Establishment and Characterization of a Human Primary Myelofibrosis Cell Line

Abstract Aims: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal proliferation of stem cells and myeloid cells. JAK2, CARL, and MPL mutations could be detected in most patients, but 15% of PMF patients could be absent of these three gene mutations. PMF has shorter median survival time and poorer prognosis when compared with closely related MPNs, polycythemia vera (PV) and essential thrombocythemia (ET). Allogenic hematopoietic stem cells (allo-HSCT) is the only curative therapy for PMF patients, however transplantation-related complications and deaths were observed in more than 50% patients. Hydroxyurea and Ruxolitinib are commonly used drugs to improve clinical symptoms of PMF patients, but lack of anti-tumor activities. These drugs could not reverse bone marrow fibrosis or induce cytogenetic remission. Basic research work of PMF is limited as to the lack of human PMF cell line. Here, we aimed to establish and characterize a patient-derived human PMF cell line, which would provide a useful tool for PMF research and screenings for novel drugs. Methods: The bone marrow cells were obtained from a 61-year-old male patient, who was diagnosed as PMF for 2 years. Mono-nuclear cells were isolated and cultured in a continuous culture system. The cells were characterized by different methods, including STR profiling, morphology observation with Giemsa staining, flow cytometry, chromosome analysis, Epstein-Barr virus (EBV) detection, mycoplasma detection, whole-genome-exon sequencing, and cell proliferation assay. Results: The cells have continued to grow in liquid culture for more than 50 passages using the same culture conditions. Doubling time was about 48-72 hours. The STR profiling identified that the cultured cells and the patient's bone marrow cells were from the same origin. The cells are free from EBV and mycoplasma. Giemsa-Wrights staining showed primitive erythrocytes (Figure A), which was consistent with CD71 positive detected by flow cytometry. Chromosome analysis revealed a hyperdiploid karyotype, which was 47, XY,+12[1]/48,idem,+9[9]. Whole-genome-exon sequencing identified mutations of ASXL1, TP53, IKZF1, IDH1, FLT3, and TET1 (Figure B). Now this cell line, designated ZYXY-M2, is collected by China center for type culture collection (CCTCC). Discussion: Established cell lines are invaluable tools in cell biology. Right now, HEL and SET-2 are two cell lines that are most widely used in MPN-related researches. HEL is an erythroleukemia cell line which was derived from a patient with leukemia transformation of Hodgkin's disease, and SET-2 is a megakaryoblastic cell line which was derived from a patient with leukemic transformation of ET. Here, we established a cell line, ZYXY-M2, derived from a PMF patient and it would prove to be a valuable model for the study of PMF. ZYXY-M2 could help to explore the pathogenesis and development of PMF, novel drug screening, and may also help to set up xenograft PMF mouse models. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Detection of Human Telomerase Reverse Transcriptase (hTERT) Protein Expression in Bone Marrow Cells of Hematological Disorder by Flow Cytometry.

Blood ◽

10.1182/blood.v104.11.4337.4337 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4337-4337

Author(s):

Hiroshi Handa ◽

Hirokazu Murakami ◽

Takafumi Matsushima

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Disease Progression ◽

Cell Line ◽

Bone Marrow Cells ◽

Telomerase Activity ◽

Expression Level ◽

Htert Expression ