Early-acting hematopoietic growth factors: biology and clinical experience.

Secreted protein growth factors that stimulate the self-renewal, proliferation, and differentiation of the most primitive stem cells are among the most biologically interesting molecules and at least theoretically have diverse applications in the evolving field of regenerative medicine. Among this class of regulators, the early-acting hematopoietic growth factors and their cellular targets are perhaps the best characterized and serve as a paradigm for manipulating other stem cell based tissues. This chapter reviews the preclinical knowledge accumulated over ~40 years, since the discovery of the first such growth factor, and the clinical applications of those that, upon testing in humans, ultimately gained regulatory approval for the treatment of various hematological diseases.

Automation, Monitoring, and Standardization of Cell Product Manufacturing.

Although regenerative medicine products are at the forefront of scientific research, technological innovation, and clinical translation, their reproducibility and large-scale production are compromised by automation, monitoring, and standardization issues. To overcome these limitations, new technologies at software (e.g., algorithms and artificial intelligence models, combined with imaging software and machine learning techniques) and hardware (e.g., automated liquid handling, automated cell expansion bioreactor systems, automated colony-forming unit counting and characterization units, and scalable cell culture plates) level are under intense investigation. Automation, monitoring and standardization should be considered at the early stages of the developmental cycle of cell products to deliver more robust and effective therapies and treatment plans to the bedside, reducing healthcare expenditure and improving services and patient care.

Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells.

Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential. Access to patient samples can also be limiting or cell numbers too low to enable large-scale assays and/or to obtain reproducible quantitative data. Here we describe a feeder cell-free and serum-free liquid culture system for the expansion of CD34+ HSPCs from CML/AML samples and healthy control tissues. Following 7 or 14 days of culture, CD34+ cells are expanded 30- to 65-fold or 400- to 800-fold, yielding a purity of ∼80% and ∼60% CD34+ cells, respectively. This system was adapted to a 96-well format to measure the sensitivity of leukemic and normal HSPCs to cytotoxic drugs after only 7 days. The assay requires only 103 cells per well to determine drug IC50 values and can be performed with uncultured and culture-expanded cells. Importantly, resulting IC50 values strongly correlate with those obtained in the classic colony-forming unit (CFU) assay. Compared with the CFU assay, this novel 96-well liquid-based assay designed specifically for leukemic and normal HSPCs is faster and simpler, with more flexible readout methods for selecting candidates for further drug development.

Interleukin-4 deficiency decreases atherosclerotic lesion formation in a site-specific manner in female LDL receptor-/- mice.

Activated lymphocytes and mast cells have been detected in human atherosclerotic lesions. Interleukin-4 (IL-4) is a prominent cytokine released during the activation of both these cell types, and its mRNA has been detected in human and mouse atherosclerotic lesions. To define the effects of IL-4 on atherogenesis, bone marrow stem cells from either IL-4-/- or IL-4+/+ mice were transplanted into lethally irradiated female low density lipoprotein (LDL) receptor-/- mice. After an interval sufficient to allow engraftment, mice were placed on a diet containing 21% saturated fat, 1.25% cholesterol, and 0.5% cholate. Hematopoietic engraftment was confirmed by the presence of the LDL receptor gene in bone marrow cells. The effect on IL-4 depletion was confirmed by quantifying cytokine release from splenocytes of reconstituted mice. The deficiency of IL-4 in bone marrow-derived cells had no effect on serum cholesterol concentrations or on the distribution of cholesterol among lipoproteins. Atherosclerotic lesion formation was not changed in the aortic root. However, deficiency of IL-4 led to reduced lesion size in the arch (9.1 +/- 1.1% versus 2.8 +/- 0.8% of intimal area, P<0.001) and the thoracic aorta (1.2 +/- 0.2% versus 0.4 +/- 0.1%, P<0.002). Therefore, IL-4 deficiency reduced atherosclerotic lesion formation in a site-specific manner in female LDL receptor-/- mice fed a high-fat diet.

Depletion of natural killer cell function decreases atherosclerosis in low-density lipoprotein receptor null mice.

