Hematopoietic Stem Cells:  A Scientific Review

Introduction
In 1917, it was postulated that undifferentiated stem cells gave rise to a plethora of blood cells via an intermediate state of progenitor cells (Huntley, et al. 2005).  In 1963, evidence was provided for the existence of hematopoietic stem cell (HSC) and the search began to isolate this stem cell (Weissman, 2005).  In 1992, scientists isolated the candidate human HSC population that self-renewed with clonal progenies to form all types of blood cells (Baum, et at. 1992).  The HSC, which give rise to either a lymphoid or myeloid lineage, are responsible for producing and maintaining the blood and immune systems.  Recent studies have also provided evidence that HSC demonstrate plasticity, indicating that they can differentiate into non-immune/blood cells: endothelial, hepatic, muscle, neural, and cardiac lineages (Sata, et al. 2002; Chan, et al. 2004).

HSCs exhibit the following characteristics: 1) self-renew, 2) differentiate into multiple cell lineages, 3) mobilize from the bone marrow into the peripheral blood, and 3) undergo apoptosis (Shizura, et al. 2005).  HSCs can be identified by phenotypic methods.  This is possible through the expression of cell surface markers that differ in mice and human (Bonde, et al. 2004; Chan, et al, 2004).  In adults, the main source of HSCs is the bone marrow where they form contact with stromal cells (Huygen, et al. 2002).  HSCs can be found in other sources as well, such as the peripheral blood in the case of adult, and in the umbilical cord in newborns (Auletta, et al. 2005).
Several disorders, such as anemia, cytopenia, and aging have been linked to dysfunction of HSCs.  Transplantation of HSCs from a closely matched donor is common for hematological disorders, including malignancies.  HSC transplantation could be from autologous or allogeneic sources from bone marrow or HSCs mobilized into the peripheral blood (Perry, et al. 1996; Lewis, 2005; Down, et al. 2003).  While HSCs shown success (Weissman, et al. 2005), there are still many challenges for effective HSC transplant, while reducing the risk of side effects (Wanko, et al. 2005).  Other disorders, such as several autoimmune diseases and severe-combined immunodeficiency, arise from a genetic basis (Beilhack, et al. 2003).  Gene therapy, although encompassing many risks, is being explored as a means of treatment for these disorders.
History
In the 19th century, Virchow and Schwann first described mammalian tissues to be composed of cells, leading to the claim that cells originate exclusively from other cells. Half a century later, in 1917, Pappenheim postulated the existence of an undifferentiated stem cell giving rise to the plethora of blood cells via an intermediate state of progenitor cells (Huntley, et al. 2005).  Later, several groups corroborated the existence of the hematopoietic stem cell (HSC) in the bone marrow. In 1961, James Till and Ernest McCulloh discovered HSCs while studying sensitivity of irradiated mouse bone marrow cells. They called this population “spleen colony forming unit” due to their ability to form a colony of cells in the spleen of a transplant recipient mouse. They were characterized by their ability of self-renewal and differentiation into all blood cell types (Fangming, et al. 2007).

HSC Niche

The main source of hematopoietic stem cells is the bone marrow.  The HSC live in tight junction with the stromal cells of the bone marrow via cell adhesion molecules—a prominent example being fibronectin (Huygen, et al. 2002). HSCs also exist in close association with osteoblasts near the trabecular bone. This niche gives signals to HSC for self-renewal survival, and quiescence (R. Welner, et al. 2007). The matrix component, osteopontin synthesized by osteoblasts, has been shown to negatively regulate the HSC compartment (Blank U, et al. 2007). It is this microenvironment that either retains the “stemness” of the HSC or advances the HSC to differentiate, proliferate, or undergo apoptosis (Wang, et al. 2005).  Research has found HSCs in other sources aside from the bone marrow, such as the peripheral blood and umbilical cord blood (Auletta, et al. 2005). 

