Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1329-1329
Author(s):  
Aleksandra Rizo ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
Jan Jacob Schuringa
Keyword(s):  
Cord Blood ◽  
Cell Expansion ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC). In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors. After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.

Study of the Mechanisms by Which Angptl5 and IGFBP2 Support Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2308-2308
Author(s):  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract We previously showed that angiopoietin-like protein 5 (Angptl5) and IGF Binding Protein 2 (IGFBP2) support dramatic ex vivo expansion of human hematopoietic stem cells (HSCs). To understand the mechanisms of their action, here we studied the effects of Angptl5 and IGFBP2 on the surface phenotype, signaling activation, self-renewal, apoptosis, differentiation, and homing of human cord blood CD34+ cells. Using immunofluorescence staining, we showed that Angptl5 and IGFBP2 activate certain signaling pathways such as MAPK and Stat5 in human cord blood CD34+ cells. IGFBP2 and Angptl5 increased the expression of transcription factors HoxB4, Bmi-1, EZH2, and survivin, measured by intracellular staining flow cytometry analysis and real-time RT-PCR. IGFBP2 and Angptl5 also inhibit expression of certain transcription factors important for differentiation of myeloid, erythroid, and lymphoid lineages. To test whether IGFBP2 and Angptl5 affect the homing of HSCs, we cultured human cord blood CD34+ cells in serum-free medium supplemented with SCF, TPO, Flt3-L, IGFBP2 or Angptl5, and transplanted them into sublethally irradiated NOD/SCID mice intraveneously or intrafemorally. Both IGFBP2 and Angptl5 support ex vivo expansion of SRCs in intrafemorally injected mice, suggesting the expansion-stimulating effects elicited by both factors are not caused by modulation of HSC homing. Interestingly, when we used intrafemoral injection, we found that Angptl5 treated HSCs have enhanced engraftment in non-injected bone marrow. This suggests Angptl5 treated HSCs further facilitate the mobilization of HSCs in vivo. We conclude that IGFBP2 and Angptl5 support self-renewal and inhibit differentiation of human cord blood HSCs. Our data also suggest that a combination of expression of transcription factors important for self-renewal, survival, and differentiation of HSCs can be used as a “stemness index” that predicts the activity of cultured human HSCs.

Opposite Effects of M1 and M2 Macrophages on Hematopoietic Stem Cell Self-Renewal and Ex Vivo Expansion

