 79 ) . TGF- b receptors are ubiquitously expressed on normal and malignant cells ( 80, 81 ) . TGF- b 1 interacts with subtypes I and II where the type 1 form is activated by type II ( 82, 83 ) . Type I signaling activates four members of Smad transcription factors ( 84– 86 ) . TGF- b is involved in development during embryogenesis and neurogenesis ( 79, 87 ) . TGF- b 1 also modulates immune responses and inhibits cell proliferation, differentiation, and apoptosis ( 88, 89 ) . TGF- b 1 exerts both tumor suppressor and oncogenic properties ( 90 ) .

This section includes another in fl ammatory mediator, TNF a . This cytokine, along with other members of the family, interacts with the TNF receptor superfamily ( 91 ) . TNF a is produced by activated macrophages and monocytes to induce cell death, and is involved in in fl ammatory disorders such as arthritis ( 92 ) . Since MSCs are suggested for in fl ammatory responses, the role of TNF a could be signi fi cant in tissue repair. It is unclear if MSCs can produce TNF a , although they can respond to TNF a ( 93, 94 ) . This suggests that their presence in an area of tissue injury could lead to an immediate cross-communication between MSCs and the microen-vironment. TNF a enhances the adhesion of MSCs ( 95, 96 ) . This role is interesting since it is possible that TNF a might be involved in mediating the attachment between MSCs and tissues. MSCs exert immune suppressive function partly through a decrease in TNF a in other immune cells ( 64 ) . Therefore, if MSCs mediate immune suppressive functions, this should decrease TNF a produc-tion. The question is whether this would be an advantage or a disadvantage, especially if TNF a is required for the MSCs to adhere and retain their location within the damaged tissue.

To understand the role of cytokines as mediators of cross talk between stem cells and the microenvironment of tissue injury requires including a discussion of miRNAs. They are single-strand RNA of approximately 19–25 nucleotides ( 97 ) . MiRNAs are derived from precursor hairpin-shaped transcripts ( 98, 99 ) and most act as a guide in posttranscriptional gene silencing by forming base pair structures with mRNA ( 97 ) . Although miRNAs repress translation of mRNA, they do not prevent mRNA from docking to polyribosomes and do not block the initiation of translation ( 89, 98, 100, 101 ) . MiRNAs are involved in the development of stem cells along distinct lineages ( 102– 104 ) . The role of miRNAs in stem cell biology has been established as their roles in neurogenesis,

131 Current Thoughts on the Therapeutic Potential of Stem Cell

including MSC-derived neurons, have been reported ( 105– 107 ) . Taken together, it is clear that cytokines are involved in the behavior of stem cells and miRNAs are involved in development. Therefore, studies are required to determine how cytokines affect stem cells through the regulation of miRNA.

IFN g is a critical cytokine in determining the immune response of MSCs, and perhaps other stem cells, from allogeneic sources. Type 1 IFN- g , although linked to viral protection ( 108 ) , has been involved in general immune responses. This cytokine is produced by T-cells, NK cells, and MSCs ( 57, 109, 110 ) . IFN g activates the type I receptor (IFN g RI) to activate JAK1 and JAK2, leading to phosphorylation and dimerization of STAT1 a ( 111 ) . IFN g can also mediate responses through intracrine mechanism ( 112– 114 ) . IFN g regulates the expression of MHC-II in a bimodal manner. At high levels, MHC-II is decreased, partly through the retention of the master regulator CIITA in the cytoplasm without any change in the IFN g receptor ( 57, 71 ) . This method of IFN g -mediated expression of MHC-II in MSCs is different in macrophages where the effect of IFN g is dose-dependent ( 57, 115, 116 ) .

The reduction in MHC-II by high IFN- g level is consistent with their immune suppressive function ( 117, 118 ) . Furthermore, even at high IFN g level, as third party cells, MSCs can be suppres-sive to graft vs. host disease ( 119 ) . Although inhibition on graft vs. host disease is a clinical bene fi t, the presence of MSCs might compromise graft vs. tumor effect ( 120 ) . The reduction in graft vs. host disease is expected to correlate with decrease in IFN g . This decrease could result in the expression of MHC-II. At this time, the outcome of this potential effect is yet to be studied. Long-term studies in animal models are required to address these questions for ef fi cient application of MSCs.

The question is what is the mechanism by which high level of IFN g cause immune suppression of MSCs? It is likely that IFN- g could induce the production of the immune suppressor TGF- b 1 ( 117, 118 ) . Also, IFN g can induce the release of indoleamine 2,3-dioxygenase from MSCs ( 121 ) . The effect of IFN g on MSC functions is complex since recent studies have indicated its role in cross presentation of antigens on MSCs ( 122 ) . This indicates that high levels of IFN g will not always exert immune suppressive pro-perties. Interestingly, TGF b 1 did not alter this cross presentation despite its changes in the intracellular molecules linked to antigen loading to the MHC molecule ( 122 ) .

8. Interferon Gamma in MSC Functions

14 P. Rameshwar

The above discussion focused on the role of cytokines in immune suppression of MSCs and as mediators for intercellular interactions. This section discusses whether cytokines can change the expression of genes linked to pluripotency. The discussion focuses on REST (Repressor Element-1 Silencing Transcription factor), also known as NRSF (Neuron Restrictive Silencing Factor). REST is a DNA-binding protein that exerts both tumor suppressor and oncogenic properties ( 123 ) . REST assembles a repressor complex to modify histone acetylation, chromosomal methylation, and DNA phos-phorylation in promoter regions of a wide array of genes ( 124– 130 ) . The cofactors utilized by REST depend on the type of cell ( 130– 133 ) , suggesting that there could be differences in the mechanisms by which REST is regulated in MSCs, and perhaps other stem cells. If there were differences by which REST is expressed in stem cells, this would indicate that stem cells, through the expression of REST, would cause varied outcomes in response to in fl ammatory mediators. This is particularly relevant to neurogenesis since REST represses the transcription of neuronal genes in nonneural tissues ( 124– 127 ) .

It is possible that REST expression could be changed by cytok-ines when the MSCs are placed at sites of tissue injury. Since REST is one of the tumor-linked genes, its expression in stem cells could be important for safety of this type of therapy. The same argument can be made for Oct - 4 , which is linked to both oncogenesis and pluripotency ( 134 ) . IL-1 can regulate the expression of other cytokines. One of these cytokines, TGF- b 1, can negatively affect in fl ammatory responses, since IL-1 a has been shown to cause a rapid decrease in REST expression in MSCs and their neuronal-induced cells ( 135 ) . While it is an advantage to have a pluripotent gene decrease for differentiation, rapid decrease in REST expression could predispose the cell to transformation, based on the reports that REST could have a tumor suppressor role ( 136– 138 ) .

