Stephen B. Marley

Characterization and Clinical Application of Human CD34+ Stem/Progenitor Cell Populations Mobilized into the Blood by Granulocyte Colony‐Stimulating Factor

A phase I study was performed to determine the safety and tolerability of injecting autologous CD34+ cells into five patients with liver insufficiency. The study was based on the hypothesis that the CD34+ cell population in granulocyte colony‐stimulating factor (G‐CSF)‐mobilized blood contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated a candidate CD34+ stem cell population from the majority of the CD34+ cells (99%) by adherence to tissue culture plastic. The adherent and nonadherent CD34+ cells were distinct in morphology, immunophenotype, and gene expression profile. Reverse transcription‐polymerase chain reaction‐based gene expression analysis indicated that the adherent CD34+ cells had the potential to express determinants consistent with liver, pancreas, heart, muscle, and nerve cell differentiation as well as hematopoiesis. Overall, the characteristics of the adherent CD34+ cells identify them as a separate putative stem/progenitor cell population. In culture, they produced a population of cells exhibiting diverse morphologies and expressing genes corresponding to multiple tissue types. Encouraged by this evidence that the CD34+ cell population contains cells with the potential to form hepatocyte‐like cells, we gave G‐CSF to five patients with liver insufficiency to mobilize their stem cells for collection by leukapheresis. Between 1 × 106 and 2 × 108 CD34+ cells were injected into the portal vein (three patients) or hepatic artery (two patients). No complications or specific side effects related to the procedure were observed. Three of the five patients showed improvement in serum bilirubin and four of five in serum albumin. These observations warrant further clinical trials.

Autologous Infusion of Expanded Mobilized Adult Bone Marrow-Derived CD34+ Cells Into Patients With Alcoholic Liver Cirrhosis

OBJECTIVES: Recent advances in regenerative medicine, including hematopoietic stem cell (HSC) transplantation, have brought hope for patients with severe alcoholic liver cirrhosis (ALC). The aim of this study was to assess the safety and efficacy of administering autologous expanded mobilized adult progenitor CD34+ cells into the hepatic artery of ALC patients and the potential improvement in the liver function.METHODS: Nine patients with biopsy-proven ALC, who had abstained from alcohol for at least 6 months, were recruited into the study. Following granulocyte colony-stimulating factor (G-CSF) mobilization and leukapheresis, the autologous CD34+ cells were expanded in vitro and injected into the hepatic artery. All patients were monitored for side effects, toxicities, and changes in the clinical, hematological, and biochemical parameters.RESULTS: On average, a five-fold expansion in cell number was achieved in vitro, with a mean total nucleated cell count (TNCC) of 2.3 × 108 pre infusion. All patients tolerated the procedure well, and there were no treatment-related side effects or toxicities observed. There were significant decreases in serum bilirubin (P < 0.05) 4, 8, and 12 wk post infusion. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) showed improvement through the study period and were significant (P < 0.05) 1 wk post infusion. The Child-Pugh score improved in 7 out of 9 patients, while 5 patients had improvement in ascites on imaging.CONCLUSION: It is safe to mobilize, expand, and reinfuse autologous CD34+ cells in patients with ALC. The clinical and biochemical improvement in the study group is encouraging and warrants further clinical trials.

The tyrosine kinase inhibitor STI571, like interferon-α, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors from patients with chronic myeloid leukemia

Abstract Objective To determine whether the compound STI571 (formerly known as CGP571418B), a selective inhibitor of the protein tyrosine kinase (PTK) activity of ABL and BCR-ABL proteins, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors (CFU-GM) from patients with chronic myeloid leukemia while sparing normal CFU-GM and to compare responses of CML and normal cells with STI571 and IFN-α. Materials and Methods Chronic phase CML and normal CFU-GM were grown with and without STI571, IFN-α, or the two agents in combination. Colonies were plucked and replated in 96-well microtiter plates. Secondary colonies were scored, and the results were expressed as the area-under-the-curve (AUC) of the distribution of secondary colony numbers per primary CFU-GM. This value gives an overall measure of the replating ability or amplification of the original CFU-GM population. Results STI571 selectively inhibits the formation of granulocyte-macrophage colony-forming cells (CFU-GM) from CML patients. It also significantly inhibits the amplification of CML CFU-GM (p = 0.002) as measured by secondary colony formation after replating primary CFU-GM colonies. In contrast, amplification of normal CFU-GM was enhanced (p = 0.001) at low concentrations (0.1 μM) of STI571 with a return to baseline at 10 μM STI571. Addition of interferon (IFN)-α to STI571 abolished the increase in normal CFU-GM amplification seen with either agent alone. There was a highly significant correlation between the in vitro response to STI571 and the in vitro response to IFN-α (r = 0.74 for CML cells, and 0.77 for normal cells). Conclusion We conclude that STI571, like IFN-α, preferentially suppresses amplification of CML CFU-GM while sparing normal CFU-GM.