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2909-2909
Author(s):  
Yi Luo ◽  
Lijian Shao ◽  
Jianhui Chang ◽  
Wei Feng ◽  
Chengcheng Li ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Serum Free Medium ◽  
Free Medium ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Expansion Medium ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Macrophages (MΦ) are professional phagocytes in the innate immune system. They are not only involved in regulation of various immune functions and inflammation, but also exhibit plasticity in modulation of tissue regeneration and repair after being polarized into M1 and M2 MΦ by different inflammatory cytokines. In addition, several recent studies show that MΦ are a new constituent of the hematopoietic stem cells (HSCs) niche and play a role in regulation of HSCs maintenance and mobilization in bone marrow (BM). However, it is not known whether MΦ can regulate HSCs self-renewal and whether the effects of MΦ on HSCs can be influenced by differential MΦ polarization. This was investigated using an ex vivo HSCs expansion model consisting of mouse bone marrow LSK (Lin-sca-1+c-Kit+) cells cultured with or without MΦ in a mouse HSCs expansion medium (StemSpanTM serum-free medium supplemented with 20ng/ml of stem cell factor [SCF] and thrombopoietin [TPO]). We found that LSK cells were expanded about 20-, 15-, and 30-fold after 6 days of co-culture with MΦ harvested from mouse BM, spleen, and peritoneal cavity, respectively, whereas there was no significant expansion after culture without MΦ or with BM Gr-1high or Gr-1low monocytes. In addition, we found that M1-MΦ polarized by INFγ were more effective than IL4-polarized M2-MΦ in promoting LSK cells expansion ex vivo (45-fold vs. 15-fold). However, the promotion of LSK cells expansion by M1-MΦ resulted in about 88% reduction in HSCs as judged by 5-week cobblestone area forming cell (CAFC) assay. In contrast, M2-MΦ significantly promoted HSCs expansion. A greater expansion of HSCs was achieved after LSK cells were co-cultured with M2-MΦ for 9 days than for 6 days (20-fold vs. 6-fold). These findings suggest that M1-MΦ are more effective than M2-MΦ in promoting LSK cells or hematopoietic progenitor cells (HPCs) expansion, at the expense of HSCs self-renewal, whereas M2-MΦ can promote HPCs expansion as well as HSCs self-renewal. This suggestion is supported by results of serial transplantation and competitive repopulation unit (CRU) assays. CRU assay showed that LT-HSCs (e.g. 4-month CRU) were increased about 13 folds relative to the starting numbers of CRU in the input after LSK cells were co-cultured with M2-MΦ for 9 days, but were barely detectable after the cells were cultured without MΦ or with M1-MΦ. The inhibitory effect of M1-MΦ on HSCs self-renewal and expansion was attenuated by inhibition of inducible nitric oxide synthase (iNOS) activity with an inhibitor or knockout iNOS. Inhibition of arginase and/or cyclooxygenase activities with an inhibitor attenuated the promotion of HSCs self-renewal and expansion by M2-MΦ. More importantly, we found that human CD34+ cells, 8-week CAFC, and SCID mice repopulating cells (SRCs) were increased 42±14, 8±2.1, and 4 folds over the input values, respectively, after human cord blood CD34+ cells were co-cultured with M2-MΦ generated from human cord blood CD34- cells for 7 days in a human HSCs expansion medium (StemSpanTM serum-free medium supplemented with 50 ng/ml of SCF, TPO, and FLT-3 ligand). These findings demonstrate that M1-MΦ and M2-MΦ have opposite effects on HSCs self-renewal, which may be important for regulation of hematopoiesis under various pathological conditions in which MΦ are differentially polarized to M1 or M2 by diverse inflammatory cytokines. In addition, M2-MΦ may be used to promote human cord blood HSCs ex vivo expansion to make human cord blood transplantation available to more patients. Disclosures No relevant conflicts of interest to declare.

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Blood ◽  
2009 ◽  
Vol 114 (24) ◽  
pp. 5044-5051 ◽  
Author(s):  
Isabelle I. Salles ◽  
Tim Thijs ◽  
Christine Brunaud ◽  
Simon F. De Meyer ◽  
Johan Thys ◽  
...  
Keyword(s):  
Cord Blood ◽  
Flow Model ◽  
Ex Vivo ◽  
Human Platelets ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34+ cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 × 103/μL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibα (GPIbα) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

scholarly journals SALL4 Is a Key Factor in HDAC Inhibitor Mediated Ex Vivo Expansion of Human Peripheral Blood Mobilized Stem/Progenitor CD34+CD90+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1566-1566 ◽  
Author(s):  
Hiro Tatetsu ◽  
Fei Wang ◽  
Chong Gao ◽  
Shikiko Ueno ◽  
Xi Tian ◽  
...  
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Cd34 Cells