OBJECTIVE: Natural killer (NK) cells are a key component of innate immunity. Despite being identified in human and mouse atherosclerotic lesions, the role of NK cells in the disease process in unknown. To determine this role, we created chimeric atherosclerosis-susceptible low-density lipoprotein (LDL) receptor null (ldl-r-/-) mice that were deficient in functional NK cells through expression of a transgene encoding for Ly49A. METHODS AND RESULTS: Bone marrow cells from Ly49A transgenic and nontransgenic littermates were used to repopulate the hematopoietic system of lethally-irradiated female ldl-r-/- mice. After a recovery period to permit sufficient engraftment, mice were placed on a diet enriched in saturated fat and cholesterol. After 8 weeks, there was no difference in either serum total cholesterol concentrations or lipoprotein cholesterol distribution in mice repopulated with nontransgenic versus Ly49A transgenic marrow cells. Using immunohistochemistry, we detected NK cells in atherosclerotic lesions of both groups of mice. However, deficiency of functional NK cells significantly reduced the size of atherosclerosis by 70% (P=0.0002) in cross-sectional analysis of the aortic root and by 38% (P=0.004) in en face analysis of the intimal surface of the aortic arch. CONCLUSIONS: These studies demonstrate that NK cells infiltrate the vessel wall and promote atherosclerotic lesion development.

Macrophage-expressed group IIA secretory phospholipase A2 increases atherosclerotic lesion formation in LDL receptor-deficient mice.

OBJECTIVE: Transgenic mice expressing human group IIA secretory phospholipase A(2) (group IIA sPLA(2)) spontaneously develop atherosclerotic lesions. The mechanism for this proatherogenic effect is likely multifactorial, because HDL-cholesterol is significantly lower and LDL/VLDL cholesterol is slightly higher in transgenic mice compared with nontransgenic littermates. In the present study, we show for the first time that elicited peritoneal macrophages from transgenic mice express human group IIA sPLA(2). This study tested whether macrophage-expressed sPLA(2) contributes to atherogenesis. METHODS AND RESULTS: Bone marrow cells from either sPLA(2) transgenic mice or control C57BL/6 mice were transplanted into LDL receptor-deficient mice. After hematopoietic engraftment, animals were fed a diet enriched with saturated fat and cholesterol for 12 weeks. Despite a lack of effect on serum lipoprotein concentrations, the presence of bone marrow-derived cells expressing human group IIA sPLA(2) resulted in a significant increase in the extent of atherosclerosis in the aortic arch (12.8+/-1.4% versus 7.4+/-0.9%; P<0.005) and aortic sinus (0.3+/-0.03 mm(2) versus 0.2+/-0.04 mm(2); P<0.05). CONCLUSIONS: Group IIA sPLA(2) can contribute to atherosclerotic lesion development through a mechanism that is independent of systemic lipoprotein metabolism.

Genetic analysis of progenitor cell mobilization by granulocyte colony-stimulating factor: verification and mechanisms for loci on murine chromosomes 2 and 11.

OBJECTIVE: It is notable that there is significant inter-individual variability in humans and inter-strain variability in mice in the ability to mobilize hematopoietic stem and progenitor cells, suggesting that there is genetic regulation of mobilization. In the murine system, loci on chromosomes 2 and 11 have been linked to an inter-strain variation in granulocyte colonystimulating factor (G-CSF)-induced stem cell mobilization proficiency. The aim of this study was to verify this linkage and to gain insight into the function of these loci. METHODS: Animals congenic for the loci on chromosomes 2 and 11 were generated by a speed-congenic approach and the function of the loci were further analyzed in doubly congenic animals and with a competitive transplantation/mobilization protocol. RESULTS: The analysis of congenic animals verified that both loci are linked to mobilization proficiency. Analysis of mobilization in doubly congenic animals suggested that both loci act in the same regulatory pathway. Mobilization experiments conducted with mice that had previously been competitively repopulated with congenic and parental-strain BM revealed that the locus on chromosome 11 operates via a progenitor cell-intrinsic mechanism. CONCLUSION: We confirmed linkage of loci on chromosomes 2 and 11 to G-CSF-induced mobilization and thus validated their role as regulators of hematopoietic progenitor cell mobilization in mice. These findings will be useful for further studies directed at identifying genes that regulate mobilization proficiency.

The marrow homing efficiency of murine hematopoietic stem cells remains constant during ontogeny.