Chronic myeloid leukaemia: stem cell derived but progenitor cell driven.

The biology of CML (chronic myeloid leukaemia) has been extensively investigated as the disease is a paradigm of neoplasms induced when a translocation results in expression of a novel fusion protein, in this instance p210(BCR-ABL). Although CML manifests itself principally as unregulated expansion of the myeloid lineage, the lesion is present in the stem cell population and it has long been assumed that disregulated stem cell kinetics must underlie the basic pathology of the disease. In this review, we present evidence that, in normal haemopoiesis, less primitive precursor cells retain considerable flexibility in their capacity to undergo self-renewal, allowing them to maintain lineage-specific homoeostasis without inflicting proliferative stress upon the stem cell population. This mechanism is dysregulated in CML and we have developed a self-renewal assay for CFU-GM (colony-forming unit-granulocyte/macrophage) which demonstrates that, in CML, the PI (proliferative index) of the myeloid progenitor cell population is increased. The ability to measure the PI as an endpoint of p210(BCR-ABL) expression gives considerable versatility to the in vitro investigation of putative therapeutic regimes in CML.

Effects of combinations of therapeutic agents on the proliferation of progenitor cells in chronic myeloid leukaemia.

Summary. Combination of STI571, a tyrosine kinase inhibitor, with other drugs may be beneficial in the treatment of chronic myeloid leukaemia (CML). We measured the effects of STI571, AG490, farnesyltransferase inhibitor (FTI), interferon alpha (IFN‐α), cytosine arabinoside (Ara‐C) and all‐trans retinoic acid (ATRA), singly and in combination, on clonogenic leukaemic cell proliferation. STI571, IFN‐α and ATRA each reduced proliferation by 50–60%; AG490, FTI and Ara‐C had less effect. Comparing the observed and expected (i.e. additive) effects of drug combinations showed STI571 + FTI, STI571 + AG490 and IFN‐α+ ATRA were additive; STI571 + IFN‐α, IFN‐α+ Ara‐C and STI571 + AG490 + FTI were less than additive. Thus, STI571 + FTI, STI571 + AG490 and IFN‐α+ ATRA may be better combination therapies for CML than STI571 +  IFN‐α, IFN‐α+ Ara‐C or STI571 + AG490 + FTI.

High frequency of fetal cells within a primitive stem cell population in maternal blood

BACKGROUND During pregnancy, fetal cells enter the maternal bloodstream resulting in fetal cell microchimerism. The fetal cells persist in the mother for decades and colonize a variety of maternal organs. They are associated with maternal autoimmune diseases and may also participate in tissue repair. The identity of the microchimeric cells is not certain but they must be able to persist long-term and have potential for multitissue differentiation. METHODS AND RESULTS Here we tested the hypothesis that the fetal microchimeric cells are primitive stem cells, represented by CD34+ adherent cells, which have a wide potential for differentiation. We isolated these stem cells from the blood of pregnant females (n = 25) and detected fetal cells of the correct gender, using fluorescence in situ hybridization, in a high proportion (71% male fetuses and 90% female fetuses; false positive rate 11%, false negative rate 29%) of cases. By RT-PCR, we demonstrated that the cells express Oct-4, Nanog and Rex-1. No fetal cells were detected in the mononuclear or total CD34+ cell populations but high frequencies (mean 11.8%) of fetal cells were detected in the adherent CD34+ cell population. CONCLUSIONS These results identify adherent CD34+ stem cells as candidate fetal microchimeric cells, which are capable of sustaining the fetal cell population in the long term and have the ability to colonize multiple tissues and organs.

Targeting primary human leukaemia cells with RNA interference: Bcr-Abl targeting inhibits myeloid progenitor self- renewal in chronic myeloid leukaemia cells

We have investigated functional outcome of challenging primary chronic myeloid leukaemia (CML) cells with Bcr‐Abl fusion sequence‐directed RNA interference (RNAi). We targeted the Bcr‐Abl b3a2 variant, by RNAi, in primary chronic phase CML cells, and detected strikingly reduced proliferation of myeloid precursor cells expressing this variant. Lack of an effect in cells expressing a distinct Bcr‐Abl variant confirmed the specificity of the response. Through the functional targeting of an oncogene in primary human tumour cells, we have demonstrated that Bcr‐Abl enhances CML progenitor cell amplification, and that RNAi may be suitable for development as a specific anti‐leukaemia treatment.