Bone marrow is considered the primary site for blood cell formation, however during fetal development the spleen and liver are sites of importance for this process. Moreover, in conditions like myelofibrosis where increase blood cell formation is required, the spleen and liver can be reactivated (R. Welner, et al. 2007). HSCs undergo massive self-renewal in the fetal liver during ontogeny. When these HSCs are transplanted into irradiated recipients, they prove to have more regenerative potential than adult HSCs. A developmental switch has been suggested by the research of Bowie et al., which changes HSCs from a cycling population into a quiescent one at the age of 3-4 weeks in mouse models. The alteration develops independently of the niche because it takes place after the migration of HSCs to the bone marrow (Blank U, et al. 2007).

The Hematopoietic Hierarchy
Figure 1

Hierarchy of Development in the Hematopoietic Stem Cell

It is difficult to isolate stem cells to study their individual biological processes because of their heterogeneity.  Most studies to date have focused on population analysis in hopes of identifying stem cells with functional and phenotypic potentials (Sharkis, et al. 2001).
Further advancement in technology gave rise to new approaches in the studies of HSC, particularly of murine origin (Punzel, et al. 2001).  The revealing of morphogenesis via germ layers in the early embryo fostered the idea of directed cellular proliferation.  To aid with isolation, a quantitative in vivo assay, like xenotransplantation, has made it possible to analyze single cells of highly defined populations of mouse HSC.  Cell surface marker characterization was aided by the development of flow cytometry machines around the late 1960s.  This is an automated technique used to identify and/or sort distinct cell populations, based on the ability of fluorescently labeled antibodies to bind cell-surface antigens (Huntly, et al. 2005).  The development in flow cytometry and clonogenic assays made cell separation possible in vitro, and helped in delineating the pivotal roles played by many of the regulatory cytokines (Punzel, et al. 2001).
Functional HSCs lack expression of a variety of cell surface markers that are normally found on differentiated blood cells. However, they display high levels of Sca1 and c-kit and are referred as lineage-, Sca1+, c-kit+. At the top of the hematopoietic hierarchy, resides a unique population of HSCs that give rise to progeny that lose self-renewal ability, become differentiated and commited to a cell lineage. HSCs are divided by their self renewal capability into long-term (LT-HSCs) and short-term (ST-HSCs) reconstituting HSCs. LT-HSCs have a broad self-renewal capability and are able to sustain lifelong hematopoiesis. In the other hand, ST-HSCs have restricted self-renewal ability, where hematopoiesis is maintained for a limited time (Blank U, et al. 2007). Multicolor flourescence-activated cell sorting system permitted isolation of the HSC lineage restricted progenitors at different developmental stages: common lymphoid progenitor (CLP) and common myeloid progenitor (CMP). The CLP lineage makes T-cells, B-cells, and natural killer (NK) cells. The CMP lineage produces a variety of functionally diverse cells, which consist of granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, erythrocytes, megakaryocytes, and mast cells.  These two lineages develop independently downstream of HSCs (Iwasaki, et al. 2007).

Scientists (1992) isolated a candidate human HSC population that self renewed and included, in their clonal progeny, all blood cell types (Baum, et at. 1992).  Several years later, the downstream progenitors of mouse and human hematopoietic lineage had been isolated.  The successful cloning of Dolly from an adult tissue cell helped spur several groups to reevaluate the differentiation capacity of adult tissue stem cells like HSC (Ho, et al. 2003).

HSCs activity is linked to exclusion of fluorescent dyes like Hoechst and Rhodamine. Isolation of human HSCs is challenging because of the lack of proper in vivoassays. The Rhodamine fraction of the Lin-CD34+CD38- population of human cord blood (CB) with HSCs at a frequency of 1 in 30 cells represents the most stringent isolation of human HSCs.  Human HSCs can be functionally evaluated using the non-obese diabetic/severe combined immune deficient (NOD/SCID) mouse models. The SCID repopulating cells correspond to the most quantifiable primitive hematopoietic population; however NOD/SCID assays are hindered by the difficulty to maintain NOD/SCID mice for extended trials and the low engraftment of human cells. Serial NOD/SCID transplantations may overcome this obstacle and allow study of even more primitive populations (Blank U, et al. 2007).