OBJECTIVE: Several recent studies have established the potential clinical utility of hematopoietic stem cells (HSCs) not only for marrow rescue but also for regenerating diseased or damaged nonhematopoietic tissues. These findings have focused renewed interest in understanding the in vivo trafficking patterns of HSCs from different sources. Previous experiments have suggested that the half-life of HSCs in the circulation is short, although the actual proportion that return to the bone marrow (BM) following transplantation has not been previously quantitated. The present study was undertaken to measure this fraction and compare the values obtained for functionally defined HSCs from adult murine BM and day-14 fetal liver (FL). METHODS: The number of HSCs that could be recovered from the BM of lethally irradiated mice 24 hours after intravenous injection of Ly-5 congenic BM or FL cells was determined by limiting-dilution competitive repopulating unit (CRU) assays in secondary mice. RESULTS: The marrow seeding efficiency of both adult BM- and FL-CRU able to produce lymphoid and myeloid progeny for 5-26 weeks posttransplant was approximately 10%. FL-CRU generated clones that were approximately threefold larger than those produced by BM-CRU. Interestingly, clones produced by "homed" HSCs were approximately twofold smaller than those produced by freshly isolated HSCs. Differences were also seen in the proportions of lymphoid vs myeloid progeny generated by fresh and homed HSCs. CONCLUSIONS: These data suggest common mechanisms regulating the BM homing of long-term repopulating HSCs throughout ontogeny despite subtle differences in the size and composition of the clones they generate.

The biology of hematopoietic stem cells.

Rarely has so much interest from the lay public, government, biotechnology industry, and special interest groups been focused on the biology and clinical applications of a single type of human cell as is today on stem cells, the founder cells that sustain many, if not all, tissues and organs in the body. Granting organizations have increasingly targeted stem cells as high priority for funding, and it appears clear that the evolving field of tissue engineering and regenerative medicine will require as its underpinning a thorough understanding of the molecular regulation of stem cell proliferation, differentiation, self-renewal, and aging. Despite evidence suggesting that embryonic stem (ES) cells might represent a more potent regenerative reservoir than stem cells collected from adult tissues, ethical considerations have redirected attention upon primitive cells residing in the bone marrow, blood, brain, liver, muscle, and skin, from where they can be harvested with relative sociological impunity. Among these, it is arguably the stem and progenitor cells of the mammalian hematopoietic system that we know most about today, and their intense study in rodents and humans over the past 50 years has culminated in the identification of phenotypic and molecular genetic markers of lineage commitment and the development of functional assays that facilitate their quantitation and prospective isolation. This review focuses exclusively on the biology of hematopoietic stem cells (HSCs) and their immediate progeny. Nevertheless, many of the concepts established from their study can be considered fundamental tenets of an evolving stem cell paradigm applicable to many regenerating cellular systems.

Quantitation of murine and human hematopoietic stem cells by limiting-dilution analysis in competitively repopulated hosts.

In designing functional assays for the various classes of hematopoietic cells described in this book, one needs to consider the properties of the cell to be measured which must be incorporated into the assay design, and the end points to allow its specific detection. The most primitive hematopoietic stem cells (HSC) in mouse and man are characterized by two cardinal properties that distinguish them from more mature clonogenic cells and their terminally differentiated progeny. Firstly, HSCs are pluripotent: they are characterized by the potential to differentiate into all of the eight major lineages of lymphoid, myeloid, and erythroid cells in vivo (1-3). Secondly, HSCs are able to self-renew, or generate daughter stem cells in vivo and in vitro that are functionally identical to the stem cell that gave rise to them (3-5). These hallmark properties of HSCs are measured empirically by their potential to regenerate and maintain lymphocytes, granulocytes, and erythrocytes upon transplantation into lethally irradiated or immunocompromised primary and secondary hosts. However, functional assays for primitive HSCs must also consider the fact that differentiated cells present in the hematopoietic organs at different times after bone marrow transplantation are derived from different types of precursors (6), and particularly at later times, cannot be assumed to be of donor origin (7). Support for this concept derives from the relatively recent demonstration in mice that most, if not all, spleen colonies detectable ≈ 2 wk after transplantation originate.

Most purported antibodies to the human granulocyte colony-stimulating factor receptor are not specific.