Abstract Hematopoietic stem cells (HSCs) possess the unique capacity to self-renew and give rise to all types of mature cells within the blood and immune systems. Despite our progress in understanding the molecular factors that support the self-renewal and differentiation of the hematopoietic system in vivo, less is known on how to modulate the factors that govern the self-renewal of hematopoietic stem/progenitor cells (HSPCs) ex vivo. Unlike in the case of embryonic stem (ES) cells, expansion of CD34+ HSPC in culture in general is at the expense of loss of “stemness”. HSPCs can be collected from cord blood (CB), mobilized peripheral blood (PBSC), and rarely bone marrow (BM) at the present practice. Due to the limited CD34+ cell number in one single cord blood unit, much of the current efforts on developing technology of ex vivo expansion of HSPC uses cord blood as a source and is clinically applied to cord blood HSPC transplants. However, there are growing needs for expanding PBSCs for transplant-related practices such as HSPC expansion from poor autologous mobilizations, gene therapy or genome-editing via TALENs or CRISPR/Cas9. Developing a technology that would allow HSPC ex vivo expansion from both CB and PBSC sources is a key step towards this goal. Several groups have reported that ex vivo culture of CB CD34+ cells with HDAC inhibitors (HDACi) can lead to expansion of a CD34+CD90+ population, which is responsible for enhanced marrow-repopulating potential. In this study, we ask whether HDACi can have a similar effect on PBSC CD34+ cells. Furthermore, we have explored the mechanism(s) mediated by HDACi in CD34+CD90+ cell expansion. First we assessed a panel of HDACi to identify the most potent molecule for CD34+CD90+ cell expansion and selected trichostatin A (TSA) for future study. Next, TSA was added to the cytokines (SCF, Flt3 ligand, IL-3 and IL-6) to further characterize its potential in PBSC CD34+CD90+ cell expansion. We observed TSA treated CD34+ cultures with cytokines yielded 4.8 times greater numbers of CD34+CD90+ cells as compared to the cultures containing cytokines with DMSO solvent control. We next examined SCID repopulating ability (SRA) to evaluate the cultured CD34+CD90+ cells in vivo. We observed that mice transplanted with 3 million CD34+ cells treated with TSA had higher degree of human cell chimerism than those treated with DMSO at 8 weeks bone marrow and peripheral blood (32% vs 18%; p < 0.05), which was further confirmed by secondary transplantation. Furthermore, these cells were capable of differentiating into cells belonging to multiple hematopoietic lineages. To investigate the molecular mechanisms responsible for the expansion of functional HSCs and progenitors that were observed following TSA treatment, we analyzed the expression levels of several HSPC related genes, which were compared between CD34+ cells treated with TSA and DMSO. Significantly higher transcript levels were detected for GATA 2 (p < 0.05), HOXB4 (p < 0.05), HOXA9 (p < 0.05), and SALL4 (p < 0.05) by real time quantitative RT-PCR in TSA expanded cells as compared with controls. To evaluate whether these transcription factors can contribute to the expansion of CD34+CD90+ cells, GATA2, HOXB4 or SALL4 shRNAs were transfected into PBSC CD34+ cells, followed by culture with TSA. Among these transcription factors, knocking down SALL4 expression led to the most significant reduction of CD34+CD90+ cell numbers (33% of reduction). In addition, overexpression of SALL4 in PBSC CD34+ cells led to an increase of CD34+CD90+ cell numbers when compared to controls (p < 0.05). Overall, our study demonstrated a novel HDACi mediated ex vivo PBSC culture technology that leads to the expansion of CD34+CD90+ cells and an increase of the marrow repopulating potential of these cells. Both gain-of-function and loss-of-function studies support that SALL4 is a key transcription factor responsible for the process. Future study on the use of HDACi or other methods to increase SALL4 expression/function will be highly beneficial to ex vivo HSPC (CB and PBSC) expansion technology. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cell cycling status of human cord blood CD34+ cells during ex vivo expansion is related to the level of very late antigen expression

2001 ◽  
Vol 16 (1) ◽  
pp. 20 ◽  
Author(s):  
Ju Young Seoh ◽  
Hae Young Park ◽  
Wha Soon Chung ◽  
Seung Cheol Kim ◽  
Myong Joon Hahn ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Antigen Expression ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Late Antigen ◽  
Cord Blood Cd34 ◽  
Cell Cycling