In addition to cytokines, REST is also linked to other mole-cules during the development of stem cells to specialized cells. MiRNAs are beginning to “surface” as a central category of mediators in the development of stem cells to specialized cells. It is expected that their roles will be tightly linked to other genes such as REST , Oct - 4 , and in fl ammatory mediators. As an exam-ple, during the development of MSCs to neurons there is a decrease in REST expression, which correlates with an increase in miR124a ( 139 ) . Indeed, the miR124a seems to promote neuro-genesis. The regulated expression of REST , in consort with miR124a expression, is an example of the mechanisms by which

9. Cytokines in Pluripotency

151 Current Thoughts on the Therapeutic Potential of Stem Cell

different categories of molecules can co-ordinate to suppress the expression of nonneuronal genes in neurons while enhancing the expression of neural genes. It is highly likely that the link between miR124 and REST depends on Oct - 4 . Computer analyses have determined the presence of multiple REST sites in the 5 ¢ regula-tory region of Oct - 4 and vice versa. This suggests that Oct - 4 and REST could regulate the expression of each other. The inclusion of miRNAs is not expected to be independent of the cytokines. For example, IL-1 stimulation can cause a decrease in REST ( 107 ) . Reduced level of REST could lead to decreased Oct-4 and increase in miR124 (Fig. 1 ). While these parameters are estab-lished as a simpli fi ed network, this interaction is complex with other mediators and interactions with cells in close proximity. Ultimately, the cross talk between the stem cells and mediators within the microenvironment could affect the functional out-come of stem cells such as MSCs.

An understanding of how cross talk between stem cells and microenvironmental factors occurred required a discussion on two neurotransmitter genes. In addition, discuss the role of REST in their expressions during the development of MSCs to mature neurons. MSCs and other stem cells are nonneural cells. Therefore, they are expected to repress neural genes. However, when stem cells mature to neurons, the neural genes are de-repressed while nonneuronal genes are expressed to be repressed. REST expression would be critical in the expression of neural and nonneural genes. Indeed, the 5 ¢ noncoding region of the neurotransmitter TAC1 gene has one functional REST binding site while the tyrosine hydroxylase

IL-1

Neurogenesis REST ¯ Oct-4 ¯

miR124 �

TGF-β

Fig. 1. A network is depicted to describe MSC-mediated neurogenesis with representative cytokines, REST, Oct-4, and miRNA. IL-1 a , which can be found at sites of tissue injury, can enhance neurogenesis partly through the decrease in REST ( 107 ) . Low levels of REST could lead to decrease in Oct - 4 expression in MSCs. The presumed interaction between Oct-4 and REST is based on bioinformatic studies. Including in the network following a decrease in REST is an increase in miR124, leading to enhanced neurogenesis ( 139 ) . The functional inhibitory effect of TGF- b on immune functions could lead to a blunting response of IL-1.

16 P. Rameshwar

gene has three sites ( 135, 140 ) . As expected, REST acts as a repressor for TAC1 transcription in nonneuronal cells ( 124, 125, 135 ) . During the development of MSCs to neurons, REST expression is gradually decreased, leading to TAC1 expression ( 135 ) . IL-1 stimulation of MSCs or the early differentiated MSCs toward neurogenesis causes a rapid decrease in REST with concomitant increase in TAC1 expression ( 135 ) . This increase in the neurotrans-mitter gene is consistent with a repressor function of REST. If this fi nding were placed in perspective with MSCs at sites of tissue injuries, the MSCs would be exposed to multiple cytokines. This would facilitate cross talk between the MSCs and cytokines. At this time, it would be dif fi cult to predict the responses unless, there, the mediators are known with a molecular understanding of neuronal genes to cytokines and other in fl ammatory mediators.

Oct - 4 , also referred to as octamer-binding transcription factor (also OCT3/4 and Pou5F1), is expressed in adult and embryonic stem cells. However, the expression of Oct - 4 comes with contro-versies since others have argued against its expression in somatic cells and in the pluripotency of adult stem cells ( 14, 121, 141– 143 ) . Oct - 4 expression is decreased during differentiation. These fi ndings, combined with other reports, support a link between Oct - 4 and pluripotency ( 14, 144, 145 ) .

In the derivation of inducible pluripotent stem cells, Oct - 4 is among the four genes that can convert adult fi broblasts to cells to embryonic-like cells ( 146 ) . However, there is no data to suggest that Oct - 4 is the sole gene in self-renewal of stem cells. Oct - 4 expression is increased in various tumors ( 147, 148 ) , and has been shown in cancer-initiating cells ( 149 ) . Its role in malignancy is in the beginning phase of studies with a role to protect breast cancer cells from undergoing apoptosis ( 150 ) .

A signi fi cant question for stem cells in a microenvironment of in fl ammation is to study if the Oct - 4 gene is regulated by cytokines, as well as by other mediators. If cytokines can regulate Oct - 4 expression, this would lead to insights on the behavior of stem cells through changes in Oct - 4 expression not only upon implantation, but also during the development of stem cells to mature specialized cells. REST and Oct - 4 are among the genes that could explain the complex functions of stem cells and/or the cells that they generate. Several questions remain unanswered regarding the mechanisms by which cytokines affect the expression of these genes, and/or if there are indirect effects on one to affect the expression of the other (Fig. 2 ). These are important questions to understand how mediators at sites of tissue injuries can affect the outcomes of stem cells through Oct - 4 and REST . Although IL-1 and TGF b are dis-cussed as in fl ammatory mediators in the model of cross talk with stem cells, IFN g and TNF a have been studied in the immune biology of MSCs.

171 Current Thoughts on the Therapeutic Potential of Stem Cell

This section discusses the use of MSCs and/or their generated dopamine (DA) neurons for diseases such as Parkinson’s (PD) or traumatic brain injury (TBI), both of which are associated with defects in the dopaminergic system. PD and TBI are selected as examples to represent examples of cross talk between an environ-ment and stem cells because it is expected that the microenviron-ment could represent distinct milieu of in fl ammatory mediators. Since PD is a chronic disease, the pathology is likely to be different from TBI during the acute phase and even the beginning of a more chronic phase when there will be an abundance of in fl ammatory cells. In this case, one would expect the cross talk between the implanted cells and microenvironment to be different.

