Edward F. Srour

Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist

Improving approaches for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is clinically important because increased numbers of these cells are needed for enhanced transplantation. Chemokine stromal cell derived factor-1 (also known as CXCL12) is believed to be involved in retention of HSCs and HPCs in bone marrow. AMD3100, a selective antagonist of CXCL12 that binds to its receptor, CXCR4, was evaluated in murine and human systems for mobilizing capacity, alone and in combination with granulocyte colony-stimulating factor (G-CSF). AMD3100 induced rapid mobilization of mouse and human HPCs and synergistically augmented G-CSF–induced mobilization of HPCs. AMD3100 also mobilized murine long-term repopulating (LTR) cells that engrafted primary and secondary lethally-irradiated mice, and human CD34+ cells that can repopulate nonobese diabetic-severe combined immunodeficiency (SCID) mice. AMD3100 synergized with G-CSF to mobilize murine LTR cells and human SCID repopulating cells (SRCs). Human CD34+ cells isolated after treatment with G-CSF plus AMD3100 expressed a phenotype that was characteristic of highly engrafting mouse HSCs. Synergy of AMD3100 and G-CSF in mobilization was due to enhanced numbers and perhaps other characteristics of the mobilized cells. These results support the hypothesis that the CXCL12-CXCR4 axis is involved in marrow retention of HSCs and HPCs, and demonstrate the clinical potential of AMD3100 for HSC mobilization.

Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

Estradiol-regulated microRNAs control estradiol response in breast cancer cells

Estradiol (E2) regulates gene expression at the transcriptional level by functioning as a ligand for estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). E2-inducible proteins c-Myc and E2Fs are required for optimal ERα activity and secondary estrogen responses, respectively. We show that E2 induces 21 microRNAs and represses seven microRNAs in MCF-7 breast cancer cells; these microRNAs have the potential to control 420 E2-regulated and 757 non-E2-regulated mRNAs at the post-transcriptional level. The serine/threonine kinase, AKT, alters E2-regulated expression of microRNAs. E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins. Dicer, a ribonuclease III enzyme required for microRNA processing, is also an E2-inducible gene. Several E2-regulated microRNA genes are associated with ERα-binding sites or located in the intragenic region of estrogen-regulated genes. We propose that the clinical course of ERα-positive breast cancers is dependent on the balance between E2-regulated tumor-suppressor microRNAs and oncogenic microRNAs. Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome.

High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years

Transplanted cord blood (CB) hematopoietic stem cells (HSC) and progenitor cells (HPC) can treat malignant and nonmalignant disorders. Because long-term cryopreservation is critical for CB banking and transplantation, we assessed the efficiency of recovery of viable HSC/HPC from individual CBs stored frozen for 15 yr. Average recoveries (± 1 SD) of defrosted nucleated cells, colony-forming unit-granulocyte, -macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, -erythrocyte, -monocyte, and -megakaryocyte (CFU-GEMM) were, respectively, 83 ± 12, 95 ± 16, 84 ± 25, and 85 ± 25 using the same culture conditions as for prefreeze samples. Proliferative capacities of CFU-GM, BFU-E, and CFU-GEMM were intact as colonies generated respectively contained up to 22,500, 182,500, and 292,500 cells. Self-renewal of CFU-GEMM was also retained as replating efficiency of single CFU-GEMM colonies into 2° dishes was >96% and yielded 2° colonies of CFU-GM, BFU-E, and CFU-GEMM. Moreover, CD34+CD38− cells isolated by FACS after thawing yielded >250-fold ex vivo expansion of HPC. To assess HSC capability, defrosts from single collections were bead-separated into CD34+ cells and infused into sublethally irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. CD45+ human cell engraftment with multilineage phenotypes was detected in mice after 11–13 wk; engrafting levels were comparable to that reported with fresh CB. Thus, immature human CB cells with high proliferative, replating, ex vivo expansion and mouse NOD/SCID engrafting ability can be stored frozen for >15 yr, can be efficiently retrieved, and most likely remain effective for clinical transplantation.

Roles of spleen and liver in development of the murine hematopoietic system

OBJECTIVE Hematopoietic stem cells (HSCs) and colony-forming progenitor cells (CFCs) are believed to migrate from liver to bone marrow (BM) around the time of birth, where they remain throughout the animals life. Although in mice the spleen is also a hematopoietic organ, neither the origin nor the contribution of spleen HSCs to hematopoietic homeostasis has been assessed relative to that of BM HSCs. To investigate these issues we quantitated CFC and HSC activity in the spleen, BM, peripheral blood, and liver of the mouse during ontogeny. METHODS CFCs were assessed by clonogenic colony formation, and HSCs by long-term reconstituting ability. RESULTS CFCs gradually increased in the BM and decreased in the liver with age. Increased prevalence of CFCs in fetal and pup blood occurred at day (d) 12 postcoitus (pc) and during the period of d16 pc to 4d postbirth, corresponding to the times when hematopoietic cells migrate from the yolk sac and/or aorta-gonad-mesonephros (AGM) to the fetal liver and from the neonatal liver to the BM, respectively. In the spleen, CFCs displayed two peaks of activity at 2d and 14d-15d postbirth. Spleen HSCs also fluctuated during this time period. Neonatal splenectomy did not alter CFC or HSC frequencies in the BM, but CFCs increased in the livers of splenectomized mice. CONCLUSIONS These data demonstrate that the liver may act as a site of extramedullary hematopoiesis in the neonate, especially in the absence of the spleen, and imply that the spleen, BM, and liver cooperatively contribute to hematopoietic homeostasis.

