Environmental Cues Can Promote Symmetrical Division of Functional Human Hematopoietic Stem Cells.

Abstract Widespread clinical use of ex-vivo expanded human umbilical cord blood (CB) grafts has been limited by lack of proper understanding of factors regulating self-renewal type of symmetric cell divisions. The expansion of the number of functional hematopoietic stem cells (HSC) ex-vivo requires the creation of an environment which favors symmetrical division. In our current studies, addition of late acting cytokines, (GM-CSF, IL-6, Epo) with early acting cytokines (thrombopoietin, SCF, Flt-3 ligand) resulted in loss of expansion of stem/progenitor cells. These data indicate that modification of HSC fate is not fully independent of external humoral influences. We have previously demonstrated that following treatment of CD34+ cells with 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA) there is a 10- fold increase in the number of SCID mouse repopulating cells (SRC). This increase of SRC, however, occurred concomitantly with an increase in absolute number of CD34+CD90+ cells as well as primitive progenitors which gives rise to colony forming unit Mix lineage (CFU-Mix). We hypothesized that if the primary CD34+ cells generates CFU-Mix/CFU-GM in a ratio of ‘X’, then to observe a higher rate of symmetric cell division we would expect to see the ratio increased (>X) in the 5azaD/TSA treated cells in comparison to cells cultured in the absence of 5azaD/TSA (< X). Interestingly, analyses of our data suggest that when 5azaD/TSA treated CD34+ cells are cultured for 5 days and assayed for colonies we observed a significant increase in the ratio of CFU-Mix/CFU-GM in contrast to cells cultured in cytokines alone, 0.373 ± 0.06 and 0.066 ± 0.032 respectively. The ratio of CFU-Mix/CFU-GM of CB CD34+ cells (day 0) was 0.262 ± 0.045. These findings indicate that 5azaD/TSA treatment promotes the ratio of CFU-Mix/CFU-GM possibly by enhancing symmetric division of CFU-Mix while in the absence of 5azaD/TSA treatment the culture condition likely induces differentiation. In addition, we have also investigated the ratio of progenitor cells/differentiated cells by assessing the ratio of human CD34+ cells/CD33+ cells in the bone marrow of immunodeficient mice following transplantation (8 weeks) of equal numbers of CD34+ cells. The ratio of CD34+ cells/CD33+ cells following transplantation of 5azaD/TSA treated cells was 0.52 ± 0.14 (n = 11) while in the absence of 5azaD/TSA the ratio dropped to 0.31± 0.16 (n = 4). The ratio following transplantation of primary CD34+ (day 0) cells was 0.62 ± 0.14 (n = 6). These data suggest that 5azaD/TSA treated cells maintain the balance of generation of CD34+ cells/CD33+ cells at a comparable rate to that of primary CD34+ cells, while the CD34+ cells generated in the absence of 5azaD/TSA promotes generation of more differentiated cells. Alternatively, it is also possible that 5azaD/TSA treatment of CD34+ cells in the culture results in inhibition of myeloid differentiation at the cost of proliferation. However, the latter possibility is unlikely, since treatment of CB cells with 5azaD/TSA results in an increase in the absolute number of progenitors including SRC possessing both myeloid and lymphoid differentiation potential. Taken together, these data support our hypothesis that chromatin modifying agents in the culture is capable of promoting self-renewal type of symmetric cell division possessing in vivo multilineage marrow repopulating potential.

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Notch-Mediated Expansion of Human Hematopoietic Stem and Progenitor Cells By Culture Under Hypoxia

Blood ◽

10.1182/blood-2020-141121 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-29

Author(s):

Daisuke Araki ◽

Stefan Cordes ◽

Fayaz Seifuddin ◽

Luigi J. Alvarado ◽

Mehdi Pirooznia ◽

...

