Search UMDNJContact GSBS

Featured Faculty Member

Featured Graduate Student


Exploring the Hematopoietic Stem Cell: the prototype
(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, 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 disorders have been linked to dysfunction of HSCs. Transplantation of HSCs from a closely matched donor is common for hematological disorders, including malignacies. 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, 2005), thre 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 immunodefiencey, 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. Throughout the 1950s, experiments showed hematopoietic recovery from transplanted bone marrow after irradiation damage in mice. Finally in 1963, Till, McCulloch, and Siminovitch provided genetic marking evidence for rare cells in mouse bone marrow that formed myeloerythroid colonies in the spleens of irradiated mice, some of which contained cells that self-renewed. This was the reasonable evidence needed to propose the existence of HSC and begin the search to isolate human HSC (Weissman, 2005) (see Table 1).

Isolation of HSCs


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).

In the late 1980s and 1990s, developments in antibody technology allowed investigators to define the ontogeny of the stem cell system, based on surface immunophenotype in humans and mice. These studies identified a large number of cell surface markers that are associated with defined lineage and developmental stages of HSC. In 1984, scientists produced anti-CD34 as a monoclonal antibody, My10, which recognized surface antigen on the myeloid leukemic cell line. Since then, CD34 has been widely used as a stem and progenitor cell marker, and clinical CD34+ stem cell transplantation has been performed for tumor purging and for prevention of graft-versus-host disease (Ando, 2002).


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).

Experimental methods


Evidence exists that showed purified HSCs, in addition to regenerate the hematopoietic system of an mdx mouse can also generate progeny that could differentiate into a non-hematopoietic cell type. Multi-organ, multi-lineage engraftment may occur by a single BM-derived stem cell with HSC phenotype (Wulf, et al. 2001). Using xenogeneic transplantation models in NOD/SCID (non-obese diabetic/ severe combine immune deficiency) mice, different classes of stem and progenitor cells responsible for long-term and short-term engraftment and differentiation potentials were identified (Punzel, et al. 2001). Transplantation experiments in mice have therefore become standard assays for the assessment of the self-renewal and reconstitution potential of normal and malignant HSC (Huntly, et al. 2005).

General

Biology/ Markers


The precursor for all hematopoietic stem cells, as well as endothelial cells, is hypothesized to be the hemangioblast (Kyba,et al. 2003). Debate exists over the earliest site of hematopoiesis being either the extra-embryonic yolk sac or the intra-embryonic aorta-gonad-mesonephros (AGM) region (Chan, et al. 2004).


The HSC exists in low quantity at 1 HSC to 104 – 105 nucleated cells in the bone marrow (Chan, et al. 2004). The HSC shares a similar morphology to that of its neighboring white blood cells; however, functionally, the HSC has four hallmark features: the ability to self-renew, to differentiate into a variety of specialized cells, to mobilize out of the bone marrow into the peripheral blood, and to undergo apoptosis (Shizura, et al. 2005). Regulation of these cellular activities is carried out by various cytokines and growth factors, both belonging to the HSC and present in the surrounding microenvironment. The use of DNA and RNA dyes has also been utilized to identify areas rich in hematopoietic stem cells. The HSC has a multiple drug resistance gene, which enables it to pump out the dye and be detected using flow cytometric analysis.


Phenotypical classification of the hematopoietic stem cell relies on the identification of cell surface markers. While no finite list yet exists, scientists have highlighted stem cell antigen-1 (Sca-1), c-kit (CD117), and Thy-1 (CD90) as the hallmark markers of the hematopoietic stem cell (Chan, et al, 2004). Cell markers are not only useful in the identification of the HSC, but also in deciphering the differentiation and maturation stage of the cell. For example, the cell surface marker CD34 is present at different levels of activation of the HSC (Bonde, et al. 2004). As the HSC differentiates, the phenotypical markers may vary in degree of expression on the cell surface. The HSC can also be classified by its various receptors, with the most prominent being CXCR4, which interacts with the chemokine SDF-1 to mobilize the HSC out of the bone marrow and into the periphery (Wright, et al. 2002).