Use of Chromatin Modifying Agents for Ex Vivo Expansion of Human Umbilical Cord Blood Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4208-4208
Author(s):  
Hiroto Araki ◽  
Nadim Mahmud ◽  
Mohammed Milhem ◽  
Mingjiang Xu ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Functional Properties ◽  
Ex Vivo ◽  
Fold Increase ◽  
Scid Mice ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The fixed number of hematopoietic stem cells (HSCs) within a single cord blood (CB) unit has limited the use of CB grafts for allogeneic transplantation in adults. Efforts to promote self-renewal and expansion of HSCs have been met with limited success. Using presently available ex-vivo culture techniques HSCs lose their functional properties in proportion to the number of cellular divisions they have undergone. We hypothesized that chromatin modifying agents, 5-aza-2′-deoxycytidine (5azaD) and histone deacetylase inhibitor, trichostatin A (TSA) could reactivate pivotal genes required for retaining the functional properties of dividing HSC. We have demonstrated previously that the fate of human bone marrow CD34+ cells could be altered by the addition of 5azaD/TSA (Milhem et al. Blood.2004;103:4102). In our current studies we hypothesized that in vitro exposure of CB CD34+ cells to chromatin modifying agents might lead to optimal HSC expansion to permit transplantation of adults. A 12.5-fold expansion was observed in the 5azaD/TSA treated CD34+CD90+ cell cultures containing SCF, thrombopoietin and FLT3 ligand (cytokines) in comparison to the input cell number. Despite 9 days of culture, 35.4% ± 5.8% (n = 10) of the total cells in the cultures exposed to chromatin modifying agents were CD34+CD90+ as compared to 1.40 % ± 0.32% in the culture containing cytokines alone. The 12.5-fold expansion of CD34+CD90+ cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold expansion of cobblestone area-forming cells (CAFC). The frequency of SCID repopulating cells (SRC) was 1 in 26,537 in primary CB CD34+CD90+ cells but was increased to 1 in 2,745 CD34+CD90+ cells following 9 days of culture in the presence of 5azaD/TSA resulting in a 9.6-fold expansion of the absolute number of SRC. In contrast, the cultures lacking 5azaD/TSA had a net loss of both CFC/CAFC as well as SRC. The expansion of cells maintaining CD34+CD90+ phenotype was not due to the retention of a quiescent population of cells since all of the CD34+CD90+ cells in the culture had undergone cellular division as demonstrated by labeling with a cytoplasmic dye. CD34+CD90+ cells that had undergone 5–10 cellular divisions in the presence of 5azaD/TSA but not in the absence still retained the ability to repopulate NOD/SCID mice. 5azaD/TSA treated CD34+CD90+ cells, but not CD34+CD90- cells were responsible for in vivo hematopoietic repopulation of NOD/SCID assay, suggesting a strong association between CD34+CD90+ phenotype and their ability to repopulate NOD/SCID mice. We next assessed the effect of 5azaD/TSA treatment on the expression of HOXB4, a transcription factor which has been implicated in HSC self-renewal. A significantly higher level of HOXB4 protein was detected by western blot analysis after 9 days of culture in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. The almost 10-fold increase in SRC achieved using the chromatin modifying agents should be sufficient to increase the numbers of engraftable HSC within a single human CB unit so as to permit these expanded grafts to be routinely used for transplanting adult recipients.

The Polycomb Gene BMI1 Collaborates with BCR-ABL in Leukemic Transformation of Human Cord Blood CD34+ Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1350-1350
Author(s):  
Aleksandra Rizo ◽  
Sandra Olthof ◽  
OS van Ronald ◽  
Bert HJ Dontje ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
B Cells ◽  
Cord Blood ◽  
Bone Marrow Microenvironment ◽  
Leukemic Transformation ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Cd34 Cells