Keyword(s):

Er Stress ◽

Progenitor Cells ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Human Adult ◽

Hypoxic Conditions ◽

Cd34 Cells ◽

Human Hscs ◽

Ex Vivo Culture ◽

Cells Cultured

Notch activation in human CD34+ hematopoietic stem/progenitor cells (HSPCs) by treatment with Delta1 ligand has enabled clinically relevant ex vivo expansion of short-term HSPCs. However, sustained engraftment of the expanded cells was not observed after transplantation, suggesting ineffective expansion of hematopoietic stem cells with long-term repopulating activity (LTR-HSCs). Recent studies have highlighted how increased proliferative demand in culture can trigger endoplasmic reticulum (ER) stress and impair HSC function. Here, we investigated whether ex vivo culture of HSPCs under hypoxia might limit cellular ER stress and thus offer a simple approach to preserve functional HSCs under high proliferative conditions, such as those promoted in culture with Delta1. Human adult mobilized CD34+ cells were cultured for 21 days under normoxia (21% O2) or hypoxia (2% O2) in vessels coated with optimized concentrations of Delta1. We observed enhanced progenitor cell activity within the CD34+ cell population treated with Delta1 in hypoxia, but the benefits provided by low-oxygen cultures were most notable in the primitive HSC compartment. At optimal coating densities of Delta1, the frequency of LTR-HSCs measured by limiting dilution analysis 16 weeks after transplantation into NSG mice was 4.9- and 4.2-fold higher in hypoxic cultures (1 in 1,586 CD34+ cells) compared with uncultured cells (1 in 7,706) and the normoxia group (1 in 5,090), respectively. Conversely, we observed no difference in expression of the homing CXCR4 receptor between cells cultured under normoxic and hypoxic conditions, indicating that hypoxia increased the absolute numbers of LTR-HSCs but not their homing potential after transplantation. To corroborate these findings molecularly, we performed transcriptomic analyses and found significant upregulation of a distinct HSC gene expression signature in cells cultured with Delta1 in hypoxia (Fig. A). Collectively, these data show that hypoxia supports a superior ex vivo expansion of human HSCs with LTR activity compared with normoxia at optimized densities of Delta1. To clarify how hypoxia improved Notch-mediated expansion of LTR-HSCs, we performed scRNA-seq of CD34+ cells treated with Delta1 under normoxic or hypoxic conditions. We identified 6 distinct clusters (clusters 0 to 5) in dimension-reduction (UMAP) analysis, with a comparable distribution of cells per cluster between normoxic and hypoxic cultures. Most clusters could be computationally assigned to a defined hematopoietic subpopulation, including progenitor cells (clusters 0 to 4) and a single transcriptionally defined HSC population (cluster 5). To assess the relative impact of normoxia and hypoxia on the HSC compartment, we performed gene set enrichment analysis (GSEA) of cells within HSC cluster 5 from each culture condition. A total of 32 genes were differentially expressed, and pathways indicative of cellular ER stress (unfolded protein response [UPR], heat shock protein [HSP] and chaperone) were significantly downregulated in hypoxia-treated cells relative to normoxic cultures (Fig. B). When examining expression of cluster 5 top differentially expressed genes across all cell clusters, we observed a more prominent upregulation of these genes within transcriptionally defined HSCs exposed to normoxia relative to more mature progenitors (Fig. C, red plots). Hypoxia lessened the cellular stress response in both progenitors and HSCs, but the mitigation was more apparent in the HSC population (Fig. C, grey plots), and decreased apoptosis was observed only within the HSC-enriched cluster 5 (Fig. D). These findings are consistent with several reports indicating that HSCs are more vulnerable to strong ER stress than downstream progenitors due to their lower protein folding capacity. In conclusion, we provide evidence that ex vivo culture of human adult CD34+ cells under hypoxic conditions enables a superior Delta1-mediated expansion of hematopoietic cells with LTR activity compared with normoxic cultures. Our data suggest a two-pronged mechanism by which optimal ectopic activation of Notch signaling in human HSCs promotes their self-renewal, and culture under hypoxia mitigates ER stress triggered by the increased proliferative demand, resulting in enhanced survival of expanding HSCs. This clinically feasible approach may be useful to improve outcomes of cellular therapeutics. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v106.11.4208.4208 ◽

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.

