scholarly journals Engraftment and Retroviral Marking of CD34+ and CD34+CD38− Human Hematopoietic Progenitors Assessed in Immune-Deficient Mice

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1243-1255 ◽  
Author(s):  
Mo A. Dao ◽  
Ami J. Shah ◽  
Gay M. Crooks ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Low Levels ◽  
Ex Vivo Culture ◽  
Daunting Challenge

Abstract Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38− cells. Comparable extents of human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38− cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38− cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38− cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3289-3296 ◽  
Author(s):  
Amnon Peled ◽  
Orit Kollet ◽  
Tanya Ponomaryov ◽  
Isabelle Petit ◽  
Suzanna Franitza ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adhesion Molecule ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
Lymphocyte Function ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Vascular Adhesion

Abstract Hematopoietic stem cell homing and engraftment require several adhesion interactions, which are not fully understood. Engraftment of nonobese/severe combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the major integrins very late activation antigen–4 (VLA-4); VLA-5; and to a lesser degree, lymphocyte function associated antigen–1 (LFA-1). Treatment of human CD34+cells with antibodies to either VLA-4 or VLA-5 prevented engraftment, and treatment with anti–LFA-1 antibodies significantly reduced the levels of engraftment. Activation of CD34+ cells, which bear the chemokine receptor CXCR4, with stromal derived factor 1 (SDF-1) led to firm adhesion and transendothelial migration, which was dependent on LFA-1/ICAM-1 (intracellular adhesion molecule–1) and VLA-4/VCAM-1 (vascular adhesion molecule–1). Furthermore, SDF-1–induced polarization and extravasation of CD34+/CXCR4+ cells through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5. Our results demonstrate that repopulating human stem cells functionally express LFA-1, VLA-4, and VLA-5. Furthermore, this study implies a novel approach to further advance clinical transplantation.

Small Molecule Screens for Stem Cell Expansion

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-42-SCI-42
Author(s):  
Michael P. Cooke ◽  
Anthony E. Boitano
Keyword(s):  
Cord Blood ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Ex Vivo Culture ◽  
Cd34 Expression

Abstract SCI-42 The identification of safe and effective methods to expand human hematopoietic stem cells (HSC) would have a major impact on the use of HSC in clinical medicine. Several features of human HSC, including the lack of a suitable cell line model and cumbersome methods for quantification, have made the identification of conditions for human HSC expansion challenging. Current culture methods using cytokine cocktails in serum-free media support the robust proliferation of CD34 positive (CD34+) cells but this is accompanied by rapid differentiation such that after 1 week of culture fewer than 20% of cells continue to express CD34. To overcome these limitations we developed a high throughput screen that uses primary human CD34+ cells and multiparameter flow cytometry to identify compounds capable of expanding human CD34 positive cells. By screening >100,000 LMW compounds we identified a molecule (SR1) that enhanced CD34 expression during ex vivo culture. Culture of CD34+ cells with cytokines and SR1 for 3 weeks leads to a >600-fold increase in the number of CD34+ cells, and a >2000-fold increase in the number of CFU compared to starting cell numbers. Importantly, cells expanded in the presence of SR1contain a 17-fold increase in the number of NOD-SCID repopulating cells compared to starting cell numbers. Mechanistic studies reveal that SR1 binds to and antagonizes the aryl hydrocarbon receptor (AHR). Knockdown of the AHR in CD34+ cells using lentiviral transduction also maintains CD34 expression. These findings suggest that AHR normally promotes HSC differentiation during ex vivo culture and that AHR antagonists can be used to promote CD34 cell expansion. To determine the clinical utility of these findings, we have begun to explore the use of SR1 to expand CD34+ cells isolated from umbilical cord blood for clinical transplantation. To this end, we have developed a GMP compatible process to manufacture CD34 positive cells expanded with SR1 for use in cord blood transplantation. In addition, we have also explored the use of SR1 to prevent HSC differentiation during HSC transduction and enable manufacturing of differentiated blood cells. These data reveal AHR antagonism and SR1 treatment as a promising method to promote HSC expansion for clinical use. Disclosures: Cooke: Novartis: Employment. Boitano:Novartis: Employment.