In the substantia nigra, DA neurons are required for motor control, hence their association with Parkinson’s disease ( 151, 152 ) . At present, it is unclear if brain disorders will be treated with MSCs and/or their generated DA-producing cells. Since this type of cellular treatment will be an alternative to fetal cells, other issues of interac-tions with in fl ammatory mediators and the possibility of rejection will need in-depth analyses before the data could be translated to patients ( 33, 151, 153 ) .

DA is a phenethylamine neurotransmitter. Its synthesis requires two enzymatic steps ( 153 ) . Tyrosine hydroxylase converts tyrosine to L-DOPA, followed by decarboxylation to DA ( 151, 153 ) . DA is stored in synaptic vesicles and upon release; it interacts with any of fi ve related G-protein-coupled receptors ( 154 ) . Similar to MSC-derived peptidergic neurons, MS-derived DA neurons also express receptors for in fl ammatory mediators ( 155 ) . It is these receptors

10. Representative Application

OCT4/REST

OCT4

/REST

OCT4OCT4RESTREST

OCT4 and REST regulatesthe expression of each other

OCT4 and REST Expression altered by cytokines (?)

Responses by Stem Cells (?)

Fig. 2. Shown are genes for Oct - 4 and REST , regulating the expression of each other at the level of transcription, in MSCs. Cytokines found at sites of tissue injuries can affect the expression of Oct - 4 and REST to affect the development of MSCs.

18 P. Rameshwar

that support communication with the regions of injuries. In fact, studies with nonhuman primates that were subjected to chemically induced PD reported on promising outcome, and suggest cross talk between the stem cells and factors in the microenvironment ( 156 ) .

Bioinformatics studies indicate that Oct - 4 and REST regulate the expression of each other (Fig. 1 ). Thus, it is expected that there will be tight control by these two genes to regulate the expression of each other. The question is what molecules regulate the expres-sion of one or both. It is highly possible that activators of cytokines could be involved in regulating Oct - 4 and/or REST in stem cells. Consequently, there could be responses by the stem cells to remain as pluripotent cells or to generate specialized cells. The outcome might depend on the milieu that is expected of injury.

This review summarizes the potential for complex interactions among cytokines and stem cells, using MSCs as an example. Despite the focus on this single type of stem cells, the information can be extrapolated to other stem cell types. The review attempts to bring to the attention of scientists that placing stem cells at any site of tis-sue injury might not provide their expected outcome. It is para-mount to consider the cytokines, other pro-in fl ammatory and anti-in fl ammatory mediators, as well as resident cells that could establish a cross talk with the implanted stem cells or their differen-tiated cells. The model presented in this review on IL-1 and TGF- b indicated the expression of receptors, based on the developmental stages of the MSCs toward neuronal formation. This information adds to the complexity of stem cell therapy since it would be dif fi cult to predict which receptors are expressed at a given time. This would indicate that the cross talk between the cells and mediators in a microenvironment could change rapidly, depending on the rate of differentiation. Furthermore, if the stem cells are dispersed within the site of tissue injury, there will be lack of synchrony with regard to the developmental stage of the stem cells, and also the types of receptors on each cells. Therefore, cross talk between the cells and mediators within the microenvironment of tissue damage would vary within a particular region of tissue damage.

The promise for successful therapy by MSCs is great even if their use would not require additional immune suppressive thera-pies to prevent rejection ( 35 ) . The versatility of MSCs is evident from studies that show their ability to be preconditioned by microenvironmental factors ( 157 ) . MSCs have been reported to integrate in brain regions of animals ( 158, 159 ) . Despite the immune suppressive properties of MSCs ( 34 ) , it appears that this might not be harmful to patients because these individuals are likely to clear a viral infection ( 110 ) .

11. Conclusion

191 Current Thoughts on the Therapeutic Potential of Stem Cell

It is important for this review to discuss the relevance of the microenvironment and stem cells with relation to spinal cord injury (SCI). Several groups, including ours, have generated ef fi cient methods to generate functional neurons and other neuronal cells from MSCs, discussed above. It is unclear if in fl ammatory media-tors could be a positive for SCI due to the potential to cause axonal regrowth. This question might not be answered at the molecular level in a complex system such as animal models. An in vitro system would allow us to answer this question in an autologous system in which neurons are placed in contact with skeletal muscle.

In an in vitro system, it would be possible to axotomize a neuron by microdissection to recapitulate injured neurons in SCI patients. Cross talk of injured neurons with in fl ammatory mediators can be studied by adding cytokines to the system. The system would allow for studies before and after injury on the responses of the injured nerve within a milieu of tissue factors. By establishing an ef fi cient system, the research could identify factors that are relevant to damage and repair. This information would allow for targeted translation of the science to patients where the treatment would take advantage of expected outcome from the development of cross talk between the stem cells and tissue factors at the site of tissue injury. The model would determine whether physicians could repair SCI through cross talk between neurons and other stem cells. Through such models, research could lead to an understanding of nerve regrowth, and synapse formation with skeletal muscle. As a fi nal point, it is possible that stem cells will need to be delivered with other drugs to direct how the stem cells would behave with their microenvironment.

Acknowledgments

This work is supported by a grant awarded by F.M. Kirby Foundation.

References

1. McCulloch EA, Till JE (2005) Perspectives on the properties of stem cells. Nat Med 11:1026–1028

2. Li L, Baroja ML, Majumdar A, Chadwick K, Rouleau A, Gallacher L, Ferber I, Lebkowski J, Martin T, Madrenas J, Bhatia M (2004) Human embryonic stem cells possess immune-privileged properties. Stem Cells 22:448–456

3. Drukker M, Katchman H, Katz G, Even-Tov FS, Shezen E, Hornstein E, Mandelboim O, Reisner Y, Benvenisty N (2006) Human embryonic stem cells and their differentiated

derivatives are less susceptible to immune rejec-tion than adult cells. Stem Cells 24:221–229

4. Swijnenburg RJ, Schrepfer S, Govaert JA, Cao F, Ransohoff K, Sheikh AY, Haddad M, Connolly AJ, Davis MM, Robbins RC, Wu JC (2008) Immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts. Proc Natl Acad Sci USA 105:12991–12996