Signaling Pathways

Wnt signaling has long been linked to the regulation of murine HSCs.  Van den Berg et al. demonstrated that Wnt signaling also expanded human HSCs in vitro, which was measured by colony and immunophenotypic assays. Recent research reports show that Notch and Wnt pathways act in synergy to sustain the HSC pool. The two signaling pathways were shown to be active at the same time in a large portion of cells in the trabecular bone. Self-renewal capability of Lt-HSCs has been linked to the activation of the Notch signaling pathway or the downstream target Hes-1. Additionally, osteoblasts stimulated to express Jagged-1 were able to enhance self-renewal ability of primitive hematopoietic cells (Blank U, et al. 2007).

Types of Cells HSCs Produce/ Lineages

The process by which generating daughter cells maintain their parental properties is known as self-renewal.  Long-term hematopoietic stem cells (Lt-HSC) are multipotent cells capable of both differentiation and self-renewal.  Differentiation of Lt-HSC gives rise to the short-term hematopoietic stem cell (St-HSC).  St-HSC retain full hematopoietic differentiation potential but retain a limited self-renewal potential, giving them the ability to proliferate but not self-renew.  Evolving from the St-HSC is the multipotent progenitor (MPP), which directs the stem cell to either a lymphoid or myeloid lineage.  Further differentiations are restricted to their respective lineages and are incapable of self-renewal.  Cells of the lymphoid lineage give rise to B-lymphocytes, T-lymphocytes, and natural killer cells of the immune system.  Memory B and T cells are mature blood cells that re-acquire the ability to undergo long term self-renewal and are the product of a controlled process of differentiation in response to immunostimulation such as pathogenic infections.  But prior to infection, naïve cells exist in very low quantities.  Upon stimulation, the antigen-inexperienced cells capable of recognizing pathogenic isotopes undergo a process of clonal expansion and differentiation.  This process leads to the generation of effector cells that have acquired the ability to combat the pathogen.  Apoptosis regulates the number of circulating cells after the initial infection has taken place.  The remaining T cells remaining after antigen exposure comprises the memory T cell subset.  In B cells, early thymus dependent responses to antigen stimulation leads to the formation of short lived, rapidly proliferating plasma cells and germinal center B cells.  The majority of these two cell types undergo apoptosis as in T cells, and the surviving cells from these two groups separate into two separate memory compartments: self-renewing memory B cells and long-lived, antibody-secreting plasma B cells (Luckey C.J., et al. 2005).

Those of myeloid lineage form monocytes, macrophages, neutrophils, erythrocytes, or megakaryocytes (Chan, et al. 2004) (see figure 1).  As expected from these final end products, the major function of the HSC is to produce and maintain cells of both the blood and immune systems.  Recent studies have also cited HSC plasticity in which endothelial, hepatic, muscle, neural, and cardiac cells have differentiated from the HSC (Sata, et al. 2002).  This plasticity may explain the proposal that demonstrates dendritic cells being produced by both common myeloid and lymphoid progenitor cells (Bryder D. et al, 2006).

A recent study mapped the BB9 marker expression in hematopoietic development and found that the most primitive HSC arising at the ventral wall of the aorta and surrounding endothelial cells are BB9+.  This research deduced that BB9 is expressed by primitive hematopoietic cells in the fetal liver and umbilical cord blood. When BB9+CD34+ UCB cells were transplanted into NOD/SCID mice, they produced 10-fold more multi-lineage blood cells than the CD34+BB9- counterparts. Additionally, these cells had a higher frequency of SCID reconstituting cells. With the use of protein micro-sequencing of the 160kDa band, which corresponds to the BB9 protein, confirmed its identity as the somatic Angiotensin Converting Enzyme (ACE). Even though the role of ACE on human HSC is yet to be determined, further research may confirm ACE as a marker of human HSC throughout hematopoietic ontogeny and adulthood (Vanta, et al. 2007).