Location of HSC and the Bone Marrow Microenvironment


The main source of hematopoietic stem cells is the bone marrow. The HSC lives in tight junction with the stromal cells of the bone marrow via cell adhesion molecules—a prominent example being fibronectin (Huygen, et al. 2002). 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).

Types of Cells HSCs Produce/ Lineages


The self-renewing, long-term hematopoietic stem cell (LT-HSC) gives rise to the short-term hematopoietic stem cell (ST-HSC), which has 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. Cells of the lymphoid lineage give rise to B-lymphocytes, T-lymphocytes, and natural killer cells of the immune system. Those of myeloid lineage form either 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).

Disorders of the Hematopoietic System


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.

Aging/Cancer/Blood Disorders


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).

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 reinfusion 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 2). 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).

Immune Reconstitution (IR)


HSCs harvested from either PB or BM are 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 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 Clinical Trials for SCID


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 which 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 a 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. (ref for study) 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).

 

References

Allsopp, R., Morin, G., Horner, J., DePinho, R., Harley, C., Weissman, I. Effect of TERT Over-Expression on the Long Term Transplantation Capacity of Hematopoietic Stem Cells. Nature Med (2003) 9: 369-371.

Ando, K. Human CD34- Hematopoietic Stem Cells: Basic Features and Clinical Relevance. Intl J Hematol (2002) 75: 370-375.

Auletta, J.J., Lazarus, H.M. Immune restoration following hematopoietic stem cell transplantation: an evolving target. Bone Marrow Transplantation (2005) 35: 835-857.

Balsam, L.B., Wagers, A.J., Christensen, J.L., Kofidis, T., Weissman, I.L., Robbins, R.C. Hematopoietic stem cells adopt mature hematopoietic fates in ischaemic myocardium. Nature (2004) 428: 668-673.

Baum, C.M., Weissman, I.L., et al. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci USA (1992) 89: 2804-2808.

Bjornson, C.R., Rietze, R.I., Reynolds, B.A., et al. Turning brain into blood: a hematopoietic fate adopted by neural stem cells in vivo. Science (1999) 283: 534-537.

Blaese, R.M., Culver, K.W., Miller, A.D., et. al., T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years. Science. (1995) 270: 475-480.

Bonde, J., Hess, DA., Nolta, JA. Recent advances in hematopoietic stem cell biology. Curr Opin Hematol (2004) 11: 392-398.

Bordignon, C., Notarangelo, L.D., Nobili, N., et. al. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients. Science (1995) 270: 470-475.

Brazelton, T.R., Rossi, F.M., Keshet, G.I., et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science (2000) 290: 1775-1779.

Cavazzana-Calvo, M., Hacein-Bey, S., De Saint, B.G., et al., Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science (2000) 288: 669-672.

Chan, RJ., Yoder, MC. The multiple facets of hematopoietic stem cells. Curr Neurovas Res (2004) 1: 197-206.

De Haan, G., Bystrykh, L.V., Weersing, E., Dontje, B., Geiger, H., Ivanova, N., Lemischka, I.R., Vellenga, E., Van Zant, G. A genetic and genomic analysis identifies a cluster of genes associated with hematopoietic cell turnover. Blood (2002) 100: 2056-62.

De Vries, E.G.E., Vellenga, E., Kluin-Nelemans, J.C., Mulder, N.H. The happy destination of frozen hematopoietic stem cells: from immature stem cells to mature applications. Eur J Cancer (2004) 40: 1987-1992.

Diaz, M.A., et al. Long-term outcome of allogeneic PBSC transplantation in pediatric patients with hematological malignancies. Bone Marrow Transpl (2005) 36: 781-785.

Down, J.D., White-Scharf, M.E. Reprogramming immune responses: enabling cellular therapies and regenerative medicine. Stem Cells (2003) 21: 21-32.

Eapen, M., et al. Higher mortality after allogeneic peripheral-blood transplantation compared with bone marrow in children and adolescents: the histocompatibility and alternate stem cell source working committee of the International Bone Marrow transplant Registry. J Clin Oncol (2004) 22: 4872-4880.

Ferrari, G., Cusella-De Angelis, G., Coletta, M., et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science (1998) 279: 1528-1530.