Abstract Previously, we demonstrated that BMI1 acts as a stem cell maintenance factor for human stem/progenitor cells. Here, we report that BMI1 collaborates with BCR-ABL in inducing leukemogenic transformation of human cord blood (CB) CD34+ cells. BMI1 and BCR-ABL were co-expressed into CB CD34+ cells (further referred as B/B cells) using a retroviral approach and cells were transplanted into NOD-SCID mice. In two out of five mice we observed leukemia within 4 months after transplantation. Chimerism levels reached 80–90% in the bone marrow and peripheral blood and morphological analysis revealed the appearance of primitive blast-like human hematopoietic cells with features that recapitulate human lymphoid leukemia. The mice were lethargic, with splenomegaly and infiltration of leukemic cells in the spleen, liver and the bone marrow and immunophenotypical analyses revealed that the cells expressed CD34 and CD19. To further understand the mechanisms underlying the leukemic transformation we performed ex-vivo long-term cultures on bone marrow stroma. We observed that the double transduced B/B cells had a strong proliferative advantage and elevated self-renewal potential as compared to controls. Expanding cultures could be maintained for over 20 weeks and Cobblestone Area Forming Cells (CAFCs) could be harvested and replated to initiate new expanding cocultures. Stem cell frequencies were determined in Long-Term Culture-Initiating Cell (LTC-IC) assays and frequencies were enhanced over 100-fold as compared to controls. Depending on the MS5 co-culture conditions, both myeloid as well as lymphoid long-term cultures could be established, indicating that extrinsic factors might dictate the lineage fate of transformed cells. To determine the necessity of a bone marrow microenvironment, we performed stroma-free liquid cultures and observed that the B/B cells were capable of expanding over 23 weeks, BMI1 cells were able to grow for 16 weeks and, importantly, BCR-ABL cells were not able to propagate long-term in stromain-dependent cultures. Thus, these data suggest that BCR-ABL cells are still dependent on cues from the bone marrow microenvironment for long-term self-renewal, and that co-expression of the intrinsic stem cell regulator BMI1 might alleviate this necessity of BCR-ABL+ cells for a microenvironment. Experiments in which B/B-transduced cells were sorted into HSC, CMP, GMP and MEP populations indicated that long-term self-renewal and expansion could particularly be imposed on the HSC population, and much less efficiently on progenitor subpopulations. In order to study whether the B/B-leukemic stem cells could be targeted by Imatinib, we applied a short pulse of Imatinib to expanding MS5 cocultures for 7 days. While the vast majority of cells in all cultures did not survive, in the B/B-transduced group a population of immature cells remained that was capable of re-initiating proliferative cultures of self-renewing CAFCs with very high frequencies (1/96 as determined by LTC-IC assays). Finally, we asked whether retroviral introduction of BMI1 in BCR-ABL+ CD34+ cells isolated from CML patients in chronic phase that expressed low endogenous BMI1 levels would affect long-term growth and self-renewal. Upon overexpression of BMI1 we observed increased proliferation capacity of the BMI1 transduced CML cells, and cultures could be maintained for much longer periods than control-transduced cultures. In conclusion, our data indicate that BMI1 collaborates with BCR-ABL in leukemic transformation, and our human-based system should provide a useful model to study the pathology of leukemias and test new drug entities.

UM171 Is a Novel and Potent Agonist Of Human Hematopoietic Stem Cell Renewal

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 798-798 ◽  
Author(s):  
Iman Fares ◽  
Jalila Chagraoui ◽  
Yves Gareau ◽  
Stephane Gingras ◽  
Ruel Rjean ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Stem Cell Population ◽  
Fed Batch ◽  
Self Renewal ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
The Impact