Disorders

HSCs are particularly sensitive to a large number of physical, chemical, and biological agents.  They are also responsible for a large number of blood pathologies and represent a high-interest target for the genetic treatment of various hematological diseases and non-hematological diseases.  Normal hematopoiesis requires several factors to maintain normal function of differentiated cell production throughout an individual’s lifetime, such as a supportive microenvironment and a highly regulated system of appropriate growth factors.  Disruption of normal production and function can lead to a variety of disorders, including anemia, neutropenia, and thrombocytopenia.  Many mechanisms of hematopoietic disorders are related to various cancers or to their treatments, either directly or indirectly.
A significant decrease in the pluripotency of HSC is proportional to the individual’s age. Telomere shortening is the sole factor limiting the capacity of normal hematopoietic cells and their indefinite division ability.  Telomere restriction fragment (TRF) length is shown to decrease with each HSC proliferation, and therefore, with age progression.  Similarly, findings in childhood leukemias indicate that leukemic blast cells have shorter TRF length than normal HSCs (Takauchi et al., 1994; Sugihara et al., 1999).

Metastases in the bone marrow cavity may cause damage to the bone marrow, resulting in a negative impact on the microenvironment.  This leads to the impairment of the production of growth factors and to the increase in cytokine production, which could cause inhibition of hematopoiesis.  The cytotoxic chemotherapeutic agents used in the treatments of most cancers have a negative impact on hematopoiesis.  When cytotoxic agents are administered at high doses for long periods of time, there is a significant depletion of hematopoietic stem cells.

Cytopenias include disorders that result from the reduction in erythrocyte, neutrophil, and platelet numbers.  In cytopenia, an increased hematopoietic cell consumption or destruction is prominent.  Many mechanisms of various cytopenias occur as a result of treatments of cancer, including, but not limited to, radiation and drug treatments.  G-CSF and GM-CSF are treatments of cytopenias, given shortly after completion of chemotherapy.
Aplastic anemia results from the failure of HSC to produce normal blood cells and megakaryocytes.  In aplastic anemia, the CD-34 cell population is severely deficient in number.  Several influencing factors include radiation, infection, drugs, chemicals, and immune mechanisms.  The first line of therapy is the attempt to stimulate the marrow with erythropoietin (EPO) and GM-CSF in order to reestablish normal hematopoiesis (Allsopp et al., 2003).
Presently, through numerous studies, there is a growing body of evidence that indicate the plasticity of adult hematopoietic marrow cells.  Research has shown that these adult stem cells are able capable of differentiating into an assortment of various cells from non-hematopoietic organs, such as from the bone, skin, muscle, liver, and lung.  In murine models, functional and anatomically normal pulmonary epithelial cells have been shown to derive from exogenous marrow cells.  Therefore, in certain human pulmonary diseases, a “normal” phenotype may be attained if defective cells were to be replaced by a small number of efficient cells (Aliota J.M, et al 2007).

Stem cells play a key role in the repair of diverse lung injuries, but many factors have contributed to the lack of understanding in this area.  Insufficient research, low rates of in-vivo cellular proliferation, as well as the structural and cellular complexity of the respiratory tract have contributed to the deficiency in lung stem cell knowledge when compared to other organ systems.  Many lineages comprise the tissues of the lung.  Cells of multiple lineages interact both during morphogenesis and maintain adult lung structure.  Recent studies have reported findings that HSC’s contribute to the repair of certain lineages (Aliotta J.M., et al 2007; Neuringer, et al. 2004).

In order to understand the lung in its healthy state, one has to examine this tissue in a diseased state.  There are different views on repair and this is where stem cells come into play.  The traditional view of repair occurs during development.  Here, self-renewing tissues are saturated with resident tissue-specific stem cells.  Since there are many different tissues that comprise the lung, it is difficult to distinguish which adult somatic cells are involved.  Recent evidence suggests another view, which is that of plasticity.  Stem cells from a variety of sources can generate tissues of other lineages as well as their own.  And this is where the hematopoietic stem cell comes into play.  Cells derived from the bone marrow may augment the resident stem cells in an area and differentiate into the tissue localized in that particular germinal lineage (Aliotta J.M. et al 2007; Neuringer, et al. 2004).

Plasticity is not completely understood as of yet, but there is evidence for and against this phenomena. Both, experimental studies in animals and clinical trials in humans, have provided evidence for and against circulatory delivery of lung progenitor cells.  Some findings are suggesting that adult stem cells from outside the bone marrow may reconstitute the hematopoietic system.  But most reports are showing a different view. Cells from the bone marrow can generate diverse non-hematopoietic cell types.  Mast cells, lymphocytes, alveolar macrophages, and dendritic cells are some of the bone marrow derived cells that migrate to the lung.  Under certain circumstances, new evidence suggests that circulating cells can apparently generate lung resident cells as well, such as epithelial, endothelial, and myofibroblast cells (Neuringer, et al. 2004).

The identification of these cells is a technically challenging process involving the co-localization of a donor cell marker.  These markers are present in sex-mismatched transplantation, a genetically engineered marker in murine experiments, and proteins that are characteristic of the differentiated cell type in the lung.  These lung proteins include collagen in fibroblasts and even keratin in epithelial cells.  Results on these findings are again contradictory, inconsistent, and dependent of many different factors, including: the methods for marker detection, amount of injury to the lung, and the starting cell population.  The mechanisms by which cells take on typical lung cell phenotypes: cell fusion, bone marrow derived cells or transdifferentiation, remains unknown and controversial.  They may all play a role in lung repair by promoting the local production of stem cells or reparative lung function of various cell types specific to the lung (Neuringer, et al. 2004).

Table 1
Unbiased evidence regarding circulating progenitor cell generation of non-hematopoietic lung cell types.

Hematopoietic Stem Cell Transplantation (HSCT)
History
The first successful infusion of allogeneic bone marrow was performed in 1958. However, graft rejection, due to lingering residual host lymphocytes post-irradiation, was a major impediment for widespread success (Perry, et al. 1996).  In 1968 and 1971, successes of bone marrow grafts were the result of HSCs being able to reconstitute the blood and immune system after myeloablation (Ho, et al. 2003).  With the discovery of the human leukocyte antigen (HLA) histocompatibility system in the mid-20th century, the first HLA-matched transplant in a child with immunodeficiency was performed in 1968.  By the 1970s, transplantations were used in patients suffering from aplastic anemia and leukemia.  Bone marrow transplants and cryopreservation techniques increased in the 1980s, and peripheral blood harvesting methods were also introduced (Perry, et al. 1996).

Sources of HSCs for Transplantation
HSCs used in transplantation can be derived from peripheral blood, bone marrow, or umbilical cord blood.  Stem cells derived from peripheral blood (PB) speed engraftment of neutrophil and platelets and display more rapid hematopoietic recovery, as compared to standard bone marrow transplant (BMT) (Lewis, 2005; Eapen, et al. 2004).  In PB, however, only few HSC are present, therefore, requiring an injection of growth factors during harvest, such as (granulocyte colony stimulating factor) G-CSF to mobilize the HSC from the BM to periphery (de Vries et al. 2004; Eapen, et al. 2004).  Harvesting of stem cells (SC) from PB is less burdensome and safer than from BM, in that the donors do not have to undergo general anesthesia or hospitalization, and is therefore, preferred especially for pediatric donors (de Vries et al. 2004; Eapen, et al. 2004; Diaz, et al. 2005). A third alternate source of HSC is umbilical cord blood, which tends to exhibit higher proliferative capacity and expansion potential, and have a higher percentage of stem cells (de Vries, et al. 2004).

Ideally, HSCs would be expanded ex vivo, so as to eliminate the need for donors, bone marrow aspirates, and harvesting; however, presently, this method does not seem attainable.  In vitro methods, such as the addition of cytokines, are insufficient in establishing the self-renewing quality of HSCs (Nakano, 2003) because once a stem cell is manipulated ex vivo; it tends to lose its “stemness.”  However, some researchers claim that ex vivo expansion may be possible under hypoxic conditions or with the use of extracellular signaling molecules (Nakano, 2003; Ivanovic, et al. 2004).