OBJECTIVE: Antibodies to human granulocyte colony-stimulating factor receptor (HuG-CSFR) are widely available and have been used in numerous studies to evaluate the expression of this protein on normal and malignant cells of hematopoietic and nonhematopoietic origin. Spurred by recent studies that demonstrated that two commonly used antibodies against the erythropoietin and thrombopoietin receptors can in fact bind to completely unrelated and more broadly expressed proteins, we screened 27 commercially available monoclonal and polyclonal antibodies with claimed specificity to HuG-CSFR to determine if they are specific to this receptor. MATERIALS AND METHODS: Antibodies were evaluated by Western blotting, flow cytometry, and immunohistochemistry using 293T cells engineered to overexpress HuG-CSFR protein, immortalized human hematopoietic cell lines expressing endogenous G-CSFR, and purified human neutrophils. RESULTS: Only two monoclonal antibodies and one polyclonal antibody could be employed using defined Western blotting or flow cytometry protocols to detect G-CSFR protein in cell lysates or on the surface of cells that express G-CSFR messenger RNA with no binding to cells that did not express the gene. None of the antibodies were suitable for immunohistochemistry. Competitive inhibition with soluble G-CSFR extracellular domain and small inhibitory RNA-mediated knock-down of G-CSFR messenger RNA further demonstrated the limited specificity of these antibodies for HuG-CSFR expressed on the cell surface. CONCLUSIONS: Most commercially available anti-HuG-CSFR antibodies do not bind specifically to this protein. These studies highlight the need for investigators to validate antibodies in their own systems to avoid the inadvertent use of nonspecifically binding antibodies that could lead, as exemplified in this case with a hematopoietic growth factor receptor, to erroneous conclusions about protein expression.

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

Haematopoietic stem and progenitor cell-targeted therapies for thrombocytopenia.

This review discusses the present outlook for new thrombocytopenia therapies that induce haematopoietic stem and progenitor cells to proliferate, differentiate and produce functional platelets. A brief overview of megakaryopoiesis and its regulation by thrombopoietin (TPO) is followed by a discussion of how early experience with recombinant TPO therapies stimulated the search for novel TPO receptor ligands. A summary is then provided of the results of Phase I clinical trials with the new small molecule and peptide TPO mimetics that are in development at present. Finally, recent developments in the ex vivo expansion of primitive haematopoietic cells and the potential enhancement of cell-based therapies by haematopoietic growth factors in vivo are briefly summarised as part of a look towards the future.

Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells.

To test the hypothesis that aging has negative effects on stem-cell homing and engraftment, young or old C57BL/6 bone marrow (BM) cells were injected, using a limiting-dilution, competitive transplantation method, into old or young Ly5 congenic mice. Numbers of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) recovered from BM or spleen were measured and compared with the numbers initially transplanted. Although the frequency of marrow competitive repopulation units (CRUs) increased approximately 2-fold from 2 months to 2 years of age, the BM homing efficiency of old CRUs was approximately 3-fold lower than that of young CRUs. Surprisingly, the overall size of individual stem-cell clones generated in recipients receiving a single CRU was not affected by donor age. However, the increased ages of HSC donors and HSC transplant recipients caused marked skewing of the pattern of engraftment toward the myeloid lineage, indicating that HSC-intrinsic and HSC-extrinsic (microenvironmental) age-related changes favor myelopoiesis. This correlated with changes after transplantation in the rate of recovery of circulating leukocytes, erythrocytes, and platelets. Recovery of the latter was especially blunted in aged recipients. Collectively, these findings may have implications for clinical HSC transplantation in which older persons increasingly serve as donors for elderly patients.

Interleukin-4 deficiency promotes gallstone formation.

Feeding interleukin-4 (IL-4) deficient C57BL/6 LDL receptor (LDLr)(-/-) mice a modified diet to investigate the role of this cytokine in cholesterol metabolism led to an unexpected phenotype. IL-4(-/-) --> LDLr(-/-) mice had enlarged gallbladders and an increased mortality that was preceded by acute body weight loss. To determine if IL-4 deficiency accounted for these findings, C57BL/6 IL-4(+/+) and IL-4(-/-) mice were fed either a normal or modified diet. IL-4 deficiency did not alter bile composition or cause liver toxicity in mice fed a fat-enriched diet. Following 8 weeks of feeding a fat-enriched diet, no gallstones were detected in IL-4(+/+) mice, and only 20% had cholesterol crystals. In contrast, IL-4(-/-) mice had a 100% incidence of gallstones and cholesterol crystals. IL-4(-/-) deficiency also increased serum concentrations of bilirubin following feeding a fat-enriched diet. Therefore, these studies revealed an unexpected finding that IL-4 deficiency predisposes to gallstone formation.

