A Platform for the Scalable Derivation of Genetically-Enhanced T and NK Lymphocytes from Naive Human Induced Pluripotent Stem Cells for Cancer Immunotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2364-2364
Author(s):  
Raedun Clarke ◽  
William Kim ◽  
Brian Groff ◽  
Ramzey Abujarour ◽  
Megan Robinson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nk Cells ◽  
Single Cell ◽  
Cancer Immunotherapy ◽  
Small Molecule ◽  
Lymphoid Cells ◽  
Scale Production ◽  
Hematopoietic Stem ◽  
Induced Pluripotent

Abstract Human induced pluripotent stem cell (hiPSC) technology enables the generation of a potentially unlimited source of therapeutically viable hematopoietic cells for the treatment of numerous hematological and non-hematological malignancies, and represents a highly promising approach for overcoming many of the challenges and limitations of patient-derived cancer immunotherapies. To advance the promise of hiPSC technology as an "off-the-shelf" source of hematopoietic cellular therapeutics, it is essential to be able to efficiently and reproducibly generate not only hematopoietic stem cells (HSCs) but also immune effector populations, including the diverse subsets of T and NK lymphoid cells, through a robust and scalable process. The in vitro derivation of HSCs and lymphocytes is complicated by the existence of at least two temporally and spatially distinct waves of blood cell formation during embryonic development: primitive and definitive. Primitive hematopoiesis initiates in the extraembryonic yolk sac and generates a transient and restricted hematopoietic repertoire consisting mainly of primitive erythroid and myeloid cells. Nascent HSCs only emerge later during the definitive wave from a specialized endothelial progenitor within the arterial vasculature termed hemogenic endothelium (HE). HE undergoes an endothelial-to-hematopoietic transition to give rise to HSCs, which then ultimately migrate to the bone marrow where they sustain multi-lineage hematopoiesis, including T and NK lymphoid cells, throughout adult life. Therefore the generation of HSCs and the formation of lymphoid effector cells from hiPSCs is dependent upon our ability to accurately recapitulate the intricate stages of early embryonic hematopoietic development towards the definitive program. While a limited number of studies have described the directed differentiation of hiPSCs to definitive HE in vitro, a major hurdle in utilizing hiPSCs for therapeutic purposes has been the requirement to initially co-culture such cells with murine-derived stromal cells in the presence of ill-defined serum-containing media in order to maintain pluripotency and induce differentiation. In addition, these protocols have employed an intermediate strategy consisting of embryoid body (EB) formation, which is difficult to scale and hindered by lack of reproducibility. We have previously demonstrated that our proprietary platform for robust and rapid derivation of clonal hiPSC lines, which utilizes small molecule reprograming and single cell selection strategies, generates cells with properties indicative of the naïve, or ground state of pluripotency. In addition to maintaining a homogeneous population of hiPSCs, our platform enables the genetic engineering of such pluripotent cells, at a single cell level, in both nuclease-dependent and -independent strategies. Here we describe a novel method for the generation of definitive HE from naïve hiPSCs in a scalable manner, void of an EB intermediate, under serum/feeder-free conditions. This platform represents a well-defined, small molecule-driven, staged protocol that can readily be translated to meet current good manufacturing practice (cGMP) requirements for the development of "off-the-shelf" hematopoietic cell-based immunotherapies. The derived HE population is definitive in nature as determine by Notch dependency and exhibits multi-lineage potential, as demonstrated through the formation of both T and NK lymphoid cells. HE generated by this protocol can be successfully cryopreserved and banked, serving as a highly-stable feedstock for subsequent derivation of various cell types for therapeutic use, including for T and NK cell-based immunotherapies. We have demonstrated that our proprietary, clinically-adaptable method for the large-scale production of definitive HE can efficiently give rise to a variety of lymphoid cell subsets. These derived lymphocytes, including NK cells, have been extensively characterized in vitro and in vivo, and we have demonstrated functionality through cytokine release and cellular cytotoxicity. Furthermore, through genetic modifications at the single cell hiPSC stage, tumor antigen-targeting and inducible caspase-mediated safety systems have been introduced into safe harbor loci to improve the specificity and safety profiles of hiPSC-derived T and NK cells for cancer immunotherapy applications. Disclosures Clarke: Fate Therapeutics Inc: Employment. Abujarour:Fate Therapeutics Inc: Employment. Robinson:Fate Therapeutics Inc: Employment. Huang:Fate Therapeutics Inc: Employment. Shoemaker:Fate Therapeutics Inc: Employment. Valamehr:Fate Therapeutics Inc: Employment.