scholarly journals Sphingolipid Perturbation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 170-170
Author(s):  
Stephanie Zhi-Juan Xie ◽  
Laura Garcia Prat ◽  
Veronique Voisin ◽  
Alex Murison ◽  
Olga I. Gan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Chromatin Accessibility ◽  
Self Renewal ◽  
Cellular Activation ◽  
Hematopoietic Stem ◽  
Risk Of Malignancy ◽  
Ex Vivo Culture

Abstract The hematopoietic stem cells (HSC) field has long been perplexed by how the blood system d (~10e12 cells produced daily) - yet hematologic malignancies remain relatively rare. The risk of malignancy is mitigated in part by a complex hierarchy in which the quiescent long-term hematopoietic stem cells (LT-HSC) with high self-renewal capacity undergo a restricted number of cell divisions. Nonetheless, such a high production demand over a lifetime raises an inherent risk of malignancy due to DNA replication errors, misfolded proteins and metabolic stress that cause cellular damage in HSC. Previously, HSC dormancy, largely thought to be controlled by transcription factor networks, was held responsible for preventing mutation acquisition. However, recent studies suggest that LT-HSC contain critical cellular networks centered around the coordination of distinct HSC metabolic programs with proteostasis, which serve as crucial decision nodes to balance persistence or culling of HSC for lifelong blood production. While HSC culling mechanisms are known, the linkage between cellular stress programs and the self-renewal properties that underlie human HSC persistence remains unknown. Here, we ask how this HSC fate choice is influenced by lipid biosynthesis - an underexplored area of HSC metabolism. We observed a distinct sphingolipid transcriptional signature in human HSC and examined the consequences of sphingolipid perturbation in human cord blood (CB) stem cells during ex vivo activation. DEGS1 (Delta 4-Desaturase, Sphingolipid 1) is the final enzyme in de novo sphingolipid synthesis, converting dihydroceramide (dhCer) to ceramide (Cer); ablation of DEGS1 either genetically or by treatment with the synthetic retinoid fenretinide/N-(4-hydroxyphenyl) retinamide (4HPR) is sufficient to activate autophagy in mouse cells and human cell lines. DEGS1 gene expression was higher in HSC than in progenitors and was significantly increased in LT-HSC following 6 hours of cytokine stimulation, suggesting that it plays a role in cellular activation. Sphingolipid composition was altered in CB cultured with 4HPR for 8 days with an increase in dhCer levels and decrease in Cer levels shown by lipidomics. Remarkably, 4HPR treatment significantly increased in vitro colony forming efficiency from LT-HSC (50% over control), but not from short-term HSC or granulocyte-macrophage progenitors. Ex vivo 4HPR treatment of CB followed by serial xenotransplantation resulted in a 2.5-fold increase in long-term repopulation cell (LTRC) frequency over control-treated cells, suggesting that 4HPR treatment affects HSC self-renewal. RNA-seq analysis showed that 4HPR activates a set of proteostatic quality control (QC) programs that coalesce around the unfolded protein response (UPR) and autophagy, the latter confirmed by immunofluorescence and flow cytometry in CB stem cells. Ex vivo culture perturbs these programs and results in loss of chromatin accessibility at sites associated with uncultured LT-HSC as determined by ATAC-Seq. Addition of 4HPR to the culture activates these proteostatic programs to sustain immunophenotypic and functional HSC. These results suggest that ceramide, the central component to all sphingolipids, may act as a "lipid biostat" for measuring cellular stress and activating stress responses. We further asked if 4HPR could synergize with known compounds to enhance HSC self-renewal. Treatment of CB with a combination of 4HPR plus CD34+ agonists UM171 and StemRegenin-1 during ex vivo culture maintains a chromatin state more similar to uncultured LT-HSC as demonstrated by ATAC-seq, and led to a 4-fold increase in serial repopulating ability in xenotransplant assays over treatment with UM171 and SR1 alone. These results suggest that sphingolipid perturbation not only activates proteostatic mechanisms that protect HSC organelles from damage incurred during cellular activation, but also regulates the landscape of chromatin accessibility in cultured HSC when combined with CD34+ agonists. This work identifies a new linkage between sphingolipid metabolism, proteostatic QC systems and HSC self-renewal, and identifies novel strategies by which to expand HSC numbers and improve HSC quality for clinical applications. Disclosures Takayama: Megakaryon co. Ltd.: Research Funding.