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Ciclopirox Ethanolamine Is a Novel Modifier of Human Hematopoietic Stem Cell Ex Vivo Expansion

Blood ◽

10.1182/blood.v126.23.36.36 ◽

2015 ◽

Vol 126 (23) ◽

pp. 36-36

Author(s):

Mehrnaz Safaee Talkhoncheh ◽

Fredrik Ek ◽

Aurelie Baudet ◽

Christine Karlsson ◽

Roger Olsson ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Cultured Cells ◽

Ex Vivo ◽

Cell Activity ◽

Cell Number ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem Cell Activity ◽

Cells Cultured

Abstract Despite extensive studies over the last decades, little is known about the mechanisms governing human hematopoietic stem cell (HSC) fate decisions. In particular, it has been challenging to define culture conditions in which HSCs can be expanded for clinical benefit. Application of small molecule screening to modulate stem cells has emerged as a useful tool for identification of new compounds with ability to expand hematopoietic stem and progenitor cells (HSPCs). Such screens have mainly relied on the expression of CD34 as predictor of stem cell activity in cultured cells. However, CD34 defines a broad repertoire of progenitor cells and does not define stem cell function. We found that the long-term repopulation potential of cultured human HSPCs is exclusively contained within a discrete cell population co-expressing CD34 and CD90, while the vast majority of progenitor cells are found in the CD34+CD90- population. Tracking the CD34+ CD90+ population is therefore a sensitive and specific tool to predict stem cell activity in cultured hematopoietic cells and provides a good basis for a screen aimed at discovering modifiers of stem cell expansion. To search broadly for novel and potential modifiers of ex vivo HSCs expansion we next developed and optimized a small molecule screen in human cord blood (CB) derived CD34+ cells. We screened >500 small molecules from 8 different annotated chemical libraries for the phenotypic expansion of CD34+ CD90+ cells following a 6-day culture in serum-free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and fms-like tyrosine kinase 3 ligand (FL). The numbers of CD34+ CD90+ cells for each molecule, tested at two different concentrations, was compared to DMSO treated controls. Following the initial screen, several candidate hits were selected and subjected to a dose response validation experiment from which we selected four top candidate molecules. Two of these molecules were histone deacetylase (HDAC) inhibitors, which recently have been reported to facilitate expansion of CB derived HSCs. One of the top candidates, Ciclopirox ethanolamine (CE), had previously not been implicated in HSC expansion. Ciclopirox ethanolamine is known as an antifungal agent and iron chelator. It has further been shown to suppress cancer cell survival through inhibition of Wnt/beta catenin signaling. We found that CB cells cultured with CE had a 4-fold increase in CD34+90+ cell number compared to DMSO treated controls following 6 days of culture. Interestingly, the total cell count was not different, suggesting a specific increase in CD34+ CD90+ cell number rather than an overall higher proliferation rate. When plated in methylcellulose, CE cultured cells generated increased numbers of myeloid colonies. Moreover, CE treated cells gave rise to multilineage colonies (CFU-GEMM) that could not be detected from the control cultures. To further test the functional capacity of cells cultured with CE, we transplanted cultured equivalents of 30,000 CB CD34+ cells (cultured with or without CE) into sub lethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human hematopoietic reconstitution in peripheral blood was determined 16 weeks later. Mice transplanted with CE cultured cells showed higher human CD45 engraftment 16 weeks post transplant compared to control cells (33.2±6.7% vs 14.6±5% p=0.04). The engrafted cells contributed to both myeloid and lymphoid lineages. This shows that Ciclopirox ethanolamine enhances the long-term engraftment capacity of ex vivo cultured HSCs and suggests that it should be considered in stem cell expansion protocols, either alone or in combination with other molecules. We are currently addressing the basis for the increased stem cell activity mediated by Ciclopirox ethanolamine using parameters for differentiation, cell cycling and apoptosis. In addition, we are comparing Ciclopirox ethanolamine with other recently defined modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

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A Chemical Approach for Ex Vivo Expansion of Human Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.481.481 ◽

2011 ◽

Vol 118 (21) ◽

pp. 481-481

Author(s):

Taito Nishino ◽

Takumi Mikashima ◽

Makiko Yui ◽

Yasuyuki Asai ◽

Hiromitsu Nakauchi ◽

...

Keyword(s):

Stem Cells ◽

Dna Microarray ◽

Ex Vivo ◽

Fold Increase ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Aryl Hydrocarbon ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Cells Cultured

Abstract Abstract 481 Hematopoietic stem cells (HSCs) have been applied to treatment of a wide variety of malignant and non-malignant blood disorders. Because of low risk of graft versus-host disease, use of human cord blood (hCB) as a source of HSCs continues to increase. However, wide application of hCB is limited by the relatively low number of HSCs obtained from a single CB unit. Numerous attempts have been made to expand hCB HSCs in cultures to acquire a sufficient number of transplantable HSCs. Chemical approaches have especially showed promise for ex vivo expansion of HSCs. As an example, Boitano et al. reported that SR1, a chemically synthesized purine derivative, induced hCB HSC expansion by antagonizing the aryl hydrocarbon receptor. In this study, we initially screened several tens of thousands of small-molecule compounds for the capability to induce STAT5 activation but obtained active compounds showed no positive effect on proliferation of hCB CD34+ hematopoietic stem and progenitor cells (HSPCs). However, a derivative of the STAT5 activating compounds proved to increase the population of CD34+CD38– cells when added to hCB CD34+ cell culture. Thus, we optimized the chemical structure of the compound and identified MISK303 as the most potent compound. We next evaluated the effects of MISK303 on the ex vivo expansion of hCB CD34+ cells in detail; hCB CD34+ cells were cultured in serum-free medium supplemented with MISK303 in addition to rhTPO and rhSCF for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assays. Although the total and CD34+ cell number when cultured with MISK303 was comparable to that cultured with DMSO (vehicle), the cultures with MISK303 exhibited a >2-fold increase in the number of CD34+CD38– cells and contained 1.7-fold more high proliferative potential colony-forming cells (HPP-CFCs; >1mm in diameter) compared to those with DMSO. Correspondingly, in the NOD/SCID repopulation assay, hCB CD34+ cells cultured with MISK303 for 7 days displayed up to 2-fold higher levels of engraftment compared to control cultures and uncultured HSPCs. These data suggest that MISK303 promotes the net expansion of hematopoietic stem and progenitor cells. Furthermore, combination of SR1 and MISK303 had additive effects; cell cultures with SR1 and MISK303 showed a >4-fold increase in the number of CD34+CD38– cells. In accordance with the data, MISK303 had no antagonizing activity against the aryl hydrocarbon receptor. We also observed that a chemically-synthesized TPO receptor (c-mpl) agonist could be substituted with rhTPO and provided better expansion of CD34+CD38– cells in combination with SR1 and MISK303. To investigate the molecular mechanism by which MISK303 increases HSPCs, we first conducted gene expression profiling of CD34+ cells cultured with MK303 by DNA microarray analysis. Treatment of CD34+ cells with MISK303 for 24 hours led to up-regulation of 343-genes (>2-fold) and down-regulation of 112 genes (<0.5-fold). We also profiled effect of MISK303 over 311 protein kinases and found no inhibitory activity. We are now validating the expression of differentially expressed genes identified by DNA microarray analysis. In conclusion, we have conducted high-throughput screening and identified MK303, a novel chemically synthesized small-molecule compound, which induces the expansion of human HSCs ex vivo. The approach using MISK303 will facilitate the development of novel and efficient technologies for hematopoietic stem cell and gene therapies. Disclosures: No relevant conflicts of interest to declare.

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Hepatoma-Derived Growth Factor Is a Novel Factor to Promote the Proliferation of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v128.22.1471.1471 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1471-1471

Author(s):

Munetada Haruyama ◽

Kozo Yamaichi ◽

Akira Niwa ◽

Megumu K Saito ◽

Tatsutoshi Nakahata

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Absolute Number ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

The Mean

Abstract Ex vivo expansion of hematopoietic stem cells (HSCs) is an attractive therapeutic strategy for many hematologic diseases and genetic disorders. Therefore, a variety of ex vivo expansion techniques have been developed, however these systems were not well done to get long term HSCs (LT-HSCs) which have a long term hematopoietic reconstitution ability. As the reasons, it is considered that the factors associating with the proliferation and self-renewal of LT-HSCs have not been clear yet. To obtain the factors to stimulate the proliferation and self-renewal of LT-HSCs, various conditioned media were evaluated. The supernatants of COS-1 cells transfected with cDNA cording for RelA (one of nuclear factor kappa B subunits) stimulated the proliferation of human CD34+ cells derived from umbilical cord blood (UCB) and increased the number of CFU-Mix strongest of all evaluated conditioned media. 60 liters of the supernatants of COS-1 cells transfected RelA genes were separated by column chromatography purifications. LC-MS/MS analysis of the final active fraction provided the information of hepatoma-derived growth factor (HDGF) as a growth factor. HDGF is a 24kD heparin-binding protein and has reported to stimulate the proliferation in various types of cells including fibroblasts, endothelial cells and hepatoma cells, its receptor(s) and signaling remain unclear, moreover, has no known function in hematopoiesis. The recombinant human HDGF indicated the ability to enhance the proliferation of CD34+ cells dose-dependently and increased the number of CFU-Mix in combination with cytokines compared to cytokines alone, especially HDGF showed the strongest synergy effect in a combination with TPO in all combinations of cytokines. Next, uncultured (UC) CD34+ cells, the cells of an equal initial number of CD34+ cells after the serum-free condition cultures in the presence of TPO alone (T), HDGF alone (H) and HDGF+TPO (HT) were transplanted into sublethally irradiated NOG (NOD/Shi-scid,IL-2RγKO) mice. HT increased the number of CD34+CD38- cells compared to UC, T and H. Analysis of CD34+CD38- cells in bone marrow cells of NOG mice 24 weeks after transplantation revealed that the mean of absolute number of CD34+CD38- cells in HT group showed about 4-fold, that in H group showed about 3-fold compared to that in UC group, however, that in T group were not detected.These results indicated that HT increased HSCs including short term and long term HSCs. In order to investigate whether HDGF could increase the number of LT-HSCs, serial transplantation experiment was carried out. Uncultured CD34+ cells and the CD34+ cells cultured with HT were transplanted into sublethally irradiated NOG mice. At 24 weeks after transplantation, the mean of absolute number of CD34+CD38- cells in HT group showed 6-fold compared to that in UC group, a half of total number of bone marrow cells from each mouse in both groups were transplanted into one secondary sublethally irradiated NOG mouse. Analysis of human hematopoietic cells in both group 20 weeks after transplantation revealed that multi-lineage human hematopoietic cells, such as CD3+ cells, CD19+ cells, CD33+ cells, CD235a+ cells, erythrocytes and platelets, were detected in all mice in HT group, but were not detected in all mice in UC group. The mean of absolute number of CD34+CD38- cells in bone marrow of HT group showed 30-fold compared to that of UC group. These results indicated that HDGF could increase the number of LT-HSCs. We showed here that the CD34+ cells cultured with HDGF can be transplanted to secondary hosts to give rise to long-term multilineage repopulation. Thus, HDGF is a novel factor to promote the proliferation of HSCs and plays an important role in hematopoiesis. HDGF will contribute the new HSCs expansion system development by using UCB for hematopoietic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

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A Novel Plant Mannose-Binding Lectin, NTL, Preserves Cord Blood Hematopoietic Stem/Progenitor Cells in Long-Term Culture and Enhances Their Ex Vivo Expansion.

Blood ◽

10.1182/blood.v110.11.1197.1197 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1197-1197

Author(s):

Karen Kwai Har Li ◽

Kam Tong Leung ◽

Vincent Eng Choon Ooi ◽

Linda Shiou Mei Ooi ◽

Carmen Ka Yee Chuen ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Mannose Binding Lectin ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Mannose Binding ◽

