Hematopoietic Stem Cell and Its Bone Marrow Niche.

Stem cells do not thrive without their niche. The bone marrow microenvironment is where hematopoietic stem cells maintain their cell state while receiving physiological input to modify their activity in response to changing physiological demands. The complexity of the bone marrow microenvironment is being unraveled and indicates that multiple different cell types contribute to the regulation of stem and progenitor cells. Further, it is becoming evident that the bone marrow represents a composite of niches with different components and different functional roles in hematopoiesis. It is now evident that alterations in specific stromal cells that comprise the bone marrow microenvironment can contribute to hematologic pathology. In this chapter, we will review the history of the niche concept, evolving information about its components and how niche dysfunction may contribute to disease.

The purinergic P2Y14 receptor axis is a molecular determinant for organism survival under in utero radiation toxicity.

In utero exposure of the embryo and fetus to radiation has been implicated in malformations or fetal death, and often produces lifelong health consequences such as cancers and mental retardation. Here we demonstrate that deletion of a G-protein-coupled purinergic receptor, P2Y14, confers potent resistance to in utero radiation. Intriguingly, a putative P2Y14 receptor ligand, UDP-glucose, phenocopies the effect of P2Y14 deficiency. These data indicate that P2Y14 is a receptor governing in utero tolerance to genotoxic stress that may be pharmacologically targeted to mitigate radiation toxicity in pregnancy.

A novel role for CCL3 (MIP-1α) in myeloma-induced bone disease via osteocalcin downregulation and inhibition of osteoblast function.

Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine, levels of which in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. In this study, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function, and therefore contributes to OB/OC uncoupling in MM.

Preclinical activity of P276-00, a novel small-molecule cyclin-dependent kinase inhibitor in the therapy of multiple myeloma.

Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. Here, we sought to identify the effects of targeting cyclin D in MM. We first confirmed cyclin D mRNA overexpression in 42 of 64 (65%) patient plasma cells. Silencing cyclin D1 resulted in >50% apoptotic cell death suggesting its validity as a potential therapeutic target. We next evaluated P276-00, a clinical-grade small-molecule cyclin-dependent kinase inhibitor as a way to target the cyclins. P276-00 resulted in dose-dependent cytotoxicity in MM cells. Cell-cycle analysis confirmed either growth arrest or caspase-dependent apoptosis; this was preceded by inhibition of Rb-1 phosphorylation with associated downregulation of a range of cyclins suggesting a regulatory role of P276-00 in cell-cycle progression through broad activity. Proliferative stimuli such as interleukin-6, insulin-like growth factor-1 and bone-marrow stromal cell adherence induced cyclins; P276-00 overcame these growth, survival and drug resistance signals. Because the cyclins are substrates of proteasome degradation, combination studies with bortezomib resulted in synergism. Finally, in vivo efficacy of P276-00 was confirmed in an MM xenograft model. These studies form the basis of an ongoing phase I study in the treatment of relapsed/refractory MM.

A niche opportunity for stem cell therapeutics.

The success of hematopoietic stem cell (HSC)-based therapies relies on the ability of the stem cells to both engraft and self-renew sufficiently in the bone marrow microenvironment. Previous studies identified that a number of components of bone contribute to the regulation of HSCs indicating that they participate in a stem cell 'niche'. This niche is a dynamic microenvironment that changes during development and with varying physiologic states. Components of it, such as the osteoblast, can be modulated through pharmacological treatment. Reasoning that the stem cell niche may be manipulated to augment the effectiveness of stem cell therapies, we demonstrated that daily treatment with parathyroid hormone (a clinically approved method for increasing osteoblast function) resulted in therapeutic benefit in three clinically relevant models of stem cell therapy. These results suggest that the niche may be a pharmacological target for altering stem cell function in settings of regenerative medicine.

Silencing p21(Waf1/Cip1/Sdi1) expression increases gene transduction efficiency in primitive human hematopoietic cells.

Adult hematopoietic and other tissue stem cells have highly constrained cell cycling that limits their susceptibility to standard gene therapy vectors, which depend upon chromosomal integration. Using cytokine cocktails to increase transduction efficiency often compromises subsequent stem cell function in vivo. We previously showed that p21(Waf1/Cip1/Sdi1) (p21) mediates stem cell quiescence in vivo and decreasing its expression ex vivo leads to an expansion of stem cell pool in vivo. Here, we report that application of p21 specific siRNA increased the gene transduction efficiency in hematopoietic stem cells while preserving cell multipotentiality. Both types of siRNA, synthesized siRNA and transcribed shRNA, reduced p21 expression in target cells by 85-98%. The effect of RNAi in these cells was transient and the level of p21 mRNA returned to base line 14-28 days after siRNA treatment. This brief interval of reduction, however, was sufficient to increase transduction efficiency to two- to four-fold in cell cultures, and followed by a seven- to eight-fold increase in mice. The RNAi treated, lentivector-transduced CD34+ cells retained multipotentiality as assessed in vitro by colony formation assay and in vivo by NOD/SCID mouse transplantation assay. Reduction of p21 resulted in an increased chromosomal integration of lentivector into target cellular DNA. Taken together, both synthesized and transcribed siRNA knocked down p21 expression in human CD34+ hematopoietic stem/progenitor cells. Silencing p21 expression increased gene transduction efficiency and vector integration while retaining stem cell multipotentiality. Thus, RNAi targeting of p21 is a useful strategy to increase stem cell gene transfer efficiency. Decreasing p21 expression transiently while increasing gene-transfer vector integration may ultimately facilitate clinical applications of gene therapy.

Regulation of hematopoietic stem cell growth.

Hematopoietic stem cells (HSC) must balance self-renewal and differentiation to provide sufficient primitive cells to sustain hematopoiesis, while generating more mature cells with specialized capabilities. The enhanced self-renewal capacity of primitive HSCs enables their ability to sustain hematopoiesis throughout decades of life and their ability to repopulate a host when used therapeutically in bone marrow transplantation. However, hematopoietic cell perturbations resulting in unchecked self-renewal participate in leukemogenesis. While mechanisms governing self-renewal are still being uncovered, they are thought to bear relationship to the malignant process in a variety of tumor types and may therefore provide useful therapeutic targets in putative cancer stem cells. This review discusses molecular mechanisms recently defined to participate in HSC governance and highlights features of stem cell interactions with the microenvironment that may help guide therapies directed at HSCs.

Osteoblastic cells regulate the haematopoietic stem cell niche.

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.

Transforming growth factor beta 1 mediates cell-cycle arrest of primitive hematopoietic cells independent of p21(Cip1/Waf1) or p27(Kip1).

The regulation of stem cell proliferation is a poorly understood process balancing rapid, massive blood cell production in times of stress with maintenance of a multipotent stem cell pool over decades of life. Transforming growth factor beta 1 (TGF-beta 1) has pleiotropic effects on hematopoietic cells, including the inhibition of primitive cell proliferation. It was recently demonstrated that the cyclin-dependent kinase inhibitors, p21(Cip1/Waf1) (p21) and p27(Kip1) (p27), can inhibit the proliferation of hematopoietic stem cells and progenitor cells, respectively. The relation of TGF-beta 1 stimulation to p21 and p27 was examined using a fine-mapping approach to gene expression in individual cells. Abundant TGF-beta 1 expression and p21 expression were documented in quiescent, cytokine-resistant hematopoietic stem cells and in terminally differentiated mature blood cells, but not in proliferating progenitor cell populations. TGF-beta 1 receptor (T beta R II) was expressed ubiquitously without apparent modulation. Cell- cycle-synchronized 32D cells exposed to TGF-beta 1 demonstrated a marked antiproliferative effect of TGF-beta 1, yet neither the level of p21 mRNA nor the protein level of either p21 or p27 was altered. To corroborate these observations in primary cells, bone marrow mononuclear cells derived from mice engineered to be deficient in p21 or p27 were assessed. Progenitor and primitive cell function was inhibited by TGF-beta 1 equivalently in -/- and +/+ littermate controls. These data indicate that TGF-beta 1 exerts its inhibition on cell cycling independent of p21 and p27 in hematopoietic cells. TGF-beta 1 and p21 or p27 participate in independent pathways of stem cell regulation, suggesting that targeting each may provide complementary strategies for enhancing stem or progenitor cell expansion and gene transduction.

Hematologic Aspects of HIV/AIDS.

