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.     

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.     

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.     

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.     

Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells

In an effort to obtain defined culture conditions for ex vivo expansion of hematopoietic stem and progenitor cells which avoid the supplementation of serum, we cultured human CD34(+) hematopoietic progenitor cells in a chemically defined, serum-free medium in the presence of hematopoietic growth factors (HGFs), stem cell factor (SCF), interleukin (IL)-1beta, IL-3, IL-6, and erythropoietin (EPO). A medium, SFM-1, was prepared according to a protocol previously optimized for semisolid progenitor cell assays containing Iscove's Modified Dulbecco's Medium (IMDM) plus cholesterol, bovine serum albumin, transferrin, nucleotides and nucleosides, insulin, and beta-mercaptoethanol. In static cultures seeded with CD34(+)-enriched progenitor cells isolated from human peripheral blood, a mean 76.6-fold expansion of total nucleated cells and a mean 4.6-fold expansion of colony-forming cells (CFC) was recorded after 14 days. Morphological analysis of the expanded cells revealed formation of myeloid, erythroid, and megakaryocytic cells. Flow cytometric analysis indicated that CD34(+) antigen expressing cells were maintained to a limited degree only, and cell populations expressing surface markers for myeloid (CD33, CD14, and CD15) and megakaryocytic (CD41a) lineages predominated. Within SFM-1, bovine serum albumin (BSA), cholesterin, and transferrin represented the most critical components needed for efficient total cell and CFC expansion. Addition of autologous patient plasma (APP) or fetal calf serum (FCS) to SFM-1 resulted in inferior cell amplification and CFC formation compared to controls in SFM-1, indicating that the components used in SFM-1 could replace exogenous serum. Four commercially available serum-free media resulted in either comparable or lower total cell and CFC yields as SFM-1. The transplantation potential of CD34(+) cells after culture in SFM-1 was assayed using limiting dilution analysis on preformed irradiated bone marrow stroma and revealed maintenance of long-term bone marrow culture initiating cell (LTCIC) levels during the culture period. These data indicate that HGF-supported multilineage ex vivo expansion of human CD34(+) hematopoietic progenitor cells is feasible using an IMDM-based culture medium which contains a restricted number of additives, resulting in analogous or improved yields of both primitive and differentiated cells compared to previously established protocols. We suggest that this culture protocol is of advantage when working with pharmaceutical-grade preparations under serum-free conditions.     

脐血造血细胞体外保存及扩增的研究

研究了脐血造血细胞在4℃冰箱保存过程中单核细胞活力、造血性能的变化以及细胞因子种类、组合、不同培养基添加成分对造血细胞扩增的影响。研究表明脐血在4℃冰箱保存应不超过3d;细胞因子组合SCF IL-6 FL TPO扩增CFU—C的效果最佳;培养基体系中添加脐血混合血浆对扩增CFU—C作用明显,脐血混合血浆的最佳浓度为25％。     

Critical appraisal of ex vivo expansion of human limbal epithelial stem cells

The stem cells (SCs) of the corneal epithelium located in the limbal basal layer are the ultimate source to maintain corneal epithelial homeostasis. Like other adult tissue-specific SCs, self renewal and fate decision of limbal SCs are regulated by a specialized in vivo microenvironment, termed "niche". Loss of limbal SCs or dysfunction of the limbal niche renders corneas with a unique clinical disease labeled limbal stem cell deficiency (LSCD). Besides transplantation of autologous or allogeneic limbal SCs or amniotic membrane, a new strategy of treating LSCD is to transplant a bio-engineered graft by expanding limbal SCs ex vivo. Herein, we conduct a critical appraisal of six protocols that have successfully been practiced in treating human patients with LSCD, and identify issues whether niche regulation has been disrupted or maintained during isolation and expansion. Consequently, we propose a future direction that may circumvent the potential pitfalls existing in these conventional protocols by preserving the interaction between limbal SCs and their native niche cells during isolation and expansion. Such an approach may one day help realize considerable promise held by adult SCs in treating a number of diseases.     

Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion

The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increasedCD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.     

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.     

An ex vivo model of hematopoietic stem cell mobilization

BACKGROUND: Mobilization of hematopoietic stem cells to the circulation facilitates their collection, thereby providing a non-marrow source of these cells for transplantation. Hematopoietic cytokine administration induces mobilization for most, but not all, donors. Because the underlying biology of mobilization is not well understood, improving the process on a rational basis is difficult. The design of an in vitro mobilization model was pursued to facilitate investigations of the process. METHODS: MS5 murine stromal cell line cells were grown to confluence on microporous transwell membranes. Murine femoral marrow plugs were placed on top of the prepared transwell membranes. The transwells were then seated in wells containing media and hematopoietic growth factors. Cells that were released from the marrow plugs over time and migrated through the stromal layer into the wells were assayed for stem cell/progenitor cell characteristics. RESULTS: Few or no GM-CSF (progenitors) were found in wells containing media alone or media plus mobilizing cytokines after 24 h. After 120 h, the numbers of cells in the cytokine-containing wells increased, as did the numbers of CD34(+) cells. Cells in the wells at the time progenitor cells were most frequent were shown to include side population (SP) hematopoietic stem cells. After 120 h in the presence of cytokines, cells pooled from the wells were transplanted to lethally irradiated mice. Eighty per cent of the transplanted mice survived 30 days or more, demonstrating that radioprotective stem cells were present in the wells. DISCUSSION: An ex vivo model has been designed that may aid investigations of the various steps of stem cell mobilization.     