5. Blum B, Benvenisty N (2008) The tumorige-nicity of human embryonic stem cells. Adv Cancer Res 100:133–158

20 P. Rameshwar

6. Parolini O, Alviano F, Bagnara GP, Bilic G, Buhring HJ, Evangelista M, Hennerbichler S, Liu B, Magatti M, Mao N, Miki T, Marongiu F, Nakajima H, Nikaido T, Portmann-Lanz CB, Sankar V, Soncini M, Stadler G, Surbek D, Takahashi TA, Redl H, Sakuragawa N, Wolbank S, Zeisberger S, Zisch A, Strom SC (2008) Concise review: isolation and charac-terization of cells from human term placenta: outcome of the fi rst international Workshop on Placenta Derived Stem Cells. Stem Cells 26:300–311

7. Dan YY, Riehle KJ, Lazaro C, Teoh N, Haque J, Campbell JS, Fausto N (2006) Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Natl Acad Sci USA 103:9912–9917

8. De Coppi P, Bartsch G, Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC, Snyder EY, Yoo JJ, Furth ME, Soker S, Atala A (2007) Isolation of amniotic stem cell lines with potential for therapy. Nat Biotech 25:100–106

9. Siegel N, Rosner M, Unbekandt M, Fuchs C, Slabina N, Dolznig H, Davies JA, Lubec G, Hengstschlager M (2010) Contribution of human amniotic fl uid stem cells to renal tissue formation depends on mTOR. Hum Mol Genet 19(17):3320–3331

10. Perin L, Sedrakyan S, Giuliani S, Da Sacco S, Carraro G, Shiri L, Lemley KV, Rosol M, Wu S, Atala A, Warburton D, De Filippo RE (2010) Protective effect of human amniotic fl uid stem cells in an immunode fi cient mouse model of acute tubular necrosis. PLoS One 5:e9357

11. Fuchs E (2008) Skin stem cells: rising to the surface. J Cell Biol 180:273–284

12. Takebe N, Ivy SP (2010) Controversies in cancer stem cells: targeting embryonic signal-ing pathways. Clin Cancer Res 16:3106–3112

13. Clement V, Dutoit V, Marino D, Dietrich PY, Radovanovic I (2009) Limits of CD133 as a marker of glioma self-renewing cells. Int J Cancer 125:244–248

14. Greco SJ, Liu K, Rameshwar P (2007) Functional similarities among genes regulated by OCT4 in human mesenchymal and embry-onic stem cells. Stem Cells 25:3143–3154

15. Woodward WA, Chen MS, Behbod F, Rosen JM (2005) On mammary stem cells. J Cell Sci 118:3585–3594

16. Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM (2001) Identi fi cation of mesenchymal stem/progeni-tor cells in human fi rst-trimester fetal blood, liver, and bone marrow. Blood 98:2396–2402

17. Castillo M, Liu K, Bonilla LM, Rameshwar P (2007) The immune properties of mesenchy-mal stem cells. Intl J Biomed Sci 3:100–104

18. Dominici M, Paolucci P, Conte P, Horwitz EM (2009) Heterogeneity of multipotent mesenchymal stromal cells: from stromal cells to stem cells and vice versa. Transplantation 87:S36–S42

19. Sakaguchi Y, Sekiya I, Yagishita K, Ichinose S, Shinomiya K, Muneta T (2004) Suspended cells from trabecular bone by collagenase digestion become virtually identical to mesen-chymal stem cells obtained from marrow aspi-rates. Blood 104:2728–2735

20. Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH (2004) Isolation of multipo-tent mesenchymal stem cells from umbilical cord blood. Blood 103:1669–1675

21. Meliga E, Strem BM, Duckers HJ, Serruys PW (2007) Adipose-derived cells. Cell Transplant 16:963–970

22. Troyer DL, Weiss ML (2008) Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells 26:591–599

23. Baksh D, Yao R, Tuan RS (2007) Comparison of proliferative and multilineage differentia-tion potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells 25:1384–1392

24. Caplan AI (1994) The mesengenic process. Clin Plast Surg 21:429–435

25. Delorme B, Ringe J, Pontikoglou C, Gaillard J, Langonne A, Sensebe L, Noel D, Jorgensen C, Haupl T, Charbord P (2009) Speci fi c lin-eage-priming of bone marrow mesenchymal stem cells provides the molecular framework for their plasticity. Stem Cells 27:1142–1151

26. Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I, Taglia fi co E, Ferrari S, Robey PG, Riminucci M, Bianco P (2007) Self-renewing osteoprogenitors in bone mar-row sinusoids can organize a hematopoietic microenvironment. Cell 131:324–336

27. Martinez C, Hofmann TJ, Marino R, Dominici M, Horwitz EM (2007) Human bone marrow mesenchymal stromal cells express the neural ganglioside GD2: a novel surface marker for the identi fi cation of MSCs. Blood 109:4245–4248

28. Deng J, Petersen BE, Steindler DA, Jorgensen ML, Laywell ED (2006) Mesenchymal stem cells spontaneously express neural proteins in culture and are neurogenic after transplanta-tion. Stem Cells 24:1054–1064

29. Padovan CS, Jahn K, Birnbaum T, Reich P, Sostak P, Strupp M, Straube A (2003) Expression of neuronal markers in differentiated

211 Current Thoughts on the Therapeutic Potential of Stem Cell

marrow stromal cells and CD133+ stem-like cells. Cell Transplant 12:839–848

30. Greco SJ, Zhou C, Ye JH, Rameshwar P (2007) An interdisciplinary approach and characterization of neuronal cells transdiffer-entiated from human mesenchymal stem cells. Stem Cells Dev 16:811–826

31. Trzaska KA, King CC, Li KY, Kuzhikandathil EV, Nowycky MC, Ye JH, Rameshwar P (2009) Brain-derived neurotrophic factor facilitates maturation of mesenchymal stem cell-derived dopamine progenitors to func-tional neurons. J Neurochem 110:1058–1069

32. Trzaska KA, Reddy BY, Munoz JL, Li KY, Ye JH, Rameshwar P (2008) Loss of RE-1 silenc-ing factor in mesenchymal stem cell-derived dopamine progenitors induces functional maturity. Mol Cell Neurosci 39:285–290

33. Trzaska KA, Kuzhikandathil EV, Rameshwar P (2007) Speci fi cation of a dopaminergic phenotype from adult human mesenchymal stem cells. Stem Cells 25:2797–2808