This review addresses various aspects of HIV infection pertinent to hematology, including the consequences of HIV infection on specific aspects of hematopoiesis and an update on the current biologic, epidemiologic and therapeutic aspects of AIDS-related lymphoma and Hodgkin's disease. The results of the expanding use of progenitor cell transplantation in HIV infected patients are also reviewed. In Section I, Dr. Scadden reviews the basis for HIV dysregulation of blood cell production, focusing on the role of the stem cell in HIV disease. T cell production and thymic function are discussed, with emphasis placed upon the mechanisms of immune restoration in HIV infected individuals. Results of clinical and correlative laboratory studies are presented. In Section II, Dr. Levine reviews the recent epidemiologic trends in the incidence of lymphoma, since the widespread availability of highly active anti-retroviral therapy (HAART). The biologic aspects of AIDS-lymphoma and Hodgkin's disease are discussed in terms of pathogenesis of disease. Various treatment options for these disorders and the role of concomitant anti-retroviral and chemotherapeutic intervention are addressed. Drs. Zaia and Krishnan will review the area of stem cell transplantation in patients with AIDS related lymphoma, presenting updated information on clinical results of this procedure. Additionally, they report on the use of gene therapy, with peripheral blood CD34+ cells genetically modified using a murine retrovirus, as a means to treat underlying HIV infection. Results of gene transfer experiments and subsequent gene marking in HIV infected patients are reviewed.

Alternative splicing determines the function of CYP4F3 by switching substrate specificity.

Diversity of cytochrome P450 function is determined by the expression of multiple genes, many of which have a high degree of identity. We report that the use of alternate exons, each coding for 48 amino acids, generates isoforms of human CYP4F3 that differ in substrate specificity, tissue distribution, and biological function. Both isoforms contain a total of 520 amino acids. CYP4F3A, which incorporates exon 4, inactivates LTB4 by omega-hydroxylation (Km = 0.68 microm) but has low activity for arachidonic acid (Km = 185 microm); it is the only CYP4F isoform expressed in myeloid cells in peripheral blood and bone marrow. CYP4F3B incorporates exon 3 and is selectively expressed in liver and kidney; it is also the predominant CYP4F isoform in trachea and tissues of the gastrointestinal tract. CYP4F3B has a 30-fold higher Km for LTB4 compared with CYP4F3A, but it utilizes arachidonic acid as a substrate for omega-hydroxylation (Km = 22 microm) and generates 20-HETE, an activator of protein kinase C and Ca2+/calmodulin-dependent kinase II. Homology modeling demonstrates that the alternative exon has a position in the molecule which could enable it to contribute to substrate interactions. The results establish that tissue-specific alternative splicing of pre-mRNA can be used as a mechanism for changing substrate specificity and increasing the functional diversity of cytochrome P450 genes.

Definition of an optimal cytotoxic T lymphocyte epitope in the latently expressed Kaposi's sarcoma-associated herpesvirus kaposin protein.

Cytotoxic T lymphocytes (CTL) recognize and kill virus-infected cells and contribute to immunologic control of viral replication. For many herpesviruses (e.g., Epstein-Barr and cytomegalovirus), virus-specific CTL responses can be readily detected in infected persons, but CTL responses against Kaposi's sarcoma-associated herpesvirus (KSHV) appear to be weak and remain poorly characterized. Using a human leukocyte antigen (HLA) binding motif-based epitope prediction algorithm, we identified 37 HLA-A*0201 binding peptides from 8 KSHV open-reading frames (ORFs). After in vitro stimulation of peripheral blood mononuclear cells from KSHV-infected persons, CTL responses against 1 peptide in the KSHV kaposin protein (ORF K12) were detected in 2 HLA-A*0201-positive subjects. The optimal CTL epitope was identified by HLA restriction analysis and peptide titration assays. These data describe a latent phase viral gene product targeted by CTL that may be relevant for KSHV immunopathogenesis.

Tissue source dictates lineage outcome of human fetal CD34(+)CD38(-) cells.

OBJECTIVE: The translocation from fetal liver hematopoiesis to secondary organs occurs during the second trimester of human gestation. It has been hypothesized that stem cells migrate and acquire lineage potential based on cues specific to the adopted microenvironment. We evaluated primitive hematopoietic cell populations in the fetal human to determine if lineage restriction precedes or follows translocation to sites of hematopoietic activity including thymus, spleen, bone marrow, and liver. METHODS: Sets of hematopoietic tissues from individual second-trimester human abortuses were used to compare and quantitate the lineage outcome of immunophenotypically primitive cells from each of the hematopoietic organs using ex vivo myeloid and lymphoid differentiation systems. RESULTS: Despite uniformity in immunophenotype, functional capabilities were highly restricted by the tissue of origin and alteration in the ex vivo differentiation context did not lead to a change in differentiation outcome. CONCLUSION: Translocation of primitive cells from fetal liver to tissues of mature hematopoietic activity is associated with tissue-specific, quantitative changes in differentiation potential that are unresponsive to alternative differentiation environments. These data suggest that multipotentiality is lost prior to or upon stem-cell migration in the developing human. It is not persistent with residence in a secondary hematopoietic organ.