Progenitor cells divide symmetrically to generate new colony-forming cells and clonal heterogeneity

Summary. Self‐renewal is the most fundamental property of haemopoietic stem and progenitor cells. However, because of the need to produce differentiated cells, not all cell divisions involve self‐renewal. We have used a colony replating assay to follow the fates of individual haemopoietic progenitor cell clones. For this, human myeloid colony‐forming cells (CFCs) were cultured by standard methodology. Onset of proliferation and growth rates were established by a video recording method. Individual colonies were replated several times to document the rate of clonal extinction, and the numbers of secondary, tertiary and quaternary CFCs. The clonogenic population exhibited similar kinetics in terms of onset of proliferation and growth rate. Clonal extinction was progressive so that only 30 ± 7% (mean ± standard error of the mean; n = 4) of the original primary colonies formed quaternary colonies after the third replating step. However, individual primary CFCs that produced colonies throughout the experiment generated, on average, 40 ± 8 secondary and tertiary CFCs overall. The values obtained in standard culture conditions were modified when granulocyte colony‐stimulating factor (G‐CSF) or G‐CSF plus interleukin 3 were used to stimulate colony growth, showing that the kinetics of colony formation respond to extrinsic regulation. Examination of the replating potential of individual secondary colonies in the clones demonstrated that they generated different numbers of tertiary colonies. The data best fit a stochastic model of haemopoietic cell development where event probabilities can be modified by extracellular factors.

Phosphatidylinositol‐3 kinase inhibitors reproduce the selective antiproliferative effects of imatinib on chronic myeloid leukaemia progenitor cells

We investigated the role of the phosphatidylinositol‐3 kinase (PI‐3K) pathway in regulating the proliferation of primary chronic myeloid leukaemia (CML) progenitor cells by using imatinib to inhibit the activity of p210Bcr‐Abl. The effect of imatinib on the expression of PI‐3K pathway proteins was investigated by kinase assays and Western blotting; PI‐3K was inhibited by wortmannin or LY294002, Jak2 by AG490 and farnesylation by FTI II; progenitor cell proliferation (self‐renewal) was measured by growing myeloid colonies in vitro, then replating them to observe secondary colony formation. Suppression of p210Bcr‐Abl with imatinib indirectly suppressed the activity of PI‐3K and its downstream targets (Erk, Akt and p70S6 kinase), thereby implicating the PI‐3K pathway in p210Bcr‐Abl‐mediated signalling in primary CML progenitor cells. The PI‐3K inhibitors, wortmannin and LY294002 reproduced the differential effects of imatinib on normal and CML progenitor cell proliferation in vitro by increasing normal cell (P = 0·001) and reducing CML cell proliferation (P = 0·0003). This differential effect was attributable to dysregulated signalling by granulocyte colony‐stimulating factor in CML. The responses of individual patients cells to wortmannin correlated with their responses to imatinib (P = 0·004) but not their responses to AG490 (Jak2 kinase inhibitor) or FTI II (farnesyltransferase inhibitor). Individual responses to wortmannin also correlated with responses to interferon α (IFNα) (P = 0·016). Imatinib‐resistant K562 cells were sensitive to LY294002. Inhibition of the PI‐3K pathway may be common to imatinib and IFNα and reflect dysregulated cytokine signalling. As imatinib‐resistant cells remained sensitive to wortmannin and LY294002, targeting the PI‐3K pathway may provide an alternative therapy for imatinib‐resistant patients.

Potential of CD34 in the Regulation of Symmetrical and Asymmetrical Divisions by Hematopoietic Progenitor Cells

The control of symmetric and asymmetric division in the hematopoietic stem/progenitor cell population is critically important for the regulation of blood cell production. Asymmetric divisions depend on cell polarization, which may be conferred by location and/or interaction with neighboring cells. In this study, we sought evidence for polarization in CD34+ cells, which interact by binding to one another. In these cells, surface molecules became redistributed by mechanisms that included transport by lipid rafts, and the interacting cells were able to communicate via gap junctions. These changes were accompanied by modulation of cell cycle regulating proteins (p16Ink4a, p27kip1, cyclins D, and the retinoblastoma pathway proteins) and a reduction in progenitor cell proliferation in vitro. These results are consistent with an increase in asymmetric cell division kinetics. Accordingly, we found that interaction between CD34+ cells influenced the plane of cell division in a way that suggests unequal sharing of Notch‐1 between daughter cell progeny. We conclude that interaction between CD34+ cells may coordinate cell function and participate in the control of hematopoietic stem/progenitor cell division kinetics.