scholarly journals Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 811
Author(s):  
Pranav Oberoi ◽  
Kathrina Kamenjarin ◽  
Jose Francisco Villena Ossa ◽  
Barbara Uherek ◽  
Halvard Bönig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

Preclinical Ex-Vivo Generation of Clinical Relevant Amounts of NK Cells from CD34+ Progenitor Cells for Immunotherapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2916-2916
Author(s):  
Jan Spanholtz ◽  
T. M. de Witte ◽  
Marleen Tordoir ◽  
Harry Dolstra
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Product ◽  
Cytotoxicity Assays ◽  
Cd34 Cells ◽  
Specific Antigens

Abstract Alloreactive donor Natural Killer (NK) cells, displaying a KIR-ligand mismatch with the recipient play a pivotal role in graft-versus-leukemia (GVL) reactivity without significant graft-versus-host disease (GVHD) following haploidentical stem cell transplantation. Therefore, infusions of haploidentical NK cells are suggested to become an attractive approach for cancer immunotherapy. So far, difficulties in isolation and expansion of peripheral NK cells resulted in only limited data about safety and clinical efficacy of purified NK cell infusions. Therefore, we have developed a novel culture system without the use of feeder cells for the ex vivo generation of NK cells from CD34+ hematopoietic progenitor cells (HPCs) isolated from cord blood (CB) or bone marrow (BM). It is based on a two-step procedure using an expansion and a differentiation step. The NK cell generation system uses mainly cytokines such as SCF, TPO, Flt3-L, IL-2, IL-7 and IL-15 and specific modified glycosaminoglycans (GAGs) to direct and control the two phases. The developmental phase and the final NK cell product is controlled and characterized by immunophenotyping using multi-colour flow cytometry and CFSE-based cytotoxicity assays against various tumor cells. Our system generates a homogeneous final cell product of CD56+/CD3- cells with a purity of >99%. A total cell expansion of more than 5×10^4 fold allows to generate 5×10^10 NK cells from 1×10^6 CB CD34+ stem and progenitor cells within 4–5 weeks of culture. For BM cells an expansion rate of more than 1×10^4 fold was detected after a 5–6 week cell culture period. During the two week expansion phase step,we expand UCB CD34+ cells more than 100 fold. Phenotypic analysis showed a decrease of stem cell-specific antigens such as CD34 and CD117 during the first three weeks, whereas antigens specific for NK cell progenitors and mature NK cells such as CD56, CD94 and CD161 are up-regulated after initiating differentiation at day 14. Furthermore, distinct cell populations can be detected reflecting NK cell developmental stages in vitro. Effective differentiation of the expanded progenitor cells into mature NK cells is characterized by the expression of NK cell-specific antigens including CD56, CD94, NKG2A, NKG2D and NCRs as well as homing receptors such as CD62L, CXCR4 and CCR7. The final NK cell product shows high expression levels of inhibitory and activating receptors as well the intrinsic capability to express KIR, which is detected by flow cytometry after 6–7 weeks of culture. Cytotoxicity assays demonstrated robust lysis of more than 90% against AML as well as melanoma tumor cell lines. This system, with its huge expansion potential to generate highly activated NK cells with homing capability, is the basis for a first clinical trial in 2009, to infuse haploidentical NK cells generated from CD34+ cells in poor-risk AML patients. The use of our defined culture conditions enables new prospects in NK cell research, regarding NK cell development and NK cell maturation, as well as new aspects for the clinical use of NK cell products derived from HPCs.

scholarly journals Functional Stability (at +4°C) of Hematopoietic Stem and Progenitor Cells Amplified Ex Vivo from Cord Blood CD34+Cells

2013 ◽  
Vol 22 (8) ◽  
pp. 1501-1506 ◽  
Author(s):  
Pascale Duchez ◽  
Jean Chevaleyre ◽  
Philippe Brunet De La Grange ◽  
Marija Vlaski ◽  
Jean-Michel Boiron ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Functional Stability ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Cord Blood Cd34

scholarly journals Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Hui Xie ◽  
Li Sun ◽  
Liming Zhang ◽  
Teng Liu ◽  
Li Chen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