CXCR-4 desensitization is associated with tissue localization of hemopoietic progenitor cells.

The chemokine stroma-derived factor (SDF)-1, and its receptor, CXCR-4, have been shown to be essential for the translocation of hemopoietic stem cells from the fetal liver to the bone marrow (BM). We hypothesized that if CXCR-4 plays a crucial role in the localization of human hemopoiesis, stem cells from distinct tissue sources should demonstrate distinct CXCR-4 expression or signaling profiles. CD34(+) cells from BM were compared with blood: either mobilized peripheral blood or umbilical cord blood. Unexpectedly, significantly higher levels of CXCR-4 surface expression on CD34(+) cells from blood sources, mobilized peripheral blood, or cord blood were observed compared with BM (p = 0.0005 and p = 0.002, respectively). However, despite lower levels of CXCR-4, responsiveness of the cells to SDF-1 as measured by either calcium flux or transmigration was proportionally greatest in cells derived from BM. Further, internalization of CXCR-4 in response to ligand, associated with receptor desensitization, was significantly lower on BM-derived cells. Therefore, preserved chemokine receptor signaling was highly associated with marrow rather than blood localization. To test the functional effects of perturbing CXCR-4 signaling, adult mice were exposed to the methionine-SDF-1beta analog that induces prolonged down-regulation/desensitization of CXCR-4 and observed mobilization of Lin(-), Sca-1(+), Thy-1(low), and c-kit(+) hemopoietic progenitor cells to the peripheral blood with a >30-fold increase compared with PBS control (p = 0.0007 day 1 and p = 0.004 day 2). These data demonstrate that CXCR-4 expression and function can be dissociated in progenitor cells and that desensitization of CXCR-4 induces stem cell entry into the circulation.

Chemotherapy for human immunodeficiency virus-associated non-Hodgkin's lymphoma in combination with highly active antiretroviral therapy.

PURPOSE: This study investigated the efficacy, toxicity, and pharmacokinetic interactions resulting from simultaneous combination chemotherapy and highly active antiretroviral therapy (HAART) for patients with human immunodeficiency virus (HIV)-associated non-Hodgkin's lymphoma (NHL). In addition, the effects on viral load, CD4 counts, and opportunistic infections were examined with the use of combination chemotherapy combined with HAART. PATIENTS AND METHODS: Sixty-five patients with previously untreated and measurable disease at any stage of HIV-associated NHL of intermediate or high grade were entered onto this study at 17 different centers. The first 40 patients entered onto the study received reduced doses of cyclophosphamide and doxorubicin, combined with vincristine and prednisone (modified CHOP [mCHOP]), whereas the subsequent 25 patients entered onto the study received full doses of CHOP combined with granulocyte colony-stimulating factor (G-CSF). All patients also received stavudine, lamivudine, and indinavir. RESULTS: The complete response rates were 30% and 48% among patients who received mCHOP and full-dose CHOP combined with HAART, respectively. Grade 3 or 4 neutropenia occurred in 25% of patients receiving mCHOP and 12% of those receiving full-dose CHOP combined with G-CSF (25% v 12%). There were similar numbers of patients with grade 3 or 4 hyperbilirubinemia (12% and 17%), constipation and abdominal pain (18% and 17%), and transaminase elevation (48% and 52%) on the modified and full-dose arms of the study, respectively. Doxorubicin clearance and indinavir concentration curves were similar among patients on this study and historical controls, whereas cyclophosphamide clearance was 1.5-fold reduced as compared with control values. Human immunodeficiency virus (HIV) load declined from a median baseline value of 29,000 copies/mL to a median minimum value on therapy of 500 copies/mL. CONCLUSION: Either modified-dose or full-dose CHOP chemotherapy for HIV-NHL, delivered with HAART, is effective and tolerable.

The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitor cells.