Fry, T.J. and Mackall, C.L. Immune reconstitution following hematopoietic progenitor cell transplantation: challenges for the future. Bone Marrow Transpl (2005) 35: S53-S57.

Gaspar, H.B., Parsley, K.L., Howe, S., et al., Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet (2004) 364: 2181-2187.

Hacein-Bey-Abina, S., Le Deist, F., Carlier, F., et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med (2002) 346: 1185-1193.

Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med (2003) 348: 255-256.

Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science (2003) 302: 415-419.

Hackney, J.A., Charbord, P., Brunk, B.P., Stoeckert, C.J., Lemischka, I.R., Moore, K.A. A molecular profile of a hematopoietic stem cell niche. Proc Natl Acad Sci USA (2002) 99:13061-6.

Ho, A.D. and Punzel, M. Hematopoietic stem cells: can old cells learn new tricks? J Leuk Biol (2003) 73: 547-555.

Hoogerbrugge, P.M., Van Beusechem, V.W., Fischer, A., et. al. Bone marrow gene transfer in three patients with adenosine deaminase deficiency. Gene Ther. (1996) 3: 179-183.

Huntly, Brian J.P. and Gilliland, Gary D. Leukemia stem cells and the evolution of cancer-stem-cell research. Nat Rev: Cancer (2005) 5: 312-321.

Huygen, S., Giet, O., Artisien, V., Di Stefano, I., Beguin, Y., and Gothot, A. Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture. Blood (2002) 100: 2744-2752.

Ivanova, N.B., Dimos, J.T., Schaniel, C., Hackney, J.A., Moore, K.A., Lemischka, I.R. A stem cell molecular signature. Science (2002) 298: 601-604.

Ivanovic Zoran, et al. Simultaneous maintenance of human cord blood SCID-repopulating cells and expansion of committed progenitors at low O2 concentration (3%). Stem Cells (2004) 22: 716-724.

Jackson, K.A., Majka, S.M., Wang, H., et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest. (2001) 107: 1395-1402.

Kohn, D.B., Weinberg, K.I., Nolta, J.A., et. al. Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nat. Med. (1995) 1: 1017-1023.

Krause, D.S., Theise, N.D., Collector, M.I., et al. Multiorgan, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell. (2001) 105: 369-377.

Kyba, M. and Daley, GQ. Hematopoiesis from embryonic stem cells: lessons from and for ontogeny. Experimental Hematology (2003) 31: 994-1006.

Lapidot, Tsvee, Dar, Ayelet, and Kollet, Orit. How do stem cells find their way home? Blood (2005) 106: 1901-1910.

Lapidot, Tsvee and Petit, Isabelle. Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol (2002) 30: 973-981.

Lemischka, I. Stem cell dogmas in the genomics era. Rev Clin Exp Hematol. (2001) 5: 15-25. Review.

Lewis, Ally. Autologous stem cells derived from peripheral blood compared to standard bone marrow transplant; time to engraftment: a systematic review. Intl J Nursing Studies (2005) 42: 589-596.

Li, Z., Dullmann, J., Schiedlmeier, B., et al. Murine leukemia induced by retroviral gene marking. Science (2002) 296: 497.

Massengale, M., Wagers, A.J., Vogel, H., Weissman, I.L. Hematopoietic cells maintain hematopoietic fates upon entering the brain. J Exp Med. (2005) 201:1579-1589.

Muul, L.M., Tuschong, L.M., Soenen, S.L., et al. Persistence and expression of the adenosine deaminase gene for 12 years and immune reaction to gene transfer components: long term results of the first clinical gene therapy trial. Blood (2003) 101: 2563-2569.

Nakano, Toru. Hematopoietic stem cells: generation and manipulation. TRENDS in Immunol (2003) 24: 589-594.

Noguchi, M., Nakamura, Y., Russell, S.M., et al. Interleukin-2 receptor gamma chain: a functional component of the interleukin-7 receptor. Science (1993) 262:1877-1880.

Orlic, D., Kajstura, J., Chimenti, S., et al. Bone marrow cells regenerate infarcted myocardium. Nature (2001) 410: 701-705.