scholarly journals B-1 lymphoid cells develop independently of Notch signaling during mouse embryonic development

2020 ◽  
Author(s):  
Nathalia Azevedo ◽  
Elisa Bertesago ◽  
Ismail Ismailoglu ◽  
Michael Kyba ◽  
Michihiro Kobayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Development ◽  
Blood Cell ◽  
Notch Signaling ◽  
Embryonic Stem ◽  
Cell Types ◽  
Lymphoid Cells ◽  
Lineage Specification ◽  
Hematopoietic Stem

AbstractThe in vitro generation from pluripotent stem cells (PSCs) of different blood cell types, in particular those that are not replenished by hematopoietic stem cells (HSCs) like fetal-derived tissue-resident macrophages and innate-like lymphocytes, is of a particular interest. In order to succeed in this endeavor, a thorough understanding of the pathway interplay promoting lineage specification for the different blood cell types is needed. Notch signaling is essential for the HSC generation and their derivatives, but its requirement for tissue-resident immune cells is unknown. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B-1 cells. Their Notch-independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling was needed for the emergence of B-2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dosage is critical for the different B-cell lineage specification and provides pivotal information for their in vitro generation from PSCs for therapeutic applications.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

In Vitro Generation of NK22 Cells From Hematopoietic Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 108-108
Author(s):  
Qin Tang ◽  
Ahn Yong-Oon ◽  
Peter Southern ◽  
Bruce R. Blazar ◽  
Jeffrey S Milller ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Nk Cells ◽  
Lymphoid Tissue ◽  
Culture System ◽  
Stage Iv ◽  
Stage Iii ◽  
Hematopoietic Stem ◽  
Secondary Lymphoid Tissue

Abstract Abstract 108 NK cells are the first lymphocytes to recover after allogeneic hematopotiec cell transplantation (allo-HCT). Rapid NK recovery after allo-HCT is associated with reduced treatment related mortality. Because NK cells elaborate inflammatory cytokines (IFN-g) and mediate cytotoxic killing of malignant cells, they are also implicated in graft vs. leukemia reactions. Curiously early after transplant, donor-derived NK cells are hypofunctional and immature. Over the past year, investigators identified a new category of NK cells, called NK22 cells. These cells are present in secondary lymphoid tissue, such as tonsils, lymph nodes and Peyer's patches. Previous investigators have not been able to identify NK22 cells in adult blood or UCB, likely due to lymphoid tissue homing receptor expression (CCR6 and CCR7). NK22 cells are CD56+/−CD117highCD94−IL-1bR+, a phenotype which overlaps with one previously used to describe NK progenitors (i.e., stage III immature NK cells). At present, it is not known whether NK22 cells are a distinct branch of the NK lineage or are NK developmental intermediates. NK22 cells are present in secondary lymphoid tissue at vanishingly small quantities (<1% of all mononuclear cells), thereby making the study of these cells challenging. Functionally, NK22 cells lack of “classical” NK functions (cytotoxicity and IFN-g production) and instead elaborate IL-22 in response to dendritic cell derived IL-1 and/or IL-23. IL-22 does not act on hematopoietic cells, but rather on mucosal tissues to induce proliferation, anti-apoptotic functions and the production of antimicrobial proteins (b defensins). NK22 cells also increase the expression of adhesion molecules on MSCs after co-culture, suggesting a role in secondary lymphoid generation and homeostasis. We have previously used a stromal cell based culture system to study NK development from hematopoietic stem cells. Briefly, CD34+ cells are cultured in the presence of IL-3 (for the first week), FLT-3L, SCF, IL-7 and IL-15 for ~4-5 weeks. At the end of this culture period, functional mature NK cells are obtained. Because this system closely recapitulates ontongeny, we hypothesized that it could be used to study NK22 development. At D28 of culture, we found that 90% (range=88-94%) of cells expressed CD56. Approximately 22% (range=16-28%) had a stage III immature NK cell phenotype (i.e., CD56+CD117highCD94−), of which ~87% (range=77-93%) also expressed IL-bR, a phenotype consistent with NK22 cells (n=5). We next purified CD56− and CD56+ cell populations in these cultures and neither showed IL-22 expression at rest. Following IL-1 and/or IL-23 stimulation, the CD56+ fraction made IL-22 transcripts (by qPCR) and protein by ELISA. We next purified the stage III (CD56+CD117highCD94−) and stage IV (CD56+CD117lowCD94+) fractions and found that only the stage III cells were capable of IL-22 production following IL-1/23 stimulation. Co-culture of NK22 cells (or their supernatant) with MSCs resulted in a >2 log increase in ICAM. Likewise, the supernatant of from activated stage III cells induced keritinocyte proliferation and production of antimicrobial compounds. In vitro derived NK22 cells were compared to freshly isolated NK22 cells from human tonsils and nearly identical staining patterns for ROR-gt, Acyl hydrocarbon receptor, NKp44, NKp46, CD127, CD161, CCR6 and CCR7 were observed. Lastly, purified CD56+CD117highCD94− cells could acquire IL-bR and then further differentiate into stage IV cells (CD56+CD117lowCD94+) in the presence of IL-15. However, this was less likely in the presence of IL-15 and IL-1b, suggesting that NK22 cells are developmental intermediates with specific functions (SLT homeostasis and maintenance of mucosal surfaces and immunity). Depending upon the environmental stimuli, these cells will either maintain their IL-22 producing capacity or develop into cytotoxic lymphocytes. These studies are the first studies to describe the generation of NK22 cells from hematopoietic stem cells. They also allow a better understanding of the developmental requirements and functions of these rare cells. Lastly, this simple culture system creates a new opportunity to use NK22 cells therapeutically to enhance SLT tissue repair and mucosal immunity after allo-HCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human Fetal Liver: AnIn VitroModel of Erythropoiesis