WT1, a transcription factor implicated in both normal kidney differentiation and tumorigenesis, is also expressed in differentiating hematopoietic progenitors. Most human acute leukemias contain high levels of the wild-type transcript, while a minority have point mutations, raising the possibility that this tumor suppressor might have a paradoxical oncogenic effect in some hematopoietic cells. Using high titer retroviral infection, we demonstrate that WT1 triggers rapid growth arrest and lineage-specific differentiation in primary hematopoietic progenitors and differentiation-competent leukemia cell lines, while it induces cellular quiescence in a primitive subset of primary precursors. Growth arrest by WT1 is associated with induction of p21(CIP1), but expression of this cyclin-dependent kinase inhibitor alone is insufficient for either cellular differentiation or primitive cell preservation. The effects of WT1 are enhanced by co-expression of its naturally occurring isoforms, and are correlated with the physiological expression pattern of WT1 in vivo. Our observations suggest a role for WT1 in the differentiation of human hematopoietic cells, and provide a functional model that supports its capacity as a tumor suppressor in human acute leukemia.

Hematopoietic stem cells in HIV disease.

The hematopoietic stem cell has long been hypothesized to be a target of human immunodeficiency virus type-1 (HIV) infection that limits the potential for compensatory immune cell production. Data have recently emerged documenting stem cell dysfunction in HIV disease and indicating that immune recovery from potent antiretroviral therapy is partly driven by new T-cell generation. Effects of HIV on stem cell physiology, however, appear to be indirect, as stem cells are highly resistant to HIV infection. Despite the presence of surface receptors for HIV, the hematopoietic stem cell is not infectible with HIV. However, stem transduction can be achieved with HIV constructs in which the envelope glycoproteins have been replaced by vesicular stomatitis virus G protein. Therefore, hematopoietic stem cells are likely participants in HIV-related cytopenias, but they are spared direct infection and can serve as a resource for cellular therapies for AIDS.

Topical treatment of cutaneous lesions of acquired immunodeficiency syndrome-related Kaposi sarcoma using alitretinoin gel: results of phase 1 and 2 trials.

OBJECTIVE: To evaluate the efficacy and safety of topical alitretinoin gel (9-cis-retinoic acid [LGD1057], Panretin gel; Ligand Pharmaceuticals, Inc, San Diego, Calif) in cutaneous Kaposi sarcoma (KS). DESIGN: Open-label, within-patient, controlled, dose-escalating phase 1 and 2 clinical trials. In all patients, 1 or more cutaneous KS lesions were treated with alitretinoin gel, and at least 2 other lesions served as untreated controls for up to 16 weeks. Alitretinoin (0.05% or 0.1% gel) was applied twice daily for the first 2 weeks and up to 4 times daily thereafter, if tolerated. SETTING: Nine academic clinical centers. PATIENTS: One hundred fifteen patients with biopsy-proven acquired immunodeficiency syndrome (AIDS)-related KS. MAIN OUTCOME MEASURES: AIDS Clinical Trials Group response criteria. RESULTS: Statistically significant clinical responses were observed in 31 (27%) of 115 patients for the group of treated index lesions compared with 13 (11%) for the group of untreated control lesions (P<.001). Responses occurred with low CD4(+) lymphocyte counts (<200 cells/microL) and in some patients with refractory response to previous systemic anti-KS therapy. The incidence of disease progression was significantly lower for treated index lesions compared with untreated control lesions (39/115 [34%] vs 53/115 [46%]; P =.02). Alitretinoin gel generally was well tolerated, with 90% of treatment-related adverse events confined to the application site and only mild or moderate in severity. CONCLUSIONS: Alitretinoin gel has significant antitumor activity as a topical treatment for AIDS-related KS lesions, substantially reduces the incidence of disease progression in treated lesions, and is generally well tolerated.

Stem cell repopulation efficiency but not pool size is governed by p27(kip1).

Sustained blood cell production requires preservation of a quiescent, multipotential stem cell pool that intermittently gives rise to progenitors with robust proliferative potential. The ability of cells to shift from a highly constrained to a vigorously active proliferative state is critical for maintaining stem cells while providing the responsiveness necessary for host defense. The cyclin-dependent kinase inhibitor (CDKI), p21(cip1/waf1) (p21) dominates stem cell kinetics. Here we report that another CDKI, p27(kip1) (p27), does not affect stem cell number, cell cycling, or self-renewal, but markedly alters progenitor proliferation and pool size. Therefore, distinct CDKIs govern the highly divergent stem and progenitor cell populations. When competitively transplanted, p27-deficient stem cells generate progenitors that eventually dominate blood cell production. Modulating p27 expression in a small number of stem cells may translate into effects on the majority of mature cells, thereby providing a strategy for potentiating the impact of transduced cells in stem cell gene therapy.