Down-Modulating p18Ink4c for Ex Vivo Expansion of Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1687-1687
Author(s):  
Tao Cheng ◽  
Hui Yu ◽  
Donna Shields ◽  
Youzhong Yuan ◽  
Hongmei Shen
Keyword(s):  
Ex Vivo ◽  
The Self ◽  
Transduction Efficiency ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Our recent study demonstrated that the cyclin-dependent kinase inhibitor (CKI) p18Ink4c (p18), also an INK4 family protein acting at early G1-phase, exerts its inhibitory role during the self-renewing division of murine hematopoietic stem cells (HSC) in vivo (Nature Cell Biology 2004). Down-modulating p18 may permit enhanced stem cell expansion in vitro, a hypothesis that is now being testing in our laboratory. To provide the proof-of-the concept, we first took advantage of the murine system by testing the in vivo reconstituting ability of cells that had been cultured under the Dexter culture condition for 19 weeks. 2–20x105 cells with non-adherent and adherent populations were transplanted into lethally irradiated hosts. 3 of 7 mice revealed long-term engraftment in the p18−/− transplanted group (0.5–33% engraftment levels) while there was no engraftment in the p18+/+ group (n=7). Moreover, a substantial level (38.6% on average) of long-term engraftments (7 months) in multilineage was achieved in secondary recipients transplanted with the p18−/− cells (n=3), demonstrating the self-renewal potential of the expanded HSCs after the extended period of long-term culture. These data strongly indicate that p18 absence is able to substantially mitigate the differentiating effect of the ex vivo culture conditions on HSCs and therefore offer a strong rationale for targeting p18 in human HSC expansion. P18 mRNA was detected by RT PCR in human CD34+ cells with a higher expression level in the more primitive subset: CD34+CD38−. To explore the possibility of targeting p18 for expanding human HSCs, we have employed the RNA interference (RNAi) technology in CD34+ cord blood cells. We screened a pool of small interfering RNA (siRNA) oligos and three of them were able to effectively reduce p18 expression by 60–80% in 48 hours as assessed by both RNA and protein analyses in human cells. Further, we tested both transient and permanent delivery methods for introducing the RNAi effect in the CD34+CD38− cells. To demonstrate whether the RNAi method would be sufficient to impact the outcome of cell division after a single or limited cell cycle(s), we chose the nucleofector technology and were able to achieve 48.30±11.66% of transduction efficiency with good viability (50.63±9.38%, n=3) in human CD34+ cells. After a single electroporation pulse, we were able to increase by 2-fold the CD34+CD38− cells associated with the same magnitude of increased colony forming activity under culture condition supplemented with SCF, TPO and Flt3. To observe the long-term effect of p18 downregulation in human HSCs, we constructed a p18 short hairpin (shRNA)-expressing lentiviral vector that was engineered to have the mouse U6 promoter upstream of a CMV-EGFP expression cassette. A transduction efficiency of 30–60% was achieved after overnight infection of the human CD34+ cells with the p18 shRNA or with control lentiviral vectors pseudotyped with the VSV-g envelope. 72–96 hours after the transduction, human p18 protein can be knocked down by the p18 siRNA lentivector at near 100% in the HeLa cell line as determined on the western blot, and at more than 50% in human primary CD34+ cells as determined by real time RT PCR. We are currently undertaking further study aimed at assessing the repopulating ability of the transduced human HSCs with lentivirus-mediated p18 shRNA in NOD/SCID mice. Together, these findings suggest that down-modulating p18 might be a feasible approach for manipulating human HSCs ex vivo.

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 ◽  
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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