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Guillaume Pourcher ◽  
Christelle Mazurier ◽  
Yé Yong King ◽  
Marie-Catherine Giarratana ◽  
Ladan Kobari ◽  
...  
Keyword(s):  
Stem Cells ◽  
Large Scale ◽  
Adult Stem Cells ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Scale Production ◽  
Cloning Efficiency ◽  
Hematopoietic Stem

We previously described the large-scale production of RBCs from hematopoietic stem cells (HSCs) of diverse sources. Our present efforts are focused to produce RBCs thanks to an unlimited source of stem cells. Human embryonic stem (ES) cells or induced pluripotent stem cell (iPS) are the natural candidates. Even if the proof of RBCs production from these sources has been done, their amplification ability is to date not sufficient for a transfusion application. In this work, our protocol of RBC production was applied to HSC isolated from fetal liver (FL) as an intermediate source between embryonic and adult stem cells. We studied the erythroid potential of FL-derived CD34+cells. In thisin vitromodel, maturation that is enucleation reaches a lower level compared to adult sources as observed for embryonic or iP, but, interestingly, they (i) displayed a dramaticin vitroexpansion (100-fold more when compared to CB CD34+) and (ii) 100% cloning efficiency in hematopoietic progenitor assays after 3 days of erythroid induction, as compared to 10–15% cloning efficiency for adult CD34+cells. This work supports the idea that FL remains a model of study and is not a candidate forex vivoRBCS production for blood transfusion as a direct source of stem cells but could be helpful to understand and enhance proliferation abilities for primitive cells such as ES cells or iPS.

scholarly journals Age-Associated Characteristics of Murine Hematopoietic Stem Cells

2000 ◽  
Vol 192 (9) ◽  
pp. 1273-1280 ◽  
Author(s):  
Kazuhiro Sudo ◽  
Hideo Ema ◽  
Yohei Morita ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Lymphoid Cells ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Functional Changes

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34−/lowc-Kit+Sca-1+ lineage marker–negative (CD34−KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34−KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34−KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34−KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

scholarly journals CD16-IL15-CLEC12A Trispecific Killer Engager (TriKE) Drives NK Cell Expansion, Activation, and Antigen Specific Killing of Cancer Stem Cells in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1454-1454 ◽  
Author(s):  
Upasana Sunil Arvindam ◽  
Paulien van Hauten ◽  
Caroline Hallstrom ◽  
Daniel A. Vallera ◽  
Harry Dolstra ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Nk Cell ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Functional Assays ◽  
Target Effects