Autologous vs. Allogeneic Transplantation
Autologous transplantation entails the re-infusion of the patient’s own stem cells, which are harvested prior to treatment, and used to repopulate the cells of the bone marrow that were killed with myeloablative or lethal chemotherapeutic agents.  Allogeneic transplantations require the obtaining of bone marrow from a donor whose HLA matches closest to that of the recipient, with the best match being from a sibling (Lewis 2005; Down, et al. 2003).  Once harvested, the peripheral blood stem cells that are to be used for autologous transplantation are cryopreserved and stored in liquid nitrogen for later use (de Vries, et al. 2004).  Donor bone marrow for allogeneic BMT does not need to be cryopreserved and can be directly infused into the patient (de Vries, et al. 2004).  The advantage of autologous transplantation is that no graft-versus-host disease (GVHD) will occur as does with allogeneic transplantation (de Vries, et al. 2004).  GVHD leads to tissue damage caused by the recruitment of cytokines and through the activation of donor T-cells and natural killer (NK) cells by recipient alloantigens (Wanko, et al. 2005). Allogeneic PBSC transplantations increase risk of chronic GVHD due to the presence of a higher number of T cells (de Vries, et al. 2004; Eapen, et al. 2004), contributing to late mortality in patients surviving beyond 2 years after transplantation (Diaz, et al. 2005). The risk of GVHD is greater with increased disparity between the donor and recipient, with regards to HLA (Down, et al, 2003).  Disadvantages of autologous transplantation, however, are that these blood products may be contaminated with tumor cells, and that no graft-versus-tumor/leukemia (GVL) effect will occur, as seen with allogeneic transplantation (de Vries, et al. 2004; Down, et al, 2003).

Homing and Engraftment of HSCs
Homing is the rapid, non-random migration of HSCs through the blood to their niche in the stroma, near the endosteum in the BM (Lapidot, 2005). Homing is regulated by the interactions between markers found on HSCs, such as CXCR4, VLA4, and CD44, with SDF-1, VCAM1, and hyaluronic acid (chemokines and adhesion molecules found in the niche), respectively (Lapidot 2005) (See figure 3).  A 7-transmembrane receptor, CXCR4, present on HSC, will bind to stromal-derived factor (SDF-1). SDF-1/CXCR4 interactions tightly regulate homing (Lapidot and Petit, 2002).  As HSCs home towards their niche, they pass through a gradient of SDF-1 (Lapidot, et al. 2005).  In the stromal niche, the concentration of CXCR4 is low and the concentration of SDF-1 is high, so as to induce engraftment (Lapidot and Petit, 2002).  However, if the patient’s niche has been destroyed via radiation, for example, engraftment will not occur because the niche cannot be obtained from the donor.  Engraftment can take months to years and requires cell division in order to successfully repopulate the blood and immune systems (Lapidot, 2005).

Figure 3

Immune Reconstitution (IR)
HSCs harvested from either PB or BM is transplanted into highly immunosuppressed individuals, and despite homing and engraftment, it may take months or years for IR (Auletta, et al. 2005). NKs, dendritic cells (DC), and granulocytes seem to recover first, within a few months after HSCT.  B-cell reconstitution generally precedes T-cell recovery; however, recovery of a complete repertoire of IgG- and IgA-producing B-cells may take as long as 2 years, further delaying T-cell recovery (Auletta, et al. 2005; Fry and Mackall, 2005).  Certain factors also influence IR, such as SC source (PB or BM), SC mobilization (G-CSF, flt3), graft manipulation (selecting for CD34+, T-cell depletion), whether an autologous or allogeneic transplant was performed, and the conditioning regimens used (Auletta, et al. 2005).

Recent findings involving DCs, NKs, and T-cell subtypes have been tested in attempt to increase IR and decrease risk of GVHD while enhancing GVL (Wanko, et al. 2005; Auletta, et al. 2005).  One such technique is the depletion of T-cells, since these, along with the aid of NKs and macrophages, are the main cells involved in graft rejections; however, T-cell depletion is also associated with engraftment delay, delayed IR, and early relapse due to impaired T-cell function and loss of GVL (Wanko, et al. 2005; Auletta, et al, 2005; Fry and Mackall, 2005).  Another method would entail depletion of naïve T-cells and inclusion of memory T-cells.  Memory T-cells should be less responsive then naïve T-cells to alloantigen because of their previous antigen exposure (Wanko, et al. 2005).  DCs are believed to be potent APCs to naïve T-cells, have anti-tumor functions, and play a role in fighting post-transplant infection (Auletta, et al. 2005).  Scientists are also looking into various immunotherapies to find ways to improve IR by preventing or minimizing damage to BM using mesenchymal stem cell, and to the thymus using keratinocyte growth factor (KGF) (Auletta, et al. 2005; Fry and Mackall, 2005).

