Scaleable Production of Hematopoietic Cells Suitable for Transfusion from Human Embryonic Stem Cells in a Stirred Bioreactor System.

Abstract Blood cell products, such as red blood cells and platelets suitable for transfusion, are attractive as a first generation human embryonic stem cell (hESC) derived therapy. Development of hESC-derived transfusion therapies will require a better understanding of how to control the differentiation of human ES cells into functional blood cells in sufficient quantities for clinical use. HESCs are known to produce mature hematopoietic cells during differentiation as embryoid bodies (EBs). Previously we have demonstrated development of both hematopoietic progenitor cells and more differentiated cell types of myeloid, lymphoid and erythroid lineages from hESC. A four-fold increase in total cell number was achieved when ESC-derived EBs were differentiated in stirred vessels compared to conventional static cultures. Spinner cultures generated EBs more uniform in size and density. Static- and spinner cultivated EBs produced equivalent percentages of hematopoietic progenitors when assayed by surface antigen expression (CD34+, CD31+ and CD45+) and colony forming potential. Hence, overall a greater yield of hematopoietic cells was generated in spinner cultures. Here we incorporate pH and oxygen control into the stirred vessel system in order to closely regulate environmental conditions at levels conducive to hematopoietic differentiation. Hematopoietic potential is compared under hypoxic and normoxic conditions. Hypoxic conditions were confirmed in the EB tissue mass by 2-nitroimidazole (hypoxyprobe) staining. We observed that the cellular response to hypoxia, monitored by the presence of HIF 1α protein, is transient. Peak levels of HIF 1α were detected within 48 hours of low oxygen culture, falling to baseline levels within 7 days. Under more severe conditions the kinetics of the hypoxic response were accelerated, HIF 1α expression peaking and subsiding earlier in cultures held at 1% dissolved oxygen compared to 5% dissolved oxygen. We show that by manipulating dissolved oxygen concentration we are able to influence the progress of differentiation. This can at least partly be attributed to the upregulation of hypoxia inducible genes, including VEGF-A and EPO, under low oxygen conditions. Expression levels of VEGF-A are dependent on dissolved oxygen concentration, being most highly expressed under 1% dissolved oxygen conditions. The transitory nature of the cellular hypoxic response suggests that short exposure to low oxygen conditions may be sufficient to gain the full beneficial impact of hypoxic signalling on hematopoietic cell generation without decreases in cell proliferation and increase in cell death associated with extended oxygen deprivation. We propose that control of culture parameters such as dissolved oxygen in conjunction with cytokines can specify the cellular microenvironment within EB to yield robust levels of hematopoietic progenitors. This work demonstrates proof-of-principle for hematopoietic cell production from human embryonic stem cells in a scaleable bioreactor system.

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A Novel Method for Efficient Production of Multipotential Hematopoietic Progenitors from Human Embryonic Stem Cells by Co-Culture with Murine Fetal Liver-Derived Stromal Cells.

Blood ◽

10.1182/blood.v106.11.4214.4214 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4214-4214

Author(s):

Feng Ma ◽

Dan Wang ◽

Sachiyo Hanada ◽

Hirohide Kawasaki ◽

Yuji Zaike ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Stromal Cells ◽

Blood Cells ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Efficient Production ◽

Hematopoietic Progenitors

Abstract Human embryonic stem cells provide a unique tool to study early events occurring in the development of human embryonic hematopoiesis, and their totipotent capability indicates a potent clinical application based on the cellular therapy and the evaluation of drug effects on hematopoietic and blood cells. To achieve efficient production of hematopoietic cells from human embryonic stem cells, we attempted to reproduce the circumstance surrounding embryonic hematopoietic cells in vitro. Since fetal liver is the predominant source of hematopoietic and blood cells in mammalian embryogenesis, we established stromal cells from mouse fetal liver at days 14 to 15 of gestation. In the co-culture of human embryonic stem cells with the established stromal cells, a number of hematopoietic progenitors were generated at around day 14 of co-culture, and this hematopoietic activity was highly enriched in the cobble stone-like cells under the stromal layer. Most of the cobble stone-like cells collected expressed CD34 and contained a variety of hematopoietic colony-forming cells, especially multilineage colony-forming cells, at a high frequency. The multipotential hematopoietic progenitors in the cobble stone-like cells produced all types of mature blood cells, including adult type hemoglobin-synthesizing erythrocytes and tryptase and chymase-bouble positive mast cells in the suspension cultiue with a cytokine cocktail. The developed co-culture system of human embryonic stem cells should offer a novel source for hematopoietic and blood cells applicable to cellular therapies and drug screening.

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Modeling CML Development and Drug Resistance Using iPSC Technology,

Blood ◽

10.1182/blood.v118.21.3767.3767 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3767-3767

Author(s):

Kran Suknuntha ◽

Yuki Ishii ◽

Kejin Hu ◽

Jean YJ Wang ◽

Igor Slukvin

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Drug Resistance ◽

Stem Cell ◽

Blood Cells ◽

Kinase Activity ◽

Hematopoietic Cells ◽

Patient Specific ◽

Hematopoietic Progenitors ◽

Abl Kinase

Abstract Abstract 3767 Reprogramming of neoplastic cells to pluripotency provides a unique tool to personalize the exploration of tumor pathogenic mechanisms and drug resistance using iPSCs with patient-specific chromosomal abnormalities. We have developed a technology to generate transgene-free iPSCs from bone marrow and cord blood cells employing episomal vectors. Using this approach we created transgene-free iPSCs from a patient with CML in the chronic phase. CMLiPSCs showed a unique complex chromosomal translocation identified in the patinet's marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. Importantly, these CMLiPSCs are devoid of genomic integration and expression of reprogramming factors, which are incompatible for modeling tumor development and drug response (Hu et al. Blood 117:e109). We have also shown that these CMLiPSCs contain the BCR-ABL oncogene without any detectable mutations in its kinase domain. By coculture with OP9, we generated APLNR+ mesodermal cells, MSCs, and lin-CD34+CD45+ hematopoietic progenitors from CMLiPSCs, and control BMiPSCs from a normal subject and analyzed the levels of BCR-ABL protein and tyrosine-phosphorylated (pTyr) cellular proteins in the different cell populations. The highest level of BCR-ABL protein expression was found in the in undifferentiated iPSCs, however, the overall cellular pTyr levels was lower than the control BMiPSCs, suggesting that BCR-ABL kinase activity was suppressed in the CMLiPScs. Consistent with these findings, imatinib does not inhibit the growth and survival of these CMLiPSCs. The levels of BCR-ABL protein decreased upon differentiation with a major reduction observed when cells became mesoderm. Following differentiation of CMLiPSC-derived mesoderm into the MSCs and lin-CD34+CD45+ hematopoietic progenitors, the levels of BCR-ABL protein did not change significantly, indicating that the major epigenetic regulation of BCR-ABL expression occurs during the transition to mesoderm. In spite of the decrease in BCR-ABL expression, the total pTyr levels significantly increased following transition of CMLiPSCs to mesoderm and blood cells, suggesting recovery of BCR-ABL kinase activity during differentiation. Interestingly, we found that imatinib had no effect on CFC potential of the most primitive lin-CD34+CD45+ hematopoietic progenitors derived from CMLiPSCs, while significant inhibition in hematopoietic CFC potential was observed when we used the patient's bone marrow cells. Following expansion of lin-CD34+CD45+ progenitors in serum-free medium with cytokines, we found that more differentiated hematopoietic cells became imatinib sensitive. The differential response of progenitors versus more differentiated cells to imatinib recapitulate the clinical observation that CML stem cells display innate resistance to imatinib but their differentiated progenies become sensitive to this BCR-ABL kinase inhibitor. The iPSC-based models provide several advantages for the study of CML pathogenesis. iPSCs can provide an unlimited supply of hematopoietic cells carrying patient-specific genetic abnormalities. Using well-defined temporal windows and surface markers, distinct cell subsets with tumor-initiating/tumor-propagating potential after transplantation in immunodeficient mice could be identified and used for drug screening. iPSC models make it possible to address CML stem-cell potential at various stages of differentiation for which it may be difficult to obtain samples from the patient, for example, at the hemangioblast stage. They also provide a unique opportunity to explore the interplays between epigenetics and oncogene function, as we have demonstrated using the CMLiPSCs. The major unsolved question is why CML stem cells are naturally resistant to imatinib, and this question can be addressed using the iPS system. Disclosures: Slukvin: CDI: Consultancy, Equity Ownership.

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Stepwise Signaling and Low Oxygen Promote Hematopoietic Progenitor Emergence From Human Pluripotent Stem Cells

Blood ◽

10.1182/blood.v118.21.1317.1317 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1317-1317 ◽

Cited By ~ 2

Author(s):

Hiroshi Endo ◽

Naoya Takayama ◽

Tomo Koike ◽

Hiromitsu Nakauchi ◽

Koji Eto

Keyword(s):

Stem Cells ◽

Growth Factor ◽

Pluripotent Stem Cells ◽

Blood Cells ◽

Early Phase ◽

Late Phase ◽

Human Pluripotent Stem Cells ◽

Low Oxygen ◽

Hematopoietic Progenitor ◽

Oxygen Conditions

Abstract Abstract 1317 Hematopoietic systems in mouse models have been well characterized based on the defined cell surface markers present on fetal and adult hematopoietic stem cells (HSCs) and their blood derivatives. In humans, by contrast, hematopoietic ontogeny and the subsequent hierarchy have not been determined. Human pluripotent stem cells (ES cells, ESCs and iPS cells, iPSCs) are embryo-type cells and a promising cell source for studying the ontogeny of blood cells within a differentiation system. We previously established an in vitro co-culture method using C3H10T1/2 mesenchymal stromal cells, whereby vascular endothelial growth factor (VEGF) promotes the appearance of CD34+ hematopoietic progenitor cells (HPCs) from human ESCs or iPSCs (Takayama et al., Blood, 2008; Takayama et al., J Exp Med, 2010). Here we demonstrate that the use of a combination of low oxygen and growth factors is suitable for cells at specific developmental stages that include CD56+CD326- mesodermal progenitors during the early phase (days 0–4), CD34+CD56+CD90+CD105+CD43-KDR- hemangioblasts during the mid-phase (days 5–7), and CD43+ hematopoietic and KDR+ endothelial cells during the late phase (days 8–10). During mid-phase, application of basic fibroblast growth factor (bFGF, 10 ng/ml) under 1% O2 significantly increased numbers of CD43+ cells by 5-fold, as compared to cells without bFGF under 21% O2. Administration of a MYC inhibitor (50 μM) to cells during the early-phase, and tumor growth factor beta (TGF-beta) receptor inhibitor (SB431542, 10 μM) during mid-phase, also stimulated generation of CD34+CD43+ HPCs (1.5-fold and 3-fold in increase respectively). Notably, although this protocol resulted in a prominent yield of mesodermal progenitors during the early phase and of HPCs during the late phase (which were 50% and 10% of all derivatives from human iPSCs, respectively), this signaling manipulation had the opposite effect at other stages. For example, bFGF or TGFbeta receptor inhibition significantly depressed HPCs during the early-phase. The signaling modulation and low oxygen conditions in our co-culture system did not require factors known to affect ex vivo human CD34+ HSC / HPC expansion from cord blood cells, which contain stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, erythropoietin, interleukin (IL)-3 and IL-6. Our novel culture protocol implicates new players in the stepwise development from a pluripotent state to blood cell generation. These players appear to be governed by circumstances resembling a developmental niche with lower oxygen conditions. Disclosures: No relevant conflicts of interest to declare.

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Induced Pluripotent Stem Cells and Erythrocyte Production

Blood ◽

10.1182/blood.v120.21.sci-38.sci-38 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-38-SCI-38

Author(s):

Igor Slukvin

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Blood Cells ◽

Embryonic Stem ◽

Erythroid Cells ◽

Hematopoietic Progenitors ◽

Adult Hemoglobin ◽

Equity Ownership ◽

Induced Pluripotent

Abstract Abstract SCI-38 Induced pluripotent stem cells (iPSCs) are somatic cells that have been turned into embryonic-like stem cells by forced expression of factors critical for establishing pluripotency. Because iPSCs can be differentiated into any type of cell in the human body, including hematopoietic cells, they are seen as a logical alternative source of red blood cells (RBCs) for transfusion. In addition, the unlimited expansion potential of iPSCs makes it easy to adopt iPSC technology for RBC biomanufacturing. iPSCs can be generated from any type of donor, including O/Rh-negative universal donors and donors with very rare blood phenotypes, which makes it possible to generate blood products to accommodate virtually all patient groups. We have developed an approach for generating large quantities of RBCs from iPSCs by inducing them to differentiate into CD34+CD43+ hematopoietic progenitors in coculture with OP9 stromal cells, followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. Erythroid cultures produced by this approach consist of leukocyte-free populations of CD235a+ RBCs with robust expansion potential and long (up to 90 days) life spans. In these cultures, up to 1.8×105 RBCs can be generated from a single iPSC. Similar to embryonic stem cells, iPSC-derived RBCs express predominantly embryonic and fetal hemoglobin, with very little adult hemoglobin. It is already feasible to adopt iPSC technologies for producing cGMP-grade RBCs using defined animal-product-free differentiation conditions. However, the induction of the complete switch from embryonic to fetal and adult hemoglobin, as well as the terminal maturation and enucleation of iPSC-derived erythroid cells, remains a significant challenge. We recently identified at least three distinct waves of hematopoietic progenitors with erythroid potential in iPSC differentiation cultures. The characterization of erythroid cells produced from these waves of hematopoiesis may help to define populations with definitive erythroid potential and facilitate the production of erythrocytes from iPSCs. Additional critical steps toward translating iPSC-based RBC technologies to the clinic include the development of bioreactor-based-technology for further scaling-up of cell production, and evaluation of the therapeutic potential and safety of human pluripotent stem cell-derived blood cells in animal models. Overall, the manufacturing of RBCs provides several advantages. It can improve the continuity of the blood supply, minimize/eliminate the risk of infection transmission, reduce the incidence of hemolytic and nonhemolytic transfusion reactions, and provide an opportunity to generate RBCs that fit specific clinical needs by using genetically engineered iPSCs or iPSCs with rare blood groups. Disclosures: Slukvin: CDI: Consultancy, Equity Ownership; Cynata: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Cellular senescence or stemness: hypoxia flips the coin

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02035-0 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Daniel Otero-Albiol ◽

Amancio Carnero

Keyword(s):

Stem Cells ◽

Cellular Senescence ◽

Physiological State ◽

Tumor Development ◽

Embryonic Stem ◽

Low Oxygen ◽

Cancer Disease ◽

Oxygen Conditions ◽

Induced Pluripotent

AbstractCellular senescence is a complex physiological state whose main feature is proliferative arrest. Cellular senescence can be considered the reverse of cell immortalization and continuous tumor growth. However, cellular senescence has many physiological functions beyond being a putative tumor suppressive trait. It remains unknown whether low levels of oxygen or hypoxia, which is a feature of every tissue in the organism, modulate cellular senescence, altering its capacity to suppress the limitation of proliferation. It has been observed that the lifespan of mammalian primary cells is increased under low oxygen conditions. Additionally, hypoxia promotes self-renewal and pluripotency maintenance in adult and embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and cancer stem cells (CSCs). In this study, we discuss the role of hypoxia facilitating senescence bypass during malignant transformation and acquisition of stemness properties, which all contribute to tumor development and cancer disease aggressiveness.

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Reprogrammed Murine Fibroblasts Differentiated into Hematopoietic Progenitors Are Able to Successfully Engraft and Repopulate the Bone Marrow

Blood ◽

10.1182/blood.v112.11.389.389 ◽

2008 ◽

Vol 112 (11) ◽

pp. 389-389 ◽

Cited By ~ 1

Author(s):

Zaida Alipio ◽

Dan Xu ◽

Jianchang Yang ◽

Louis M. Fink ◽

Wilson Xu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Es Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Ips Cells ◽

Embryoid Bodies ◽

Hematopoietic Progenitors

Abstract Cellular therapy using embryonic stem cells has always been an area of great interest due to the pluripotent characteristics of stem cells. In 2006, Takahashi and Yamanaka (Cell 126, 663–676) demonstrated that somatic cells can be reprogrammed into a stem cell-like state, termed induced pluripotent stem (iPS) cells, by ectopic expression of Oct4, Sox2, Klf4 and c Myc. A later report (Nakagawa et al. Nat. Biotechnol.26:101–106, 2008) showed that iPS cells can be produced in the absence of the c Myc oncogene. We have used this latter strategy to successfully reprogram somatic cells derived from C57BL/6 mouse tail fibroblast to iPS cells. Retrovirus infected fibroblasts exhibited stem cell-like morphology by 14 days post infection. These iPS cells were then infected with a retrovirus that expressed HOXB4. Recombinant leukemia inhibitor factor (LIF) supplement was removed from media at this time and the cells allowed to differentiate into embryoid bodies. These cells were screened for specific differentiation stem cell markers, such as Oct4, Nanog, Sall4 and SSEA-1. iPS cells were converted into embryonic bodies and then infected with retroviruses expressing HOXB4. Embryoid bodies stably expressing HOXB4 were induced to hematopoietic differentiation by treatment of thrombopoietin (TPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), interferon gamma (IFNg) and fms-like tyrosine kinase (FLT3 ligand). Evaluation of iPS-derived hematopoietic cells on smears show strikingly similarity in morphology to the W4 mouse embryonic stem (ES) cells differentiated into hematopoietic cells as a control. Flow cytometry analysis of iPS-derived hematopoietic cells after 1 week exposure to cytokines revealed 7% B220+ cells (B cells), 11% Ter119+ cells (erythroid), and 13% Gr-1+ cells (granulocytes) similar to W4 ES cells. The iPS-derived hematopoietic cells were transplanted into irradiated immunodeficient mice via lateral tail vein injection. Transplantation of these iPS-derived hematopoietic progenitors tagged with GFP into irradiated SCID mice revealed that the hematopoietic progenitors were able to home to the bone marrow after 1 week of transplantation. Importantly, after 1 month, GFP+ engrafted cells remained in the bone marrow suggesting a long-term engraftment. This long term engraftment of the iPS-derived hematopoietic cells to the bone marrow constitutes an important step toward potential therapy of numerous patient-specific blood based diseases.

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