A Novel Triple Umbilical Cord Blood Transplant (UCBT) Strategy To Promote NK Cell Immunotherapy (Unit 1) with a Fully Ablative Preparative Regimen Followed by a Double UCBT in Patients with Refractory AML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3111-3111 ◽  
Author(s):  
Jeffrey S. Miller ◽  
Claudio G. Brunstein ◽  
Sarah Cooley ◽  
Michael R. Verneris ◽  
Angela Panoskaltsis-Mortari ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Treatment Options ◽  
Hematopoietic Stem ◽  
Preparative Regimen ◽  
Neutrophil Engraftment ◽  
Refractory Aml

Abstract Treatment options for refractory AML are usually ineffective. Previously, we tested adoptive transfer of haploidentical peripheral blood (PB) derived NK cells without transplantation and demonstrated correlation between in vivo NK cell expansion and those who achieved a complete remission. This therapy is limited by: the inability to expand NK cells in most patients, prolonged neutropenia in some patients and inconsistent efficacy. UCB, in contrast to adult PB, is rich in NK precursors with CD34+/CD7−, CD34+/CD7+ and CD34−/CD7+ phenotypes. We hypothesized that UCB-derived NK cells may show better in vivo expansion than adult derived NK cells after cytoreduction. Therefore, we tested our triple UCBT strategy in patients with refractory relapsed AML who were <45 years old, without active infection and eligible for myeloablative conditioning. The UCB NK product (unit 1) was CD3 depleted (using a CliniMacs system) and activated with IL-2 (1000U/ml for 16–20 hours). The UCB-derived NK cells (matched at 3 HLA loci and KIR-ligand mismatched when possible) were infused on day -12 after conditioning with cyclophosphamide 120 mg/kg, fludarabine 125 mg/m2 and TBI 1320 cGy on days -19 to -13. Subcutaneous IL-2 (10 MU) was was given on days -12, -10, -8, -6, -4 and -2 to facilitate in vivo NK cell expansion. On day 0, two UCB units (≥4/6 match) were transplanted for hematopoietic rescue and followed by mycophenolate mofetil and cyclosporine for GVHD prophylaxis. Compared to pre-treatment levels, endogenous IL-15 was markedly increased after the preparative regimen at the time of the NK UCB unit. The NK UCB units contained both precursor and mature NK cells. Three product samples were cultured for 28 days in limiting dilution on a murine stromal feeder, demonstrating cloning frequencies of 1:5, 1:9 and 1:12 infused UCB cells giving rise to NK progeny. Two of the 3 patients had partial chimerism derived from the NK product on day -1. Unexpectedly, these same two patients demonstrated prompt neutrophil engraftment on days 3 and 7 after hematopoietic stem cell rescue. In both instances, chimerism was achieved from the NK product. Of the non-NK and non-T cells in the NK UCB units from these 2 patients there were 9.6% and 5.3% CD34+/CD7− cells. In the third patient, the NK UCB unit had only 2% CD34+/CD7− cells and they did not contribute to neutrophil engraftment which occurred on day 36 after UCBT. Of note, 3/3 tolerated the NK infusion without toxicity and were leukemia-free at the time of engraftment. Two remain alive (one died of TRM) with one relapse before day 100. These data suggest UCB NK cells may be administered safely and, despite CD3 depletion and IL-2 activation (ex vivo and in vivo), provide long term engraftment potential that may dominate over unmanipulated UCB infused subsequently. In summary, UCB is a rich source of NK precursors capable of in vivo expansion which are potentially better suited than adult NK cells for use in treatment of patients with refractory AML.