Binding Lectin

Abstract Ex vivo expansion of hematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. In this study, we investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis on prolonged maintenance and expansion of cord blood CD34+ cells. Enriched CD34+ cells (1 x 105/mL, n=5) or mononuclear cells (1 x 106/mL, n=8) were cultured in X-VIVO-10 medium for 14, 21, 28 and 35 days without supplementary cytokine or medium changing. Our results showed that the presence of NTL (200 ng/mL) or FL-3 ligand (FL, 40 ng/mL) significantly preserved populations of early stem/progenitor cells (total CFU, BFU/CFU-E, CFU-GM, CFU-GEMM) in these cultures, compared with respective controls at various time points. In the ex vivo expansion study (n=16), the presence of stem cell factor (S, 50 ng/mL), thrombopoietin (T, 50 ng/mL), FL (F, 80 ng/mL) effectively expanded total nucleated cells (TNC) at day 8 (116 ± 20.2 fold) and day 12 (424 ± 68.8 fold), as well as all subsets of progenitor cells as demonstrated by flow cytometry and CFU assays. The presence of NTL (200 ng/mL) significantly increased TNC (148 ± 24.5 fold at day 8; 572 ± 91.9 fold at day 12; P < 0.01) and expansion of early progenitor cells (CD34+, CD34+CD38−, CFU-GEMM) and committed CFU of the myeloid (CFU-GM), erythroid (BFU/CFU-E) and the megakaryocytic lineage (CFU-MK) (P < 0.01 compared with respective TSF cultures). There was also slight but consistent increase of CD61+CD41+ cells in the presence of NTL (8.58 ± 2.14 x 105 vs. 7.30 ± 1.82 x 105 cells/mL, P < 0.001). Significantly, the increased expansion was not only contributed by the higher TNC, but also by the increase in the proportion of CD34+ cells, CD34+CD38− cells and the density of differential CFU. Six weeks after enriched CD34+ cells at day 0 or expanded cells at day 12 were infused into sub-lethally irradiated NOD/SCID mice, human CD45+ cells were detectable in the BM, spleen and PB of the mice. In the BM, there were engraftments of human hematopoietic cells of the early (CD34+), myeloid (CD33+, CD14+), B-lymphoid (CD19+) and megakaryocytic (CD61+) lineages. In animals that received day 12 expanded cells in the TSF + NTL group, there was a significant increase of human CD45+ cells in the BM (19.3% vs. 11.5%, P = 0.03, n = 15) when compared with those only exposed to TSF, and a trend of increased engraftment in their spleen (P = 0.07, n = 14). Comparison of the complete amino acid sequences of NTL and FRIL (a dicot mannose-binding lectin shown to preserve hematopoietic stem cells, PNAS, 96, 646–650, 1999) showed 10.2% identity and both peptides contain putative functional/structural sites such as those for N-myristoylation, casein kinase II phosphorylation, protein kinase C phosphorylation and N-glycosylation. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34+ cells for transplantation.

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Ex Vivo Expansion and Transplantation of Hematopoietic Stem/Progenitor Cells Supported by Mesenchymal Stem Cells from Human Umbilical Cord Blood

Cell Transplantation ◽

10.3727/000000007783465073 ◽

2007 ◽

Vol 16 (6) ◽

pp. 579-585 ◽

Cited By ~ 48

Author(s):

Guo-Ping Huang ◽

Zhi-Jun Pan ◽

Bing-Bing Jia ◽

Qiang Zheng ◽

Chun-Gang Xie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Umbilical Cord Blood ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Cd34 Cells

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTC-ICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colony-forming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45–55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.

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Fibronectin Enhances Ex Vivo Expansion and Maturation of Megakaryocyte Progenitors from Umbilical Cord Blood.

Blood ◽

10.1182/blood.v108.11.698.698 ◽

2006 ◽

Vol 108 (11) ◽

pp. 698-698 ◽

Cited By ~ 4

Author(s):

Varda Deutsch ◽

Einav Hubel ◽

Kay Sigi ◽

Ariel Many ◽

Elizabeth Naparstek ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Target Cells ◽