Dipeptidylpeptidase 4 negatively regulates colony-stimulating factor activity and stress hematopoiesis

Enhancement of hematopoietic recovery after radiation, chemotherapy, or hematopoietic stem cell (HSC) transplantation is clinically relevant. Dipeptidylpeptidase (DPP4) cleaves a wide variety of substrates, including the chemokine stromal cell-derived factor-1 (SDF-1). In the course of experiments showing that inhibition of DPP4 enhances SDF-1–mediated progenitor cell survival, ex vivo cytokine expansion and replating frequency, we unexpectedly found that DPP4 has a more general role in regulating colony-stimulating factor (CSF) activity. DPP4 cleaved within the N-termini of the CSFs granulocyte-macrophage (GM)-CSF, G-CSF, interleukin-3 (IL-3) and erythropoietin and decreased their activity. Dpp4 knockout or DPP4 inhibition enhanced CSF activities both in vitro and in vivo. The reduced activity of DPP4-truncated versus full-length human GM-CSF was mechanistically linked to effects on receptor-binding affinity, induction of GM-CSF receptor oligomerization and signaling capacity. Hematopoiesis in mice after radiation or chemotherapy was enhanced in Dpp4−/− mice or mice receiving an orally active DPP4 inhibitor. DPP4 inhibition enhanced engraftment in mice without compromising HSC function, suggesting the potential clinical utility of this approach.

Impact of interactions of cellular components of the bone marrow microenvironment on hematopoietic stem and progenitor cell function

Hematopoietic stem (HSC) and progenitor (HPC) cell fate is governed by intrinsic and extrinsic parameters. We examined the impact of hematopoietic niche elements on HSC and HPC function by analyzing the combined effect of osteoblasts (OBs) and stromal cells (SCs) on Lineage(-)Sca-1(+)CD117(+) (LSK) cells. CFU expansion and marrow repopulating potential of cultured Lineage(-)Sca-1(+)CD117(+) cells were significantly higher in OB compared with SC cultures, thus corroborating the importance of OBs in the competence of the hematopoietic niche. OB-mediated enhancement of HSC and HPC function was reduced in cocultures of OBs and SCs, suggesting that SCs suppressed the OB-mediated hematopoiesis-enhancing activity. Although the suppressive effect of SC was mediated by adipocytes, probably through up-regulation of neuropilin-1, the OB-mediated enhanced hematopoiesis function was elaborated through Notch signaling. Expression of Notch 2, Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, and Deltex was increased in OB cultures and suppressed in SC and OB/SC cultures. Phenotypic fractionation of OBs did not segregate the hematopoiesis-enhancing activity but demonstrated that this function is common to OBs from different anatomic sites. These data illustrate that OBs promote in vitro maintenance of hematopoietic functions, including repopulating potential by up-regulating Notch-mediated signaling between HSCs and OBs.

Breast Cancer Stem Cells and Intrinsic Subtypes: Controversies Rage On

Heterogeneity is a well-documented phenomenon in breast cancer; one of the explanations for this phenomenon is the presence of cancer stem cells (CSCs) with the capacity to differentiate along divergent pathways. These CSCs undergo asymmetric and symmetric division resulting in both expansion of the stem cell pool and the production of morphologically and functionally distinct differentiated daughter cells. Breast cancer cells that express the cell surface molecule CD44 but lack the expression of CD24 have been described as CSCs. Breast cancer cells expressing elevated levels of Aldehyde Dehydrogenase 1 (ALDH1) are also described as CSCs with ALDH1+/CD44+/CD24- subpopulation displaying highest tumorigenic potential in NOD/SCID models. The CSC hypothesis for tumor heterogeneity raises three important questions. First, in unrelated gene expression studies, breast cancers have been classified to five intrinsic subtypes; luminal type A, luminal type B, basal type, ErbB2/HER2-positive and normal-like. Therefore, do these intrinsic subtypes of breast cancer have distinct CSCs of their own or are ALDH1+ or CD44+/CD24- cells common CSCs for all intrinsic subtypes? Secondly, do ALDH1+ or CD44+/CD24- CSCs originate from normal cells of same phenotype or can differentiated cancer cells acquire ALDH1 or CD44+/CD24- status due to mutagenic events? Third, do ALDH1+, ALDH1-, CD44+/CD24- and non-CD44+/CD24- cancer cells differ in their ability to metastasize and respond to chemotherapy? In this review, we present our views on these questions based on studies conducted by several laboratories including ours and present evidence for a strong association of CD44+/CD24- phenotype with basal-like or mesenchymal-like cancer cells.

Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells: Are We There Yet?

Ex vivo expansion of hematopoietic stem and progenitor cells is a very ambitious idea that would have major implications in the areas of stem cell transplantation and somatic gene therapy. However, successful ex vivo expansion has evaded and frustrated scientists for a number of years. The goal of ex vivo expansion is to induce cell division and proliferation of stem cells while maintaining their primary functional characteristic, namely, their ability to engraft and sustain long-term hematopoiesis. Only when a balance between these two requirements is reached can ex vivo expansion of stem cells be considered successful. Establishing such a balance has not been easy. However, many lessons have been learned along the way, and today we have a more profound understanding of the potential obstacles facing ex vivo expansion than we did only a few years ago. In this review, we discuss these obstacles and evaluate the current status of ex vivo expansion of stem and progenitor cells both from the perspective of basic stem cell biology and from the viewpoint of clinical utility of these cells in transplantation.