Abstract Our group developed a 161533 trispecific killer engager (TriKE) molecule to target acute myeloid leukemia (AML) cells using Natural Killer (NK) cells. This molecule contains an anti-CD16 camelid nanobody to activate NK cells, an anti-CD33 single chain variable fragment (scFv) to engage cancer targets, and an IL-15 molecule that drives NK cell priming, expansion and survival. Using an earlier version of this molecule, we have shown that the CD33 TriKE is effective at activating NK cells against AML targets in vitro and in vivo. This preclinical data has lead to the establishment of a clinical trial in refractory AML patients at the University of Minnesota, set to open Q3 2018. While these previous studies have validated the use of TriKEs as an effective strategy of harnessing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen. The high mortality and poor five-year survival rates (26%) for AML patients can be attributed to chemotherapy resistance and disease relapse. A majority of chemotherapy resistant leukemia stem cells (LSCs), that are hypothesized to facilitate relapse, do not express CD33. In addition, all hematopoietic stem cells and normal myeloid cells express CD33, thus targeting this antigen can lead to severe defects in hematopoiesis and on-target/off-tumor toxicity. To address these limitations, we developed a TriKE that targets CLEC12A or C-type lectin-like molecule 1 (CLL-1). CLEC12A is highly expressed on AML cells and over 70% of CD33 negative cells express CLEC12A. It has been attributed as a stem cell marker in AML, being selectively overexpressed in LSCs. CLEC12A is expressed by CD34+/CD38- LSCs but not normal CD34+/CD38- hematopoietic stem cells in regenerating bone marrow, thus minimizing off-target effects. The CLEC12A TriKE was developed in a mammalian cell system to ensure that appropriate post-translational modifications are present. We confirmed that the TriKE binds specifically to HL-60 and THP-1 target cells that express CLEC12A compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with the CLEC12A TriKE drives a significant increase in NK cell specific proliferation over 7 days as measured by CellTrace dilution compared to treatment with a CLEC12A scFv or IL-15 alone (69.7 ± 6.7% vs 11.9 ± 2.5% vs 38.4 ± 7.3%) (Figure 1A). To measure NK cell killing, we conducted an IncuCyte Zoom assay. Here, HL-60 target cells were labeled with a caspase 3/7 reagent where a color change indicates target cell death. The CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15 as measured by number of live target cells at the end of the 48 hour assay (53.9 ± 1.9% vs 103.3 ± 3.4% vs 71.1 ± 1.4%). The CLEC12A TriKE induces an increase in NK cell degranulation, measured by CD107a expression against HL-60 AML tumor targets in a 4 hour functional assay compared to treatment with CLEC12A scFv or IL-15 alone (62.3 ± 1.1% vs 19.4 ± 3.8% vs 27.5 ± 4.9%). In this assay, there is also an increase in cytokine production, measured by IFNg and TNFa respectively (16.7 ± 4.2% vs 2.3 ± 1.5% vs 4.7 ± 1.9% and 18.0 ± 5.1% vs 2.5 ± 1.7% vs 4.6 ± 2.5%) (Figure 1B). We observe a similar enhanced functional response with THP-1 AML tumor targets. In these functional assays, treatment with the CLEC12A TriKE produced less background activation compared to the CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, we tested the efficacy of the CLEC12A TriKE against primary AML targets. AML blasts were identified as SSC low, CD45 intermediate and CD34 high cells. Out of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC12A (78.1 ± 5.2%). In functional assays with these samples, the CLEC12A TriKE was able to induce greater CD107a and IFNg expression, and enhanced killing of tumor targets as measured by a live/dead stain compared to CLEC12A scFv or IL-15 (Figure 1C). In these assays, the efficacy of the CLEC12A TriKE was comparable to the CD33 TriKE. Our data demonstrates that the CLEC12A TriKE drives NK cell specific proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. Apart from AML, CLEC12A is expressed on cancer cells and LSCs in patients with myelodysplastic syndromes (MDS). These findings highlight the clinical potential of the CLEC12A TriKE individually or in combination with the CD33 TriKE for the treatment of MDS and AML. Figure 1. Figure 1. Disclosures Vallera: GT Biopharma: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding.

scholarly journals UM171 Regulates the Hematopoietic Differentiation of Human Acquired Aplastic Anemia-Derived Induced Pluripotent Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2500-2500
Author(s):  
Tellechea Maria Florencia ◽  
Flavia S. Donaires ◽  
Tiago C. Silva ◽  
Lilian F. Moreira ◽  
Yordanka Armenteros ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Induced Pluripotent

Aplastic anemia (AA) is characterized by a hypoplastic bone marrow associated with low peripheral blood counts. In acquired cases, the immune system promotes hematopoietic stem and progenitor cell (HSPC) depletion by the action of several pro-inflammatory Th1 cytokines. The current treatment options for severe cases consist of sibling-matched allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) with anti-thymocyte globulin, cyclosporine, and eltrombopag. However, most patients are not eligible for HSCT and, although about 85% of patients respond to IST with eltrombopag, a proportion of patients eventually relapse, requiring further therapies. Failure to respond adequately to immunosuppression may be attributed to the scarcity of HSPCs at the time of diagnosis. Induced pluripotent stem cells (iPSCs) are potentially an alternative source of patient-specific hematopoietic cells. Patient-specific HSPCs derived from in vitro iPSC differentiation may serve as a tool to study the disease as well as a source of hematopoietic tissue for cell therapies. The pyrimidoindole molecule UM171 induces ex vivo expansion of HSCs of human cord and peripheral blood and bone marrow, but the pathways modulated by this molecule are not well understood. Here we evaluated the hematopoietic differentiation potential of iPSCs obtained from patients with acquired AA. We further determined the effects of UM171 on this differentiation process. First, we derived iPSCs from 3 patients with acquired AA after treatment (1 female; average age, 31 years; 2 partial responders, 1 complete responder) and 3 healthy subjects (3 females; average age, 61 years) and induced differentiation in vitro through the embryoid body system in cell feeder and serum-free medium supplemented with cytokines. The hematopoietic differentiation of healthy-iPSCs yielded 19% ± 8.1% (mean ± SEM) of CD34+cells after 16 days in culture, in contrast with 11% ± 4.9% of CD34+cells obtained from the differentiation of AA-iPSCs, which corresponds to a 1.7-fold reduction in CD34+cell yield. The total number of erythroid and myeloid CFUs was lower in the AA-iPSC group as compared to healthy-iPSCs (12±4.2 vs.24±7.2; respectively; p<0.03). These findings suggest that erythroid-derived AA-iPSC have an intrinsic defect in hematopoietic differentiation. Next, we tested whether UM171 modulated hematopoietic differentiation of AA-iPSCs. We found that UM171 significantly stimulated the differentiation of both healthy and AA-iPSCs. In the healthy-iPSC group, the percentage of CD34+cells was 1.9-fold higher when treated with UM171 compared to controls treated with DMSO (37% ± 7.8% vs.19% ± 8.1%; respectively; p<0.03) and in AA-iPSCs the increase was 3.9-fold (45% ± 11% vs. 11% ± 4.9%; p<0.07). The clonogenic capacity of progenitors to produce erythroid and myeloid colonies also was augmented in both groups in comparison to DMSO (28±11 vs. 23±7.2) for healthy-iPSCs and for AA-iPSCs (23±8.5 vs. 12±4.2, p<0.06). We then investigated the molecular pathways influenced by UM171. The transcriptional profile of differentiated CD34+cells showed that UM171 up-regulated genes involved in early hematopoiesis from mesoderm (BRACHYURY and MIXL1) and primitive streak specification (APELA and APLNR), to hemangioblasts and primitive hematopoietic progenitor commitment (TDGF1, SOX17, and KLF5). We also observed the up-regulation of pro-inflammatory NF-kB activators (MAP4K1, ZAP70, and CARD11) and the anti-inflammatory gene PROCR, a marker of cultured HSCs and an NF-kB inhibitor. This balanced network has been previously suggested to be modulated by UM171 (Chagraoui et. al. Cell Stem Cell 2019). Taken together, our results showed that acquired AA-iPSCs may have intrinsic defects that impair hematopoietic differentiation in vitro. This defect may be atavic to the cell or, alternatively, the consequence of epigenetic changes in erythroid precursors provoked by the immune attack. In addition, our findings demonstrate that UM171 significantly stimulate the hematopoietic differentiation of AA-iPSCs and identified a novel molecular mechanism for UM171 as an enhancer of early hematopoietic development programs. These observations may be valuable for improving the achievement of de novo hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differentiation of human induced pluripotent stem cells into erythroid cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohsen Ebrahimi ◽  
Mehdi Forouzesh ◽  
Setareh Raoufi ◽  
Mohammad Ramazii ◽  
Farhoodeh Ghaedrahmati ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Promising Alternative ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractDuring the last years, several strategies have been made to obtain mature erythrocytes or red blood cells (RBC) from the bone marrow or umbilical cord blood (UCB). However, UCB-derived hematopoietic stem cells (HSC) are a limited source and in vitro large-scale expansion of RBC from HSC remains problematic. One promising alternative can be human pluripotent stem cells (PSCs) that provide an unlimited source of cells. Human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are self-renewing progenitors that can be differentiated to lineages of ectoderm, mesoderm, and endoderm. Several previous studies have revealed that human ESCs can differentiate into functional oxygen-carrying erythrocytes; however, the ex vivo expansion of human ESC-derived RBC is subjected to ethical concerns. Human iPSCs can be a suitable therapeutic choice for the in vitro/ex vivo manufacture of RBCs. Reprogramming of human somatic cells through the ectopic expression of the transcription factors (OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG) has provided a new avenue for disease modeling and regenerative medicine. Various techniques have been developed to generate enucleated RBCs from human iPSCs. The in vitro production of human iPSC-derived RBCs can be an alternative treatment option for patients with blood disorders. In this review, we focused on the generation of human iPSC-derived erythrocytes to present an overview of the current status and applications of this field.