Abstract Various combinations of soluble factors have been reported to promote the ex vivo proliferation of human HSCs but few of them are capable of promoting self-renewal. Characterization of StemRegenin (SR1), an aryl hydrocarbon receptor (AhR) antagonist that promotes expansion of CD34+ cells ex vivo, provided a proof of principle that low molecular weight (LMW) compounds have the ability to modulate cellular pathways possibly leading to HSC expansion. In an attempt to identify novel putative agonists of HSC self-renewal, we tested a library comprising > 5,000 LMW molecules in a phenotypical screen based on in vitro expansion of CD34+CD45RA- cells. Eight hits were identified, the majority of which were AhR antagonists. Validation studies identified one hit synthesized in our institute which potently expanded human CD34+CD45RA- cord blood cells ex vivo. Importantly this molecule does not antagonize AhR. Structure activity relationship (SAR) generated a compound called UM171 which is 14-25 folds more potent than the starting molecule with an EC50 of 8-15 nM on CD34+ human cord blood (CB). Using the optimal dose of this analog, CD34+, CD34+CD45RA-, and CD34+CD38-CD90+CD45RA-CD49f+ were expanded 115±15, 62±3 and 121±6 fold respectively over the input values after 12 day culture in fed-batch system. The expansion of these phenotypic HSC populations was enhanced, in an additive manner by including SR1. Washout of UM171 led to a rapid loss of the CD34+CD45RA- population suggesting that its effect is reversible. UM171 is not a mitogen and cannot compensate for the absence of cytokines added to the media (Flt3L, SCF, and TPO). Our cell division studies revealed no effect of the compound on division rate compared to control indicating that UM171 cultured cells retain the CD34+CD45RA- phenotype. Apoptosis and cell phonotype analysis showed significantly less cell differentiation and cell death for UM171 cultured cells compared to controls or SR1 supplemented cultures suggesting that the compound acts by anti-differentiation/apoptosis. Similar to SR1, UM171 treated cells showed a 75±20 fold net increase in the numbers of multilineage colony forming cell (CFU-GEMM) and 150±20 fold when both compounds were present in the 12 day culture.To further understand the impact of UM171 on more primitive cell compartments, we transplanted 100, 500, 1000 and 10,000 uncultured CB CD34+ cells and their progeny after a 12-day culture (with or without UM171) in NSG mice and verified the human hematopoietic reconstitution in their bone marrow 20 weeks later. Mice transplanted with UM171 expanded cells showed a much higher level of human CD45 engraftment compared to uncultured or control expanded cells. The NSG Repopulating Cells (NRC) showed a net expansion ex vivo only in the presence of UM171 with values ranging from 3.1 to 5.5 fold increase over input value in 6 independent experiments. Interestingly, the co-treatment with SR1 did not significantly enhance the impact of UM171 on NRC expansion. In these fed-batch conditions SR1 treatment maintained the NRC population at input values. Importantly, we observed a linear relationship between the number of UM171-treated cells transplanted in vivo and level of human CD45 engraftment. Moreover, at limit dilution, these cells were capable of human myeloid and lymphoid differentiation in NSG BM environment suggesting that UM171 expanded cells remain multipotent. Together, our studies have identified a novel compound which truly expands human progenitors while amplifying the more primitive stem cell population. UM171 has successfully passed toxicity studies and is in the final phase of GMP production for a phase I study which will be launched shortly. In addition, preliminary evidence suggests that UM171 strikingly enhances gene transfer efficiency to primitive human CD34 CB cells. Disclosures: No relevant conflicts of interest to declare.

Conditioned Medium Represents a Useful Solution to Increase the Expansion of Multipotent Progenitors with Strong Platelet Engraftment Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4276-4276
Author(s):  
Ahmad Abu-Khader ◽  
Gwendoline Bugnot ◽  
Manal Alsheikh ◽  
Roya Pasha ◽  
Nicolas Pineault
Keyword(s):  
Cell Expansion ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Background Level ◽  
Erythroid Progenitors ◽  
Cellular Mechanisms ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Cd34 Cells