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".     

Cytokine effect on ex vivo expansion of haemopoietic stem cells from different human sources

Human pluripotential stem cells (PSC) are currently the target for transplantation attempts and genetic manipulation. We have therefore investigated the frequency and the expansion potential of PSC’s in different types of blood samples. CD 34⁺ cells were thus obtained from human bone marrow (BM), as well as from peripheral blood (PB) and cord blood (CB) samples. After immuno-magnetic separation the highest yields of CD 34⁺ cells were from BM (1.08–2.25%) and CB (0.42–1.32%) while PB samples gave much lower values. Suspension cultures of PSC’s from the three sources were then set up, in the presence of combinations of haemopoietic growth factors. A remarkable amplification of the nucleated cell pool was observed reaching a maximum between 10 and 15 days of culture; earliest and maximum expansion (up to 220-fold) was achieved when Erythropoietin (Epo) was added to the culture medium, but this resulted in reduction of colony-forming cells and differentiation into erythroid progenitors. Clonogenic tests for BFU-E’s derived colonies showed a peak value at 5 days of liquid culture. Further studies are advisable to establish the best cytokine combination for a valuableex vivo expansion, coupled with preservation of stem cell properties.     

Large-scale production and directed induction of functional dendritic cells ex vivo from serum-free expanded human hematopoietic stem cells

BackgroundDendritic cells (DCs) that are derived from hematopoietic stem cells (HSCs) are the most potent antigen-presenting cells and play a pivotal role in initiating the immune response. Hence, large-scale production and direct induction of functional DCs ex vivo from HSCs are crucial to HSC research and clinical potential, such as vaccines for cancer and immune therapy.MethodsIn a previous study, we developed a serum-free HSC expansion system (SF-HSC medium) to expand large numbers of primitive HSCs ex vivo. Herein, a DC induction and expansion medium (DC medium) was proposed to further generate large numbers of functional DCs from serum-free expanded HSCs, which were developed and optimized by factorial design and the steepest ascent method.ResultsThe DC medium is composed of effective basal medium (Iscove's modified Dulbecco's medium [IMDM]) and cytokines (2.9 ng/mL stem cell factor [SCF], 2.1 ng/mL Flt-3 ligand, 3.6 ng/mL interleukin [IL]-1β, 19.3 ng/mL granulocyte-macrophage colony-stimulating factor [GM-CSF] and 20.0 ng/mL tumor necrosis factor-α [TNF-α]). After 10-day culture in DC medium, the maximum fold expansion for accumulated CD1a⁺CD11c⁺ DCs was more than 4000-fold, and the induced DCs were characterized and confirmed by analysis of growth kinetics, surface antigen expression, endocytosis ability, mixed lymphocyte reaction, specific cytokine secretion and lipopolysaccharide stimulation.DiscussionIn conclusion, the combination of DC medium and SF-HSC medium can efficiently induce and expand a large amount of functional DCs from a small scale of HSCs and might be a promising source of DCs for vaccine and immune therapy in the near future.     

Potential for clinical ex vivo expansion of cord blood haemopoietic stem cells using non-haemopoietic factor supplements

The establishment of culture systems that promote haemopoietic stem cell (HSC) self-renewal and expansion ex vivo will increase the clinical potential of umbilical cord blood (CB) HSC transplantation. Studies defining key signalling pathways that regulate development and expansion of HSC in vivo have greatly facilitated development of protocols for expanding HSC in ex vivo culture. Recently a number of soluble factors with novel stem cell expansion activity have been identified as part of pathways associated with mesodermal induction, or as factors produced by supportive stroma. These have been reported to support, to varying degrees, HSC self-renewal under in vitro conditions. Here we review the activities of these new factors and consider their future potential as components in ex vivo expansion culture for CB HSC. Finally we discuss the challenges associated with applying these factors to clinically relevant culture systems.     

Proliferative senescence in hematopoietic stem cells during ex-vivo expansion

The good outcome of hematopoietic stem cell (HSC) transplantation is hampered by low doses of CD34+ cell infusion. Transplanted HSCs undergo a replicative stress that causes accelerated senescence due to rapid telomere shortening. The expansion of human cord blood HSCs is instrumental in obtaining a large number of "good quality" cells, in terms of telomere length and telomerase activity compared to adult HSCs.