Cytokines and hematopoietic stem cell mobilization

Hematopoietic stem cell transplantation (HSCT) has become the standard of care for the treatment of many hematologic malignancies, chemotherapy sensitive relapsed acute leukemias or lymphomas, multiple myeloma; and for some non-malignant diseases such as aplastic anemia and immunodeficient states. The hematopoietic stem cell (HSC) resides in the bone marrow (BM). A number of chemokines and cytokines have been shown in vivo and in clinical trials to enhance trafficking of HSC into the peripheral blood. This process, termed stem cell mobilization, results in the collection of HSC via apheresis for both autologous and allogeneic transplantation. Enhanced understanding of HSC biology, processes involved in HSC microenvironmental interactions and the critical ligands, receptors and cellular proteases involved in HSC homing and mobilization, with an emphasis on G-CSF induced HSC mobilization, form the basis of this review. We will describe the key features and dynamic processes involved in HSC mobilization and focus on the key ligand-receptor pairs including CXCR4/SDF1, VLA4/VCAM1, CD62L/PSGL, CD44/HA, and Kit/KL. In addition we will describe food and drug administration (FDA) approved and agents currently in clinical development for enhancing HSC mobilization and transplantation outcomes.     

Optimization of ex vivo hematopoietic stem cell expansion in intermittent dynamic cultures

For the ex vivo expansion of CD34⁺ cells, culture conditions were optimized using cytokine cocktails and media change methods. In addition, static, orbital-shake, and stirred cultures were compared. After cultivation, total cell expansion, immunophenotypes, clonogenic ability, and metabolite concentration in media were analyzed. Optimized media change methods enhanced the number of total nucleated cells (TNCs) by 600-fold (from 10⁴ to 6 × 10⁶ cells) in static cultures. Furthermore, intermittent orbital-shake cultures gave the highest fold increase of TNCs and CD34⁺/CD38⁻ cells. These results imply that proliferation of CD34⁺ cells in intermittent shake cultures was more efficient than that in static cultures under optimized culture conditions.     

Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells

Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3(+) cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.     

Modelling of ex vivo expansion/maintenance of hematopoietic stem cells

In this study, we described the modelling of the expansion/maintenance of human hematopoietic stem/progenitor cells from adult human bone marrow. CD 34(+)-enriched cell populations from bone marrow were cultured in the presence and absence of human stroma in serum-free media containing bFGF, SCF, LIF and Flt-3 ligand for several days. The cells in the culture were analysed for expansion and phenotype by flow cytometry. Although significant expansion of bone marrow cultures occurred in the presence and absence of human stroma, the results of expansion were effectively better in the presence of a stromal layer. In both situations the phenotypic analysis demonstrated a great expansion of CD 34(+)38(-) cells. The differentiative potential of bone marrow CD 34(+) cells co-cultured with human stroma was primarily shifted towards the myeloid lineage with the presence of CD 15 and CD 33.     

Human umbilical cord Wharton's jelly stem cells and its conditioned medium support hematopoietic stem cell expansion ex vivo

Bone marrow mesenchymal stromal cells (BMMSCs) have been used as feeder support for the ex vivo expansion of hematopoietic stem cells (HSCs) but have the limitations of painful harvest, morbidity, and risk of infection to the patient. This prompted us to explore the use of human umbilical cord Wharton's jelly MSCs (hWJSCs) and its conditioned medium (hWJSC-CM) for ex vivo expansion of HSCs in allogeneic and autologous settings because hWJSCs can be harvested in abundance painlessly, are proliferative, hypoimmunogenic, and secrete a variety of unique proteins. In the presence of hWJSCs and hWJSC-CM, HSCs put out pseudopodia-like outgrowths and became highly motile. Time lapse imaging showed that the outgrowths helped them to migrate towards and attach to the upper surfaces of hWJSCs and undergo proliferation. After 9 days of culture in the presence of hWJSCs and hWJSC-CM, MTT, and Trypan blue assays showed significant increases in HSC numbers, and FACS analysis generated significantly greater numbers of CD34(+) cells compared to controls. hWJSC-CM produced the highest number of colonies (CFU assay) and all six classifications of colony morphology typical of hematopoiesis were observed. Proteomic analysis of hWJSC-CM showed significantly greater levels of interleukins (IL-1a, IL-6, IL-7, and IL-8), SCF, HGF, and ICAM-1 compared to controls suggesting that they may be involved in the HSC multiplication. We propose that cord blood banks freeze autologous hWJSCs and umbilical cord blood (UCB) from the same umbilical cord at the same time for the patient for future ex vivo HSC expansion and cell-based therapies.     

Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansion

BACKGROUND: Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells. METHODS: The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed. RESULTS: We demonstrate that conditions supporting in vitro stem cell self-renewal efficiently promote oncoretroviral-mediated gene transfer to multipotent adult bone marrow stem cells, without prior in vivo conditioning. Despite using optimized culture conditions, transduction resulted in striking losses of repopulating activity, translating into low numbers of gene marked cells in competitively repopulated mice. Subjecting transduced HSCs to an ex vivo expansion protocol following the transduction procedure could partially reverse this loss. CONCLUSIONS: These studies suggest that loss of repopulating ability of transduced HSCs rather than low gene transfer efficiency might be the main problem in clinical gene therapy protocols, and that a clinically feasible ex vivo expansion approach post-transduction can markedly improve reconstitution with gene marked stem cells.     

Regulation of hematopoietic stem and progenitor cell mobilization by cholesterol efflux pathways

Intact cholesterol homeostasis helps to maintain hematopoietic stem and multipotential progenitor cell (HSPC) quiescence. Mice with defects in cholesterol efflux pathways due to deficiencies of the ATP binding cassette transporters ABCA1 and ABCG1 displayed a dramatic increase in HSPC mobilization and extramedullary hematopoiesis. Increased extramedullary hematopoiesis was associated with elevated serum levels of G-CSF due to generation of IL-23 by splenic macrophages and dendritic cells. This favored hematopoietic lineage decisions toward granulocytes rather than macrophages in the bone marrow leading to impaired support for osteoblasts and decreased Cxcl12/SDF-1 production by mesenchymal progenitors. Greater HSPC mobilization and extramedullary hematopoiesis were reversed by raising HDL levels in Abca1(-/-)Abcg1(-/-) and Apoe(-/-) mice or in a mouse model of myeloproliferative neoplasm mediated by Flt3-ITD mutation. Our data identify a role of cholesterol efflux pathways in the control of HSPC mobilization. This may translate into therapeutic strategies for atherosclerosis and hematologic malignancies.     

人脐带血来源造血干细胞体外扩增的研究进展

造血干细胞（HSCs）是血液系统中的一类成体干细胞群,具有自我更新和多谱系分化两个基本特征。造血干细胞移植（HSCT）可以治疗退行性疾病和多种血液系统疾病。脐带血来源造血干细胞（CB HSCs）是降低HLA配型要求的突破点,但单份脐带血中HSCs数量不能满足使用要求,为了获得足够数量的CB HSCs,体外扩增是一种可行的方法。近几年,学者们探索了多种体外扩增方法,包括优化细胞生长因子混合物、与基质细胞共培养及加入小分子化合物（SMCs）激动剂等。目前应用细胞因子联合小分子的扩增方法在多个临床试验中获得成功。本文对目前体外扩增CB HSCs的研究进展做一综述。     

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.     

Improved hematopoietic stem cell engraftment following ex vivo expansion of murine marrow cells with SCF and Flt3L

BACKGROUND: In vitro incubation of murine BM cells with IL-3, IL-6, IL-11 and SCF induces expansion of HPC but fails to preserve 'engraftability' in comparison with normal untreated marrow cells. We studied how culturing marrow cells for 48 and 72 h with a combination of the cytokines SCF and Flt3L influences cell expansion and engraftability. METHODS: Competitive repopulation of lethally irradiated C57BL/6 mice was used to examine engraftability of ex vivo cytokine-expanded Ptprc chimeric BM. A methylcellulose in vitro assay was used to determine the expansion of substitute progenitors. RESULTS: Both cytokine combinations successfully expanded progenitor populations when assayed in methylcellulose culture in vitro. After 72 h, the colony numbers of the expansion cultures increased 61% with IL-3, IL-6, IL-11 and SCF stimulation and 96% with SCF and Flt3L stimulation. Engraftment of competitively transplanted cells, cultured with IL-3, IL-6, IL-11 and SCF, consistently dropped to levels below 16%. However, 48 h culture with SCF and Flt3L resulted in 53.5+/-1.6% engraftment at 17 days and 64+/-3.7% engraftment at 19 weeks post-transplantation. Extending the cytokine exposure to 72 h resulted in 70+/-4.4% short-term engraftment at 17 days, and 64+/-2.4% engraftment at 19 weeks post-transplantation. DISCUSSION: The data demonstrate the ability of SCF and Flt3L cytokine-stimulated BM cells to maintain short- and long-term engraftability. We conclude that these cytokines play a crucial role in maintaining engraftment of hematopoietic progenitors.     