34. Potian JA, Aviv H, Ponzio NM, Harrison JS, Rameshwar P (2003) Veto-like activity of mes-enchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens. J Immunol 171:3426–3434

35. Blanc KL, Pittenger MF (2005) Mesenchymal stem cells: progress toward promise. Cytotherapy 7:36–45

36. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesen-chymal stem cells. Science 284:143–147

37. Delorme B, Ringe J, Gallay N, Le Vern Y, Kerboeuf D, Jorgensen C, Rosset P, Sensebe L, Layrolle P, Haupl T, Charbord P (2008) Speci fi c plasma membrane protein phenotype of culture-ampli fi ed and native human bone marrow mesenchymal stem cells. Blood 111:2631–2635

38. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang L, Norotte C, Teng PN, Traas J, Schugar R, Deasy BM, Badylak S, Buhring HJ, Giacobino JP, Lazzari L, Huard J, Peault B (2008) A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3:301–313

39. Caplan AI (2008) All MSCs are pericytes? Cell Stem Cell 3:229–230

40. Takashima Y, Era T, Nakao K, Kondo S, Kasuga M, Smith AG, Nishikawa SI (2007) Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 129:1377–1388

41. Brohlin M, Mahay D, Novikov LN, Terenghi G, Wiberg M, Shawcross SG, Novikova LN (2009) Characterisation of human mesenchy-mal stem cells following differentiation into Schwann cell-like cells. Neurosci Res 64:41–49

42. Cho KJ, Trzaska KA, Greco SJ, McArdle J, Wang FS, Ye JH, Rameshwar P (2005) Neurons derived from human mesenchymal stem cells show synaptic transmission and can be induced to produce the neurotransmitter substance P by interleukin-1{alpha}. Stem Cells 23:383–391

43. Katarzyna AT, Eldo VK, Pranela R (2007) Speci fi cation of a dopaminergic phenotype from adult human mesenchymal stem cells. Stem Cells 25:2797–2808

44. Nemeth K, Leelahavanichkul A, Yuen PST, Mayer B, Parmelee A, Doi K, Robey PG, Leelahavanichkul K, Koller BH, Brown JM, Hu X, Jelinek I, Star RA, Mezey E (2009) Bone marrow stromal cells attenu-ate sepsis via prostaglandin E2-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med 15:42–49

45. Bocelli-Tyndall C, Barbero A, Candrian C, Ceredig R, Tyndall A, Martin I (2006) Human articular chondrocytes suppress in vitro proliferation of anti-CD3 activated peripheral blood mononuclear cells. J Cell Physiol 209:732–734

46. Jones BJ, McTaggart SJ (2008) Immuno-suppression by mesenchymal stromal cells: from culture to clinic. Exp Hematol 36:733–741

47. Zhao S, Wehner R, Bornhañuser M, Wassmuth R, Bachmann M, Schmitz M (2010) Immunomodulatory properties of mesenchy-mal stromal cells and their therapeutic conse-quences for immune-mediated disorders. Stem Cells Dev 19:607–614

48. Wagner J, Kean T, Young R, Dennis JE, Caplan AI (2009) Optimizing mesenchymal stem cell-based therapeutics. Curr Opin Biotechnol 20:531–536

49. Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, Lanino E, Sundberg B, Bernardo ME, Remberger M, Dini G, Egeler RM, Bacigalupo A, Fibbe W, Ringdqn O (2008) Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 371:1579–1586

50. Nemeth K, Keane-Myers A, Brown JM, Metcalfe DD, Gorham JD, Bundoc VG, Hodges MG, Jelinek I, Madala S, Karpati S, Mezey E (2010) Bone marrow stromal cells

22 P. Rameshwar

use TGF- b to suppress allergic responses in a mouse model of ragweed-induced asthma. Proc Natl Acad Sci USA 107:5652–5657

51. Lim PK, Patel SA, Gregory LA, Rameshwar P (2010) Neurogenesis: role for microRNAs and mesenchymal stem cells in pathological states. Curr Med Chem 17(20):2159–2167

52. Ko IK, Kim BG, Awadallah A, Mikulan J, Lin P, Letterio JJ, Dennis JE (2010) Targeting improves MSC treatment of in fl ammatory bowel disease. Mol Ther 18:1365–1372

53. Richardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A (2010) Mesenchymal stem cells in regenerative medi-cine: opportunities and challenges for articu-lar cartilage and intervertebral disc tissue engineering. J Cell Physiol 222:23–32

54. da Silva ML, Caplan AI, Nardi NB (2008) In search of the in vivo identity of mesenchymal stem cells. Stem Cells 26:2287–2299

55. Greco SJ, Rameshwar P (2007) Enhancing effect of IL-1 on neurogenesis from adult human mesenchymal stem cells: implication for in fl ammatory mediators in regenerative medicine. J Immunol 179:3342–3350

56. Schinkothe T, Bloch W, Schmidt A (2008) In vitro secreting pro fi le of human mesenchymal stem cells. Stem Cells Dev 17:199–206

57. Chan JL, Tang KC, Patel AP, Bonilla LM, Pierobon N, Ponzio NM, Rameshwar P (2006) Antigen-presenting property of mes-enchymal stem cells occurs during a narrow window at low levels of interferon-gamma. Blood 107:4817–4824

58. Heng BC, Cowan CM, Davalian D, Stankus J, Duong-Hong D, Ehrenreich K, Basu S (2009) Electrostatic binding of nanoparticles to mesenchymal stem cells via high molecular weight polyelectrolyte chains. J Tissue Eng Regen Med 3:243–254

59. Romieu-Mourez R, Francois M, Boivin MN, Stagg J, Galipeau J (2007) Regulation of MHC class II expression and antigen processing in murine and human mesenchymal stromal cells by IFN-{gamma}, TGF-beta, and cell density. J Immunol 179:1549–1558

60. Campeau PM, Rafei M, Francois M, Birman E, Forner KA, Galipeau J (2008) Mesenchymal stromal cells engineered to express erythropoi-etin induce anti-erythropoietin antibodies and anemia in allorecipients. Mol Ther 17:369–372

61. Pistoia V, Raffaghello L (2010) Potential of mesenchymal stem cells for the therapy of autoimmune diseases. Expert Rev Clin Immunol 6:211–218

62. Rameshwar P, Qiu H, Vatner SF (2010) Stem cells in cardiac repair in an in fl ammatory

microenvironment. Minerva Cardioangiol 58:127–146

63. Patel SA, Sherman L, Munoz J, Rameshwar P (2008) Immunological properties of mesen-chymal stem cells and clinical implications. Arch Immunol Ther Exp 56:1–8