Macrophage-specific expression of class A scavenger receptors in LDL receptor(-/-) mice decreases atherosclerosis and changes spleen morphology.

Class A scavenger receptors (SR-A) have been implicated in the atherogenic process, although there have been conflicting reports as to their specific effect on the development of lesions. In part, this discord may arise because of the variable contribution of SR-A in the several cell types known to express this protein. To determine the effects of macrophage-specific SR-A expression in the atherogenic process, transgenic mice were created using the chicken lysozyme (lyso) promoter to drive expression of bovine SR-A (bSR-A). To express this gene in an atherosclerosis-susceptible strain, bone marrow cells from transgenic and non-transgenic littermates were used to repopulate lethally-irradiated female LDL receptor (LDLr)(-/-) mice. Following hematopoietic engraftment, mice were placed on a diet enriched in saturated fat and cholesterol. After 8 weeks, there was a modest, but statistically significant reduction in serum total cholesterol in LDLr(-/-) mice repopulated with lyso-bSR-A transgenic cells, due to decreased LDL-cholesterol. The extent of atherosclerosis was reduced in both cross-sectional analysis of the aortic root and en face analysis of the intimal surface of the aortic arch. In addition to changes in atherosclerosis, lyso-bSR-A repopulated LDLr(-/-) mice had a marked increase (3.6x) in spleen weights and a disruption of spleen white pulp formation. Therefore, macrophage-specific overexpression of SR-A resulted in reduced atherosclerosis in two vascular beds, reduced serum cholesterol concentrations, and changed the morphology of the spleen.

Abdominal aortic aneurysms: fresh insights from a novel animal model of the disease.

Abdominal aortic aneurysms (AAA) have a high prevalence in aged populations and are responsible for a large number of deaths. Despite the widespread nature of the disease, relatively little is known regarding mechanisms for formation and progression of aortic aneurysms. In part, this lack of knowledge is attributable to a paucity of animal models for this disease. This review summarizes the available animal models of AAA and focuses on a novel model of reproducible AAA generated by infusion of angiotensin II (AngII) into mice rendered hyperlipidemic by the absence of either apolipoprotein E or low-density lipoprotein receptors. AAA generated by AngII infusion have many characteristics of the human disease including marked luminal expansions, perimedial remodeling, inflammation, thrombosis and a link to hyperlipidemia. As in the human disease, male mice are more susceptible to the development of AAA than females. The vascular pathology occurs from the effects of AngII at AT1 receptors present on bone marrow-derived cells. Studies are ongoing to define the mediators responsible for AngII-induced inflammation and degradation of the medial layer of the vascular wall. The AngII-induced model of AAA has the potential to provide novel insights into the underlying mechanisms of this disease and assist with the development of pharmacological therapies.

Preferential induction of apoptosis for primary human leukemic stem cells.

Acute myelogenous leukemia (AML) is typically a disease of stem progenitor cell origin. Interestingly, the leukemic stem cell (LSC) shares many characteristics with normal hematopoietic stem cells (HSCs) including the ability to self-renew and a predominantly G(0) cell-cycle status. Thus, although conventional chemotherapy regimens often ablate actively cycling leukemic blast cells, the primitive LSC population is likely to be drug-resistant. Moreover, given the quiescent nature of LSCs, current drugs may not effectively distinguish between malignant stem cells and normal HSCs. Nonetheless, based on recent studies of LSC molecular biology, we hypothesized that certain unique properties of leukemic cells could be exploited to induce apoptosis in the LSC population while sparing normal stem cells. In this report we describe a strategy using treatment of primary AML cells with the proteasome inhibitor carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (MG-132) and the anthracycline idarubicin. Comparison of normal and leukemic specimens using in vitro culture and in vivo xenotransplantation assays shows that the combination of these two agents induces rapid and extensive apoptosis of the LSC population while leaving normal HSCs viable. Molecular genetic studies using a dominant-negative allele of inhibitor of nuclear factor kappaB (IkappaBalpha) demonstrate that inhibition of nuclear factor kappaB (NF-kappaB) contributes to apoptosis induction. In addition, gene-expression analyses suggest that activation of p53-regulated genes are also involved in LSC apoptosis. Collectively, these findings demonstrate that malignant stem cells can be preferentially targeted for ablation. Further, the data begin to elucidate the molecular mechanisms that underlie LSC-specific apoptosis and suggest new directions for AML therapy.