Generation Of Clinical Grade, Highly Cytotoxic NK Cells Using Feeder Cell Free, Particle-Based Technology

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4503-4503
Author(s):  
Jerremiah Oyer ◽  
Igarashi Y Robert ◽  
Colosimo Dominic ◽  
Melhem M. Solh ◽  
Yasser Khaled ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
Free Particle ◽  
Leukemia Cell ◽  
High Dose ◽  
Feeder Cells ◽  
Peripheral Blood Mononuclear

NK cell immunotherapy shows exciting promise, but inconsistency and variability remain as a significant challenge. Since NK cells comprise a small fraction (∼5%) of the peripheral blood mononuclear cell fraction, expansion of NK cells in vivo or ex vivo is a critical requirement to attain therapeutically effective dosages and to observe consistent positive clinical outcomes. Most of currently developed ex vivo expansion protocols depend on co-culture with various engineered and/or cancer derived stimulator/feeder cells to induce the proliferation of NK cells. The use of accessory cells poses significant challenges to clinical transfer. Our laboratory has developed a nanoparticle-based expansion technology that utilizes particles, few hundred nanometers in size, derived from the plasma membrane (PM) of K562 feeder cells expressing IL-15 and 41BBL on their surface (PM-mb15-41BBL). These particles in combination with low concentration of IL-2 induce selective and efficient expansion of NK cells within human peripheral blood mononuclear cells (PBMC). When PBMC are stimulated with PM-mb15-41BBL over 21 days the NK cell numbers increase exponentially between days 6 and 18 of culture. The numbers of NK cell increased on average 200 fold (range 104-557, n=11, 4 donors) after 12-13 days of culture in the presence of PM-mb15-41BBL particles (at 200 µg of membrane protein/mL). The expansions with the PM particles are comparable to those in the presence of live feeder cells that gave ∼200 fold (79-895, n=11, 4 donors). The PM-particle based NK expansion is far better in comparison to NK stimulation with soluble purified 41BBL, IL-15 and IL-2, at matching concentrations, that yielded only 3 fold (1-4, n=6, 3 donors) increase in NK cells. Furthermore, the NK cells expand selectively under these conditions where they initially consisted only about 10% of the population of PBMC isolated from fresh peripheral blood, but increased to more than 95% of the cell suspension after 14 days in culture. The extent of expansion and NK cell content on day 12 of culture was dependent on the concentration of PM particles used with 200 µg of PM protein/mL being the optimal dose. Thus, PM nanoparticles can expand NK cells as efficiently and selectively as feeder cells. Furthermore, the PM-particle based expansion is more reproducible between trials and with different donors as compared to NK cell expansion induced with feeder cells (coefficient of variation 63% vs. 88%, respectively). The NK cells expanded in presence of PM-particles were highly cytotoxic against several leukemia cell lines and also against patient derived AML blasts. Expanded NK cells were 4 to 9 times more potent against AML cell lines K562, KG1 and HL-60 as compared to freshly isolated NK cells that were pre-activated with a high dose of IL-2. The PM-particle expanded NK cells also were selectively cytotoxic where they efficiently killed patient derived CD34+ leukemia blasts while sparing healthy CD34- peripheral blood cells. The expanded NK cells were observed to have an increase in the expression of major activating receptors such as NKG2D, NKp44, NKp30 and of the death receptor ligand FasL. This expression difference corresponds well with the activated cytotoxic phenotype and is likely responsible for their increased cytotoxicity against AML cells. Pilot trials in NSG mice are currently ongoing. Disclosures: Solh: Celgene: Speakers Bureau.

scholarly journals Adaptive NK cells can persist in patients with GATA2 mutation depleted of stem and progenitor cells

Blood ◽  
2017 ◽  
Vol 129 (14) ◽  
pp. 1927-1939 ◽  
Author(s):  
Heinrich Schlums ◽  
Moonjung Jung ◽  
Hongya Han ◽  
Jakob Theorell ◽  
Venetia Bigley ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Nk Cell ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Stem And Progenitor Cells

Key Points GATA-2 expression is restricted to hematopoietic stem and progenitor cells, leading to NK-cell progenitor deficiency in GATA2 patients. A long-lived or self-renewing pool of adaptive NK cells can persist in patients with GATA2 mutation.

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

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

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

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