Short Term ◽

Hematopoietic Stem ◽

Platelet Production ◽

Cd34 Cells

Abstract Following cord blood (CB) transplant and bone marrow (BM) protracted thrombocytopenia remains a serious clinical problem. Platelet production following transplant depends on the availability of adequate numbers of cytokine responsive stem and megakaryocyte progenitor cells (MK-p). Thrombopoietin (TPO), had no clinical impact on thrombopoiesis when given to patients post BMT due to the paucity of MK-p in the grafts. If expanded, Mk-p would supply the appropriate target cells to maximize the effect of TPO and provide efficient earlier platelet engraftment. We propose a novel strategy to facilitate thrombopoiesis, by expanding MK-p from CB mononuclear cells (MNC) prior to transplantation in short term cultures. While CB CD34+ cells can be expanded by several reported methods, isolation of CD34+ cells from the fresh CB is not practical due to the limited number of stem and progenitor cells in the CB units. Additionally, MK expansion from purified stem cells requires long culture periods which are inappropriate for transplantation. We aimed to improved techniques for enrichment and ex-vivo expansion of MK-p and hematopoietic stem cells, from small aliquots of whole CB, using 7–10 days cultures and new growth conditions. CB progenitors were enriched by separation of MNC from RBC on gelatin followed by centrifugation on ficoll, as we previously reported (1). MNC were expanded on fibronectin (FN) coated dishes in the presence of autologous plasma with various new cytokine combinations. These included r-hu-TPO (10 ng/ml), b- FGF (10 ng/ml), r-hu-SCF (50 ng/ml) and ARP a peptide derived from the stress variant of acetylcholinesterase (AChE-R) recently discovered to have potent hematopoietic stem cell and MK growth factor activity (2). The cell populations, MK and MK-p were characterized by high resolution flow cytometry on day 0 and 10 of culture using SSC, CD41 and CD34. True MK expansion was assessed by appropriate gating out of granulocyte and monocytes, which acquire CD41+ adherent platelets in culture. FN alone, without any other growth supplement increased the viability of cells in culture and expansion of MK-p (CD41high, SSClow and FSClow) by 2.8±1.1 (P < 0.05) fold. The combination of FN with TPO enhanced MK-p number by 4.8±2.7 and the addition of either SCF or b-FGF or ARP further stimulated the expansion of MK-p all producing about a 6 fold increase (P < 0.05). Further analysis was performed on the maturing MKs which were characterized as CD41high, CD45low/negative, CD34negative. Increased Mk ploidy was found when either b-FGF or ARP were added to cultures containing TPO, grown on FN coated plates. Significant MK maturation, as measured by GPIIb/IIIa expression using real time quantitative PCR, was also found. The combination of FN and TPO increased the MK colony forming progenitors in culture by 9 fold and up to 35 fold when other supplements were added. We demonstrate that short term expansion of enriched MK-p from a small fraction of the CB unit is feasible and easy to perform and can comply with GTP regulations. This approach may lead to the development of more effective cell therapy modalities to facilitate platelet production and decrease the time of thrombocytopenia in severely myelosuppressed patients during the extended nadir before platelet engraftment.

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

Blood ◽

10.1182/blood.v112.11.2308.2308 ◽

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.

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Long-term maintenance of human hematopoietic stem/progenitor cells by expression of BMI1

Blood ◽

10.1182/blood-2007-08-106666 ◽

2008 ◽

Vol 111 (5) ◽

pp. 2621-2630 ◽

Cited By ~ 76

Author(s):

Aleksandra Rizo ◽

Bert Dontje ◽

Edo Vellenga ◽

Gerald de Haan ◽

Jan Jacob Schuringa

Keyword(s):

Progenitor Cells ◽

Ex Vivo ◽

Scid Mice ◽

Polycomb Group ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Colony Forming Capacity ◽

Term Maintenance

The polycomb group (PcG) gene BMI1 has been identified as one of the key epigenetic regulators of cell fates during different stages of development in multiple murine tissues. In a clinically relevant model, we demonstrate that enforced expression of BMI1 in cord blood CD34+ cells results in long-term maintenance and self-renewal of human hematopoietic stem and progenitor cells. Long-term culture-initiating cell frequencies were increased upon stable expression of BMI1 and these cells engrafted more efficiently in NOD-SCID mice. Week 5 cobblestone area-forming cells (CAFCs) were replated to give rise to secondary CAFCs. Serial transplantation studies in NOD-SCID mice revealed that secondary engraftment was only achieved with cells overexpressing BMI1. Importantly, BMI1-transduced cells proliferated in stroma-free cytokine-dependent cultures for more than 20 weeks, while a stable population of approximately 1% to 5% of CD34+ cells was preserved that retained colony-forming capacity. Whereas control cells lost most of their NOD-SCID engraftment potential after 10 days of ex vivo culturing in absence of stroma, NOD-SCID multilineage engraftment was retained by overexpression of BMI1. Thus, our data indicate that self-renewal of human hematopoietic stem cells is enhanced by BMI1, and we classify BMI1 as an intrinsic regulator of human stem/progenitor cell self-renewal.

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