64. Kim J, Hematti P (2009) Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages. Exp Hematol 37:1445–1453

65. Dominici M, Le BK, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for de fi ning multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position state-ment. Cytotherapy 8:315–317

66. Pfeifer JD, Wick MJ, Roberts RL, Findlay K, Normark SJ, Harding CV (1993) Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature 361:359–362

67. Stagg J (2007) Immune regulation by mesen-chymal stem cells: two sides to the coin. Tissue Antigens 69:1–9

68. Stagg J, Pommey S, Eliopoulos N, Galipeau J (2006) Interferon-{gamma}-stimulated mar-row stromal cells: a new type of nonhe-matopoietic antigen-presenting cell. Blood 107:2570–2577

69. Fujiwara N, Kobayashi K (2005) Macrophages in in fl ammation. Curr Drug Targets In fl amm Allergy 4:281–286

70. Herrero C, Sebastian C, Marquqs L, Comalada M, Xaus J, Valledor AF, Lloberas J, Celada A (2002) Immunosenescence of macrophages: reduced MHC class II gene expression. Exp Gerontol 37:389–394

71. Tang KC, Trzaska KA, Smirnov S, Kotenko SV, Schwander SK, Ellner JJ, Rameshwar P (2008) Down-regulation of MHC-II in mes-enchymal stem cells at high IFN- can be partly explained by cytoplasmic retention of CIITA. J Immunol 180:1826–1833

72. Castillo MD, Trzaska KA, Greco SJ, Ponzio NM, Rameshwar P (2008) Immunostimulatory effects of mesenchymal stem cell-derived neu-rons: implications for stem cell therapy in allogeneic transplantations. Clin Transl Sci 1:27–34

73. Allan SM, Tyrrell PJ, Rothwell NJ (2005) Interleukin-1 and neuronal injury. Nat Rev Immunol 5:629–640

74. Moore MA (2002) Cytokine and chemokine networks in fl uencing stem cell proliferation, differentiation, and marrow homing. J Cell Biochem Suppl 38:29–38

231 Current Thoughts on the Therapeutic Potential of Stem Cell

75. Laver J, Moore MAS (1989) Clinical use of recombinant human hematopoietic growth factors. J Natl Cancer Inst 81:1370–1382

76. Dinarello CA (2005) Blocking IL-1 in systemic in fl ammation. J Exp Med 201:1355–1359

77. Dinarello CA (1996) Biologic basis for inter-leukin-1 in disease. Blood 87:2095–2147

78. Bagby GC (1989) Interleukin-1 and hematopoiesis. Blood Rev 3:152–161

79. Roberts AB, Sporn MB (1993) Physiological actions and clinical applications of trans-forming growth factor-beta (TGF-beta). Growth Factors 8:1–9

80. Massague J, Andres J, Attisano L, Cheifetz S, Lopez-Casillas F, Ohtsuki M, Wrana JL (1992) TGF-beta receptors. Mol Reprod Dev 32:99–104

81. Massague J, Weis-Garcia F (1996) Serine/threonine kinase receptors: mediators of transforming growth factor beta family signals. Cancer Surv 27:41–64

82. Shi Y, Massague J (2003) Mechanisms of TGF-[beta] signaling from cell membrane to the nucleus. Cell 113:685–700

83. Wrana JL (2000) Regulation of Smad activity. Cell 100:189–192

84. Massague J, Wotton D (2000) Transcriptional control by the TGF-beta/Smad signaling system. EMBO J 19:1745–1754

85. Sato M, Muragaki Y, Saika S, Roberts AB, Ooshima A (2003) Targeted disruption of TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fi brosis induced by unilateral ureteral obstruction. J Clin Invest 112:1486–1494

86. Mehra A, Wrana JL (2002) TGF-beta and the Smad signal transduction pathway. Biochem Cell Biol 80:605–622

87. Golestaneh N, Mishra B (2005) TGF-beta, neuronal stem cells and glioblastoma. Oncogene 24:5722–5730

88. Sokol JP, Schiemann WP (2004) Cystatin C antagonizes transforming growth factor beta signaling in normal and cancer cells. Mol Cancer Res 2:183–195

89. Downing JR (2004) TGF-{beta} signaling, tumor suppression, and acute lymphoblastic leukemia. N Engl J Med 351:528–530

90. Kim SJ, Letterio J (2003) Transforming growth factor-beta signaling in normal and malignant hematopoiesis. Leukemia 17:1731–1737

91. Tracey D, Klareskog L, Sasso EH, Salfeld JG, Tak PP (2008) Tumor necrosis factor antago-nist mechanisms of action: a comprehensive review. Pharmacol Ther 117:244–279

92. Locksley RM, Killeen N, Lenardo MJ (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104:487–501

93. van den Berk LCJ, Jansen BJH, Siebers-Vermeulen KGC, Roelofs H, Figdor CG, Adema GJ, Torensma R (2009) Mesenchymal stem cells respond to TNF but do not pro-duce TNF. J Leukoc Biol 87:283–289

94. Romieu-Mourez R, Francois M, Boivin MN, Bouchentouf M, Spaner DE, Galipeau J (2009) Cytokine modulation of TLR expres-sion and activation in mesenchymal stromal cells leads to a proin fl ammatory phenotype. J Immunol 182:7963–7973

95. Fu X, Han B, Cai S, Lei Y, Sun T, Sheng Z (2009) Migration of bone marrow-derived mesenchymal stem cells induced by tumor necrosis factor-alpha and its possible role in wound healing. Wound Repair Regen 17:185–191

96. Kim YS, Park HJ, Hong MH, Kang PM, Morgan JP, Jeong MH, Cho JG, Park JC, Ahn Y (2009) TNF-alpha enhances engraft-ment of mesenchymal stem cells into infarcted myocardium. Front Biosci 14:2845–2856

97. Kim VN (2005) MicroRNA biogenesis: coor-dinated cropping and dicing. Nat Rev Mol Cell Biol 6:376–385

98. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297

99. Ambros V, Bartel B, Bartel DP, Burge CB, Chen X, Carrington JC, Dreyfuss G, Eddy SR, Grif fi th-Jones S, Marshall M, Matzke M, Ruvkun G (2003) A uniform system for microRNA annotation. RNA 9:277–279