scholarly journals GATA2 deficiency and human hematopoietic development modeled using induced pluripotent stem cells

2018 ◽  
Vol 2 (23) ◽  
pp. 3553-3565 ◽  
Author(s):  
Moonjung Jung ◽  
Stefan Cordes ◽  
Jizhong Zou ◽  
Shiqin J. Yu ◽  
Xavi Guitart ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Nk Cells ◽  
Pluripotent Stem Cells ◽  
Bone Marrow Failure ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Gata2 Deficiency ◽  
Induced Pluripotent

Abstract GATA2 deficiency is an inherited or sporadic genetic disorder characterized by distinct cellular deficiency, bone marrow failure, various infections, lymphedema, pulmonary alveolar proteinosis, and predisposition to myeloid malignancies resulting from heterozygous loss-of-function mutations in the GATA2 gene. How heterozygous GATA2 mutations affect human hematopoietic development or cause characteristic cellular deficiency and eventual hypoplastic myelodysplastic syndrome or leukemia is not fully understood. We used induced pluripotent stem cells (iPSCs) to study hematopoietic development in the setting of GATA2 deficiency. We performed hematopoietic differentiation using iPSC derived from patients with GATA2 deficiency and examined their ability to commit to mesoderm, hemogenic endothelial precursors (HEPs), hematopoietic stem progenitor cells, and natural killer (NK) cells. Patient-derived iPSC, either derived from fibroblasts/marrow stromal cells or peripheral blood mononuclear cells, did not show significant defects in committing to mesoderm, HEP, hematopoietic stem progenitor, or NK cells. However, HEP derived from GATA2-mutant iPSC showed impaired maturation toward hematopoietic lineages. Hematopoietic differentiation was nearly abolished from homozygous GATA2 knockout (KO) iPSC lines and markedly reduced in heterozygous KO lines compared with isogenic controls. On the other hand, correction of the mutated GATA2 allele in patient-specific iPSC did not alter hematopoietic development consistently in our model. GATA2 deficiency usually manifests within the first decade of life. Newborn and infant hematopoiesis appears to be grossly intact; therefore, our iPSC model indeed may resemble the disease phenotype, suggesting that other genetic, epigenetic, or environmental factors may contribute to bone marrow failure in these patients following birth. However, heterogeneity of PSC-based models and limitations of in vitro differentiation protocol may limit the possibility to detect subtle cellular phenotypes.

scholarly journals Experimental Limitations Using Reprogrammed Cells for Hematopoietic Differentiation

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Katharina Seiler ◽  
Motokazu Tsuneto ◽  
Fritz Melchers
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Clinical Settings ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Induced Pluripotent

We review here our experiences with thein vitroreprogramming of somatic cells to induced pluripotent stem cells (iPSC) and subsequentin vitrodevelopment of hematopoietic cells from these iPSC and from embryonic stem cells (ESC). While, in principle, thein vitroreprogramming and subsequent differentiation can generate hematopoietic cell from any somatic cells, it is evident that many of the steps in this process need to be significantly improved before it can be applied to human cells and used in clinical settings of hematopoietic stem cell (HSC) transplantations.

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