Otsu, M., Steinberg, M., Ferrand, C., et al. Reconstitution of lymphoid development nad function in ZAP-70 deficient mice following gene transfer into bone marrow cells. Blood (2002) 100: 1248-1256.

Parent-Massin, D. Relevance of clonogenic assays in hematotoxicology. Cell Biol Toxicol (2001) 17: 87-94.

Perry, A.R. and Linch, D.C. The history of bone marrow transplantation. Blood Rev (1996) 10:215-219.

Phillips, R.L., Ernst, R.E., Brunk, B., Ivanova, N., Mahan, MA., Deanehan, J.K., Moore, K.A., Overton, G.C., Lemischka, I.R. The genetic program of hematopoietic stem cells. Science (2000) 288: 1635-40.

Punzel, M. and Ho, A.D. Divisional History and Pluripotency of Human Hematopoietic Stem Cells. Ann NY Acad Sci (2001) 938: 72-82.

Sata, M., et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med (2002) 9: 403.

Sharkis, S.J., Neutzel, S., et al. Phenotype and Function of Hematopoietic Stem Cells. Ann. NY Acad. Sci. (2001) 938: 191-195.

Sherwood, R.I., Christensen, J.L., Weissman, I.L., Wagers, A.J. Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells. Stem Cells (2004) 22: 1292-1304.

Shizuru, Judith A., Negrin, Robert S., and Weissman, Irving L. Hematopoietic stem and progenitor cells: clinical and preclinical regeneration of the hematolymphoid system. Annu. Rev. Med. (2005) 56: 509-538.

Sugamura, K., Asao, H., Kondo, M., et al. The common gamma-chain for multiple cytokine receptors. Adv Immunol (1995) 59:225-277.

Sugihara, A., Adachi, Y., Inaba, H. Age-Dependant Abnormalities of Hematopoietic Stem Cells in (NZW X BXSB)F1 Mice. Stem Cell (1999) 17: 357-365.

Takauchi, K., Tashiro, S., Ohtaki, M., Kamada, N. Telomere Reduction of Specific Chromosome Translocation in Acute Myelocytic Leukemia. Jpn J Cancer Res. (1994) 85: 127-130.

Taylor, N., Bacon, K.B., Smith, S., et al. Reconstitution of T cell receptor signaling in ZAP-70 deficient cells by retroviral transduction of the ZAP-70 gene. J Exp Med (1996) 184: 2031-2036.

Thrasher, A.J., Hacein-Bey-Abina, S., Gaspar, H.B. et al. Failure of SCID-X1 gene therapy in older patients. Blood (2005) 105: 4255-4257.

Wagers, A.J., Sherwood, R.I., Christensen, J.L., Weissman, I.L. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science (2002) 297: 2256-2259.

Wagers, A.J., Weissman, I.L. Plasticity of adult stem cells. Cell (2004) 116:639-648.

Wang, J., Zhao, HP., Lin, G., Xie, CQ., Nie, DS., Wang, QR., and Lu, GX. In vitro hematopoietic differentiation of human embryonic stem cells induced by co-culture with human bone marrow stromal cells and low dose cytokines. Cell Biol Intl (2005) 29: 654-661.

Wanko, Sam O. and Nelson, Chao J. Non-pharmacologic approaches to graft-versus-host prevention. Blood Rev (2005) 19: 203-211.

Weissman, Irving. Stem cell research: Paths to cancer therapies and regenerative medicine. J Am Med Assoc (2005) 294: 1359-1366.

Wright, DE., Bowman, EP., Wagers, AJ., Butcher, EC., and Weissman, IL. Hematopoietic stem cells are uniquely selective in their migratory response to chemokines. J Exp Med (2002) 195: 1145-1154.

Wulf, G.G., Jackson, K.A., and Goodell, M.A. Somatic stem cell plasticity: Current evidence and emerging concepts. Exp Hematol (2001) 29: 1361-1370.

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, Jaclyn Fox, Erica Salerno, Steve Tsurumoto (in alphabetical order).

Teaching Assistant:  Edward Garay

The review was edited by two stem cell biologists.