Additional HSC therapies
HSC Therapies for Autoimmune Diseases
Several autoimmune diseases, such as type 1 diabetes mellitus (DM) and multiple sclerosis, arise from a genetic basis.  Using donor HSCs expressing “disease-resistance” genes may be effective in alleviating the dysfunctional phenotype.  These cells would engraft into the recipient’s bone marrow and give rise to progeny that would eliminate the progression of the autoimmune process, therefore abolishing the destruction of islet β cells and inflammatory and cytotoxic T cells in type I DM (Beilhack et al., 2003). However by the time patients undergo bone marrow transplantation, much destruction has already occurred.  Often times, the islet β cells of the pancreas are completely destroyed in Type I DM, and excessive oligodendrocyte damage, apparent as motor and sensory loss, has occurred in patients with multiple sclerosis.  These patients may require not only donor HSCs but also other types of donor stem cells for tissues they have lost. Unfortunately, claims of plasticity of HSCs (Brazelton et al, 2000; Ferrari et al, 1998; Jackson et al, 2001; Krause et al, 2001) have not been able to be reproduced (Bjornson et al, 1999; Wagers et al, 2002; Sherwood et al, 2004; Massengale et al, 2005; Balsam et al, 2004) and thus much more work is needed to clarify the role of HSCs as a multiple-lineage tissue source.

Gene Therapy
Childhood immunological diseases are great candidates for the benefits of gene therapy using HSCs. A unique quality of HSCs is that they can be removed from the body, modified ex vivo and then re-transplanted while preserving their function. The basic idea is that gene therapy using autologous HSCs with the corrected gene will have a favorable effect on blood cell production and function, without the possible immune rejection to allogenic HSC transplantation. When a normal gene is needed to be added to HSC, the use of vectors like a retrovirus is necessary to integrate the gene into the cell’s chromosome. Presently, patients with XSCID, ADA-deficient SCID and chronic granulomatous disease have shown to benefit from gene therapy (D B Kohn, et al. 2007).
In many cases of severe combined immunodeficiency or SCID, HLA-matched donors of HSCs cannot be found.  In these cases, host/donor human leukocyte antigen disparity may trigger destruction of recipient tissues by GVHD.  These patients may benefit from the use of gene transfer into autologous HSCs, eliminating the risk of GVHD.  The development of several successful gene therapy strategies for two forms of SCID were begun in the early 1990s (Blaese et al, 1995; Bordignon et al, 1995; Hoogerbrugge et al, 1996; Kohn et al, 1995).  The first clinical trials for patients with adenosine deaminase (ADA) deficiency were, in general, without success.  A major reason for this was continuation of exogenous replacement enzyme, which negated the selective advantage cells that the transgene would have enjoyed.  Adenosine deaminase deficiency accounts up to 20% of cases and results in an autosomal inheritance pattern (ADA-SCID) while X-linked SCID (SCID-X1 accounts for 40-50% of all patients with SCID.

Gene therapy clinical trials in SCID-X have consisted of two main trials.  The first inserted the gamma chain of the common cytokine receptor using a retroviral vector pseudotyped with the amphotropic viral envelope.  In total, CD34+ HSCs from eleven children have been treated with this protocol, from which nine have had successful reconstitution of the immune system.  New T lymphocytes in the thymus emerged in 10-12 weeks and proliferative responses of T cells to antigen stimulation were normal.  TCR rearrangements were detected in the new thymic nodes and functional humoral immunity was restored as well (Cavazzano-Calvo et al, 2000; Hacein-Bey-Abina et al, 2002).  A similar trial using an MFG-based retroviral vector pseudotyped in the gibbon ape leukemia virus (GALV) envelope was initiated and had similar immuno-repopulation kinetics in the seven children who were enrolled (Gaspar et al, 2004).  All children in these two trials responded with immune recovery; however, in older patients undergoing the same protocol, there was no evidence of immune recovery, possibly because of intrinsic host-dependent restrictions to permanent expression of the transgene or in the failure of initiation of normal thymopoiesis at a later age (Thrasher et al, 2005).