Interleukin-3 and ex vivo maintenance of hematopoietic stem cells: facts and controversies

Although the utilization of IL-3 in the ex vivo expansion of hematopoietic stem cells has been considered as an attractive possibility, its mode of action remains unclear and controversial. Some reports show that IL-3 maintains or even enhances primitive stem cell activity, whereas others show the opposite. The presence of serum in culture media enhances the pro-differentiating effect of IL-3 on stem cells. Conversely, addition of IL-3 to serum-free cultures improves the capacity of TPO, SCF and Flt3-ligand to promote the self-renewal of primitive stem cells. The presence or absence of serum or of some serum substitutes (in serum-free cultures), as well as other culture parameters are probably responsible for these contrasting effects of IL-3 on stem cells. However, none of the data presently evaluated bring a clear, definitive explanation to this apparent paradox. Those data that appear to be the most informative are presented and discussed in this "technical review".     

Functional compensation between hematopoietic stem cell clones in vivo

In most organ systems, regeneration is a coordinated effort that involves many stem cells, but little is known about whether and how individual stem cells compensate for the differentiation deficiencies of other stem cells. Functional compensation is critically important during disease progression and treatment. Here, we show how individual hematopoietic stem cell (HSC) clones heterogeneously compensate for the lymphopoietic deficiencies of other HSCs in a mouse. This compensation rescues the overall blood supply and influences blood cell types outside of the deficient lineages in distinct patterns. We find that highly differentiating HSC clones expand their cell numbers at specific differentiation stages to compensate for the deficiencies of other HSCs. Some of these clones continue to expand after transplantation into secondary recipients. In addition, lymphopoietic compensation involves gene expression changes in HSCs that are characterized by increased lymphoid priming, decreased myeloid priming, and HSC self‐renewal. Our data illustrate how HSC clones coordinate to maintain the overall blood supply. Exploiting the innate compensation capacity of stem cell networks may improve the prognosis and treatment of many diseases.     

Top-down motif engineering of a cytokine receptor for directing ex vivo expansion of hematopoietic stem cells

The technique to expand hematopoietic stem cells (HSCs) ex vivo is eagerly anticipated to secure an enough amount of HSCs for clinical applications. Previously we developed a scFv-thrombopoietin receptor (c-Mpl) chimera, named S-Mpl, which can transduce a proliferation signal in HSCs in response to a cognate antigen. However, a remaining concern of the S-Mpl chimera may be the magnitude of the cellular expansion level driven by this molecule, which was significantly less than that mediated by endogenous wild-type c-Mpl. In this study, we engineered a tyrosine motif located in the intracellular domain of S-Mpl based on a top-down approach in order to change the signaling properties of the chimera. The truncated mutant (trunc.) and an amino-acid substitution mutant (Q to L) of S-Mpl were constructed to investigate the ability of these mutants to expand HSCs. The result showed that the truncated and Q to L mutants gave higher and considerably lower number of the cells than unmodified S-Mpl, respectively. The proliferation level through the truncated mutant was even higher than that of non-transduced HSCs with the stimulation of a native cytokine, thrombopoietin. Moreover, we analyzed the signaling properties of the S-Mpl mutants in detail using a pro-B cell line Ba/F3. The data indicated that the STAT3 and STAT5 activation levels through the truncated mutant increased, whereas activation of the Q to L mutant was inhibited by a negative regulator of intracellular signaling, SHP-1. This is the first demonstration that a non-natural artificial mutant of a cytokine receptor is effective for ex vivo expansion of hematopoietic cells compared with a native cytokine receptor.     