100. Zamore PD, Haley B (2005) Ribo-gnome: the big world of small RNAs. Science 309:1519–1524

101. Kim J, Krichevsky A, Grad Y, Hayes GD, Kosik KS, Church GM, Ruvkun G (2004) Identi fi cation of many microRNAs that copu-rify with polyribosomes in mammalian neu-rons. Proc Natl Acad Sci USA 101:360–365

102. Chen CZ, Li L, Lodish HF, Bartel DP (2004) MicroRNAs modulate hematopoietic lineage differentiation. Science 303:83–86

103. Ramkissoon SH, Mainwaring LA, Ogasawara Y, Keyvanfar K, Philip McCoy J, Sloand EM, Kajigaya S, Young NS (2006) Hematopoietic-speci fi c microRNA expression in human cells. Leuk Res 30:643–647

104. Harfe BD (2005) MicroRNAs in vertebrate development. Curr Opin Genet Dev 15:410–415

24 P. Rameshwar

105. Miska E, varez-Saavedra E, Townsend M, Yoshii A, Sestan N, Rakic P, Constantine-Paton M, Horvitz HR (2004) Microarray analysis of microRNA expression in the devel-oping mammalian brain. Genome Biol 5:R68

106. Schaefer A, O’Carroll D, Tan CL, Hillman D, Sugimori M, Llinas R, Greengard P (2007) Cerebellar neurodegeneration in the absence of microRNAs. J Exp Med 204:1553–1558

107. Greco SJ, Rameshwar P (2007) miRNAs regulate synthesis of the neurotransmitter substance P in human mesenchymal stem cell-derived neuronal cells. Proc Natl Acad Sci USA 104:15484–15489

108. Garcia-Sastre A, Biron CA (2006) Type 1 interferons and the virus-host relationship: a lesson in detente. Science 312:879–882

109. Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan S, Loudon R, Sherman F, Perussia B, Trinchieri G (1989) Identi fi cation and puri fi cation of natural killer cell stimula-tory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 170:827–845

110. Kang HS, Habib M, Chan J, Abavana C, Potian JA, Ponzio NM, Rameshwar P (2005) A paradoxical role for IFN-[gamma] in the immune properties of mesenchymal stem cells during viral challenge. Exp Hematol 33:796–803

111. Stark GR, Kerr IM, Williams BRG, Silverman RH, Schreiber RD (1998) How cells respond to interferons. Annu Rev Biochem 67:227–264

112. Ahmed CMI, Burkhart MA, Mujtaba MG, Subramaniam PS, Johnson HM (2003) The role of IFN{gamma} nuclear localization sequence in intracellular function. J Cell Sci 116:3089–3098

113. Subramaniam PS, Green MM, Larkin J III, Torres BA, Johnson HM (2001) Nuclear trans-location of IFN-gamma is an intrinsic require-ment for its biologic activity and can be driven by a heterologous nuclear localization sequence. J Interferon Cytokine Res 21:951–959

114. Subramaniam PS, Johnson HM (2004) The IFNAR1 subunit of the type I IFN receptor complex contains a functional nuclear local-ization sequence. FEBS Lett 578:207–210

115. Bergmann CC, Parra B, Hinton DR, Chandran R, Morrison M, Stohlman SA (2003) Perforin-mediated effector function within the central nervous system requires IFN-{gamma}-mediated MHC up-regula-tion. J Immunol 170:3204–3213

116. Boss JM (1997) Regulation of transcription of MHC class II genes. Curr Opin Immunol 9:107–113

117. English K, Barry FP, Mahon BP (2008) Murine mesenchymal stem cells suppress den-dritic cell migration, maturation and antigen presentation. Immunol Lett 115:50–58

118. English K, Ryan JM, Tobin L, Murphy MJ, Barry FP, Mahon BP (2009) Cell contact, prostaglandin E(2) and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4+CD25(High) forkhead box P3+ regula-tory T cells. Clin Exp Immunol 156:149–160

119. Polchert D, Sobinsky J, Douglas G, Kidd M, Moadsiri A, Reina E, Genrich K, Mehrotra S, Setty S, Smith B, Bartholomew A (2008) IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease. Eur J Immunol 38:1745–1755

120. Capitini CM, Herby S, Milliron M, Anver MR, Mackall CL, Fry TJ (2009) Bone mar-row de fi cient in IFN-{gamma} signaling selectively reverses GVHD-associated immu-nosuppression and enhances a tumor-speci fi c GVT effect. Blood 113:5002–5009

121. DelaRosa O, Lombardo E, Beraza A, Mancheno-Corvo P, Ramirez C, Menta R, Rico L, Camarillo E, Garcia L, Abad JL, Trigueros C, Delgado M, Buscher D (2009) Requirement of IFN-gamma-mediated indoleamine 2,3-dioxygenase expression in the modulation of lymphocyte proliferation by human adipose-derived stem cells. Tissue Eng Part A 15:2795–2806

122. Francois M, Romieu-Mourez R, Stock-Martineau S, Boivin MN, Bramson JL, Galipeau J (2009) Mesenchymal stromal cells cross-present soluble exogenous antigens as part of their antigen-presenting cell proper-ties. Blood 114:2632–2638

123. Coulson JM (2005) Transcriptional regulation: cancer, neurons and the REST. Curr Biol 15:R665–R668

124. Fiskerstrand CE, Newey P, McGregor GP, Gerrard L, Millan F, Quinn JP (2000) A role for Octamer binding protein motifs in the regulation of the proximal preprotachykinin-A promoter. Neuropeptides 34:348–354

125. Quinn JP, Bubb VJ, Marshall-Jones ZV, Coulson JM (2002) Neuron restrictive silencer factor as a modulator of neuropeptide gene expression. Regul Pept 108:135–141

126. Su X, Kameoka S, Lentz S, Majumder S (2004) Activation of REST/NRSF target genes in neural stem cells is suf fi cient to cause neuronal differentiation. Mol Cell Biol 24:8018–8025

127. Yeo M, Lee SK, Lee B, Ruiz EC, Pfaff SL, Gill GN (2005) Small CTD phosphatases function

251 Current Thoughts on the Therapeutic Potential of Stem Cell

in silencing neuronal gene expression. Science 307:596–600

128. Bruce AW, Donaldson IJ, Wood IC, Yerbury SA, Sadowski MI, Chapman M, Gottgens B, Buckley NJ (2004) Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes. Proc Natl Acad Sci USA 101:10458–10463

129. Wood IC, Belyaev ND, Bruce AW, Jones C, Mistry M, Roopra A, Buckley NJ (2003) Interaction of the repressor element 1-silenc-ing transcription factor (REST) with target genes. J Mol Biol 334:863–874

130. Belyaev ND, Wood IC, Bruce AW, Street M, Trinh JB, Buckley NJ (2004) Distinct RE-1 silencing transcription factor-containing complexes interact with different target genes. J Biol Chem 279:556–561

131. Zhang P, Pazin MJ, Schwartz CM, Becker KG, Wersto RP, Dilley CM, Mattson MP (2008) Nontelomeric TRF2-REST interac-tion modulates neuronal gene silencing and fate of tumor and stem cells. Curr Biol 18:1489–1494

132. Greenway DJ, Street M, Jeffries A, Buckley NJ (2007) RE1 silencing transcription factor maintains a repressive chromatin environment in embryonic hippocampal neural stem cells. Stem Cells 25:354–363

133. Sun YM, Greenway DJ, Johnson R, Street M, Belyaev ND, Deuchars J, Bee T, Wilde S, Buckley NJ (2005) Distinct pro fi les of REST interactions with its target genes at different stages of neuronal development. Mol Biol Cell 16:5630–5638

134. Trosko JE (2006) From adult stem cells to cancer stem cells: Oct-4 Gene, cell-cell com-munication, and hormones during tumor promotion. Ann N Y Acad Sci 1089:36–58

135. Greco SJ, Smirnov S, Rameshwar P (2007) Synergy between RE-1 silencer of transcrip-tion (REST) and NFêB in the repression of the neurotransmitter gene Tac1 in human mesenchymal stem cells: implication for microenvironmental in fl uence on stem cell therapies. J Biol Chem 282:30039–30050

136. Weissman AM (2008) How much REST is enough? Cancer Cell 13:381–383

137. Majumder S (2006) REST in good times and bad: roles in tumor suppressor and oncogenic activities. Cell Cycle 5:1929–1935

138. Reddy BY, Greco SJ, Patel PS, Trzaska KA, Rameshwar P (2009) RE-1-Çosilencing tran-scription factor shows tumor-suppressor func-tions and negatively regulates the oncogenic TAC1 in breast cancer cells. Proc Natl Acad Sci USA 106:4408–4413

139. Conaco C, Otto S, Han JJ, Mandel G (2006) Reciprocal actions of REST and a microRNA promote neuronal identity. Proc Natl Acad Sci USA 103:2422–2427

140. Kim SM, Yang JW, Park MJ, Lee JK, Kim SU, Lee YS, Lee MA (2006) Regulation of human tyrosine hydroxylase gene by neuron-restrictive silencer factor. Biochem Biophys Res Commun 346:426–435

141. Berg JS, Goodell MA (2007) An argument against a role for Oct4 in somatic stem cells. Cell Stem Cell 1:359–360

142. Lengner CJ, Welstead GG, Jaenisch R (2008) The pluripotency regulator Oct4: a role in somatic stem cells? Cell Cycle 7:725–728

143. Saxe JP, Tomilin A, Scholer HR, Plath K, Huang J (2009) Post-translational regulation of Oct4 transcriptional activity. PLoS One 4:e4467

144. Ji KH, Xiong J, Hu KM, Fan LX, Liu HQ (2008) Simultaneous expression of Oct4 and genes of three germ layers in single cell-derived multipotent adult progenitor cells. Ann Hematol 87:431–438

145. Liedtke S, Stephan M, Kogler G (2008) Oct4 expression revisited: potential pitfalls for data misinterpretation in stem cell research. Biol Chem 389:845–850

146. Tweedell KS (2008) New paths to pluripotent stem cells. Curr Stem Cell Res Ther 3:151–162

147. Karoubi G, Gugger M, Schmid R, Dutly A (2009) OCT4 expression in human non-small cell lung cancer: implications for therapeutic intervention. Interact Cardiovasc Thorac Surg 8:393–397

148. Sotomayor P, Godoy A, Smith GJ, Huss WJ (2009) Oct4A is expressed by a subpopula-tion of prostate neuroendocrine cells. Prostate 69:401–410

149. Levings PP, McGarry SV, Currie TP, Nickerson DM, McClellan S, Ghivizzani SC, Steindler DA, Gibbs CP (2009) Expression of an exogenous human Oct-4 promoter identi fi es tumor-initiating cells in osteosar-coma. Cancer Res 69:5648–5655

150. Hu T, Liu S, Breiter DR, Wang F, Tang Y, Sun S (2008) Octamer 4 small interfering RNA results in cancer stem cell-like cell apop-tosis. Cancer Res 68:6533–6540

151. Perrier AL, Studer L (2003) Making and repairing the mammalian brain—in vitro pro-duction of dopaminergic neurons. Semin Cell Dev Biol 14:181–189

152. Lange KW, Mecklinger L, Walitza S, Becker G, Gerlach M, Naumann M, Tucha O (2006) Brain dopamine and kinematics of graphomo-tor functions. Hum Mov Sci 25:492–509

26 P. Rameshwar

153. Trzaska KA, Rameshwar P (2007) Current advances in the treatment of Parkinson’s disease with stem cells. Curr Neurovasc Res 4:99–109

154. Shigetomi S, Fukuchi S (1994) Recent aspect of the role of peripheral dopamine and its receptors in the pathogenesis of hypertension. Fukushima J Med Sci 40:69–83

155. Tansey MG, McCoy MK, Frank-Cannon TC (2007) Neuroin fl ammatory mechanisms in Parkinson’s disease: potential environmental triggers, pathways, and targets for early thera-peutic intervention. Exp Neurol 208:1–25

156. Sanberg PR (2007) Neural stem cells for Parkinson’s disease: to protect and repair. Proc Natl Acad Sci USA 104:11869–11870

157. Gregory CA, Ylostalo J, Prockop DJ (2005) Adult bone marrow stem/progenitor cells (MSCs) are preconditioned by microenvi-ronmental “Niches” in culture: a two-stage hypothesis for regulation of MSC fate. Sci STKE 2005:e37

158. Honma T, Honmou O, Iihoshi S, Harada K, Houkin K, Hamada H, Kocsis JD (2006) Intravenous infusion of immortalized human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat. Exp Neurol 199:56–66

159. Shyu WC, Lee YJ, Liu DD, Lin SZ, Li H (2006) Homing genes, cell therapy and stroke. Front Biosci 11:899–907