SCID is not the only disease that could employ gene therapy as a potential therapy.  Other indications include mutation of the receptor tyrosine kinase JAK-3.  Preclinical studies in ZAP-70 deficiency show reconstitution of ZAP-70 activity in T cells restored by retroviral gene transfer in HSCs (Otsu et al, 2002; Taylor et al, 1996).  These studies show promise and such preclinical studies may allow development of clinical trials in the near future.

Risks of Gene Therapy
Unfortunately, insertional mutagenesis poses a risk, exemplified by the development of clonal proliferation of T cells three years after the initiation of therapy in three of eleven children (Li et al, 2002).  The study demonstrated that all three proliferations resulted from a retroviral insertion into the proto-oncogene LMO2 and resulted in enhanced activity of the oncogene.  A murine model of this class of insertional mutagenesis confirmed that oncogenes, such as Evi-1, are targets for mutagenesis and can lead to myeloid leukemia in mice (Li et al, 2002).  Such models provide a useful platform to study the process of insertional mutagenesis and to develop strategies to avoid oncogenesis from retroviral vectors.

Future of HSC Research and concluding remarks
HSCs continue to be the prototype stem cell, being studied to further understand the mechanisms and functions of other kinds of stem cells.  New technologies have recently come into play to study stem cell characteristics, particularly of the HSC.  In order to characterize which genes play the role of attributing “stemness” to HSCs, studies have been undertaken to characterize the molecular expression profiles of murine HSCs (Ivanova et al, 2002; de Haan et al, 2002; Phillips et al, 2000).  Using expression profile arrays commercially available by companies like Affymetrix, Inc., the total RNA is taken from HSCs and labeled probes are synthesized that entails reverse transcription into cDNA then in vitro transcription once again into cRNA with biotinylated uridine.  The labeled cRNA is then hybridized to an array with all the genes in the organism’s genome spotted in distinct pixels and the brightness of each pixel is quantified and clustered into genes that portray the characteristics that define “stemness”, i.e., asymmetric division, self-renewal, etc.  The molecular mechanisms that regulate self-renewal and cell fate are still poorly understood and may be elucidated with expression profiling (Hackney et al, 2002).  The utility of microarrays can also reveal whether HSCs truly share the molecular signature required to transdifferentiate into liver, muscle, or neural cells (Lemischka et al, 2001).  Knowing which genes are upregulated and downregulated in cancer stem cells, myelodysplastic syndromes, and even gene-manipulated HSCs may lead to new insights into the biology of the hematopoietic stem cell, its niche, and its utility in therapies.

To date, only HSCs have really been transplanted in humans to successfully regenerate tissue (Weissman, 2005).  Scientists and doctors are faced with many challenges toward improving HSC transplant effectiveness, while reducing risk of side effects, such as eliminating GVHD, while still retaining GVL (Wanko, et al. 2005).  Several approaches have been suggested by various investigators, such as knocking out the MHC genes, creating a “null cell,” which could then have MHC genes inserted to match those of the recipient.  Another approach would be to reprogram the recipient’s immune system by establishing hematopoietic chimerism between donor and recipient, so as to recognize foreign antigen as self (Down, et al. 2003).  The possible attainment of durable chimerism that permits long-term organ tolerance, via engraftment of donor HSC with donor-matched heart, is currently under early-stage clinical trials (Shizuru, et al. 2005).

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Acknowledgements
This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey:
Karen Caneth, Amrita Ghosh, Gladys Huaylla, Jaclyn Fox, Pamela Orellana, Paul Paez, Erica Salerno, Steve Tsurumoto (in alphabetical order).
Teaching Assistant:  Marcelo Taborga
The review was edited by two stem cell biologists.