A novel monoclonal antibody of human stem cell factor inhibits umbilical cord blood stem cell ex vivo expansion

Autologous stem cell transplantation (ASCT) is the gold standard therapy for suitable multiple myeloma (MM) patients after induction with high dose therapy. To date, the evidence of a reliable marker of prognosis in these cases remains scarce. Our aim was to evaluate appearance of unrelated atypical serum immunofixation patterns (ASIPs) as a marker of prognosis in MM patients submitted to ASCT. We retrospectively analysed data from 65 patients. Interestingly, we observed that presence of ASIPs was associated with longer progression-free survival and longer overall survival. Our results suggested that presence of ASIPs could be a novel marker of good prognosis in MM patients submitted to ASCT.     

The human hematopoietic stem cell in vitro and in vivo.

A quantitative assay for a primitive human hematopoietic cell has been developed. The cell identified has been assigned the operational designation of long-term culture (LTC)-initiating cell based on its ability when cultured on supportive fibroblast monolayers to give rise to daughter cell(s) detectable by standard in vitro colony assays. Three lines of evidence support the view that the LTC-initiating cell assay may allow the relatively specific enumeration of totipotent cells with in vivo reconstituting potential. These involve the demonstration: (1) that conditions in analogous murine long-term cultures stimulate the extensive amplification (self-renewal) of some totipotent long-term repopulating cells, (2) that most of the LTC-initiating cells in normal human bone marrow are phenotypically different from most of the colony-forming cells present in the same cell suspensions in their possession of a number of characteristics specifically associated with transplantable stem cells; and (3) that cultured marrow cells from patients with chronic myeloid leukemia which, after maintenance under LTC conditions for 10 days contain some normal LTC-initiating cells but no detectable leukemic LTC-initiating cells, can after autografting reconstitute the hematopoietic system with normal cells.     

Characterization of hematopoietic stem cell subsets from patients with multiple myeloma after mobilization with plerixafor

Background aimsPrevious studies have demonstrated that the combination of granulocyte–colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34⁺ hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets.MethodsWe characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation, and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor.ResultsThe number of aldehyde dehydrogenase (ALDH)^bright and CD34⁺ cells was significantly higher after plerixafor treatment (1.2–5.0 and 1.5–6.0 times; both P < 0.01) and an enrichment of the very primitive CD34⁺ CD38^? and ALDH^bright CD34⁺ CD38^? HSC subsets was detectable. Additionally, two distinct ALDH⁺ subsets could be clearly distinguished. The small ALDH^high subset showed a higher number of CD34⁺ CD38^? cells in contrast to the total ALDH^bright subpopulation and probably represented a very primitive subpopulation of HSC.ConclusionsA combined staining of ALDH, CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34⁺ cells but was also able to increase the proportion of more primitive stem cell subsets.     

Elevated granulocyte-colony stimulating factor and hematopoietic stem cell mobilization in Niemann-Pick type C1 disease

Niemann-Pick type C1 (NPC1) disease is a progressive lysosomal storage disorder caused by mutations of the NPC1 gene. While neurodegeneration is the most severe symptom, a large proportion of NPC1 patients also present with splenomegaly, which has been attributed to cholesterol and glycosphingolipid accumulation in late endosomes and lysosomes. However, recent data also reveal an increase in the inflammatory monocyte subset in the Npc1^nih mouse model expressing an Npc1 null allele. We evaluated the contribution of hematopoietic cells to splenomegaly in NPC1 disease under conditions of hypercholesterolemia. We transplanted Npc1^nih (Npc1 null mutation) or Npc1^wt bone marrow (BM) into Ldlr^?/? mice and fed these mice a cholesterol-rich Western-type diet. At 9 weeks after BM transplant, on a chow diet, the Npc1 null mutation increased plasma granulocyte-colony stimulating factor (G-CSF) by 2-fold and caused mild neutrophilia. At 18 weeks after BM transplant, including 9 weeks of Western-type diet feeding, the Npc1 mutation increased G-csf mRNA levels by ～5-fold in splenic monocytes/macrophages accompanied by a ～4-fold increase in splenic neutrophils compared with controls. We also observed ～5-fold increased long-term and short-term hematopoietic stem cells (HSCs) in the spleen, and a ～30–75% decrease of these populations in BM, reflecting HSC mobilization, presumably downstream of elevated G-CSF. In line with these data, four patients with NPC1 disease showed higher plasma G-CSF compared with age-matched and gender-matched healthy controls. In conclusion, we show elevated G-CSF levels and HSC mobilization in the setting of an Npc1 null mutation and propose that this contributes to splenomegaly in patients with NPC1 disease.