Abstract Delayed neutrophil and platelet engraftment is a significant issue of cord blood (CB) transplantation. Ex vivo expansion of CB hematopoietic stem and progenitor cells (HSPC) before infusion has been shown to accelerate hematopoietic recovery in patients. Recently, we reported that serum free medium (SFM) conditioned with osteoblasts derived from human bone marrow (BM) mesenchymal stromal cells, referred as M-OST CM, was superior to SFM or MSC CM for the expansion of CB CD34+ cells, and that HSPC expanded in M-OST CM provided better platelet engraftment. Since large number of expanded cells were transplanted in the original study, it was not possible to estimate the increased expansion of HSPC with short-term (ST) and long-term (LT) thrombopoietic and BM engraftment activities. The objectives herein were to investigate these shortcomings using limit dilution analysis (LDA) in transplantation assay and to investigate the cellular mechanisms at play. M-OST CM was prepared by conditioning SFM with immature M-OST overnight. CB CD34+ cells were expanded in M-OST CM or in SFM (defined as control) for 7 days with SCF, FL and TPO. CB cell expansion was significantly greater in M-OST CM cultures vs. SFM control (2.4 ±0.9 fold, mean ± SD, n=4, p=0.01). LDA transplantation assays were done by infusing the progeny of 500-8000 CD34+ cells in NSG mice. First, we compared the ST (< 31 days) and LT (˃ 100 days) thrombopoietic activities of expanded HSPC by measuring circulating human platelets (hPLT). The threshold for hPLT engraftment was set above the mean background level measured in control mice + 1SD. The median ST levels of hPLT in M-OST mice tended to be greater (2.5-fold, p˃0.05) in M-OST recipients (21 mice/condition, n=2). The frequency of ST hPLT HSPC estimated by LDA was 3.4 ±0.2 fold higher in M-OST CM cultures though the difference vs. control was not significant (p=0.11). LT hPLT levels were significantly greater in M-OST recipients (median 33 vs. 8 hPLT/uL blood, p=0.0027). Consistent with this, the frequency of HSPC with LT hPLT engraftment was increased in M-OST CM cultures (3.5±0.8 fold, p<0.04). Considering the differences in cell expansion, the net expansion of HSPC with ST and LT hPLT engraftment were raised by 5.5 ±1.7 and 6.0 ±3.4 fold in M-OST CM cultures vs. control (n=2). Next, LT human BM engraftment was analyzed at week 16. Preliminary results (13 mice/condition) suggest that the frequency of LT Scid repopulating cells (SRC) was increased by 27% in the M-OST CM culture vs. SFM control (frequency of 1/2878 vs. 1/3626 of day 0 starting cell). Next, we set to determine how M-OST CM increases the thrombopoietic activity of expanded CB HSPC. First, cytometry analysis (CD34, CD38, CD45RA, CD90 and CD123) revealed that M-OST CM preferentially increased the expansion of common myeloid progenitors (CMP, 8-fold, p=0.2, n=3), megakaryocyte-erythroid progenitors (MEP, 7-fold, p=0.02) and granulocyte-macrophage progenitors (GMP, 9-fold, p=0.02) vs. SFM control. Expansion of HSC-enriched cells was unchanged while that of multipotent progenitors (MPP) was reduced 2-fold (p<0.05). We set to confirm these results by culturing purified primary CB HSPC subsets in M-OST CM or SFM; M-OST CM induced greater expansions of MEP (3-fold), GMP (˃10-fold) while expansion in MPP cultures was greater with SFM control (1.5-fold). No growth was noted with the HSC and CMP cultures likely due to low sort yields. To complement these findings, we measured the expansion of myeloid CFU progenitors and long term culture-initiating cells (LTC-IC) by LDA. The total number of CFU was increased 2.4-fold (<0.02, n=4) by M-OST CM due mostly to increased expansion of CFU-G/GM colonies (2-fold, p<0.05) and BFU-E (2-fold, p=0.05). M-OST CM also sustained a 3.4-fold increase in LTC-IC expansion vs. SFM culture, though this finding remains to be confirmed in ongoing experiments. Finally, we investigated the effect of M-OST CM on the chemotaxis of HSPC toward SDF-1 since we previously reported increased expression of its receptor CXCR4 on CB cells in M-OST CM cultures. M-OST CM HSPC showed a modest 15% increase in migration vs. SFM control (n=4, p=0.10). In conclusion, our results demonstrate that the ST and LT hPLT engraftment activities of ex vivo expanded CB HSPC can be increased 5-6 folds by the use of M-OST CM due to the expansion of immature CB HSPC subsets including perhaps LT SRC. Whether M-OST CM can also modulate the homing activity of HSPC remains unclear. Disclosures No relevant conflicts of interest to declare.

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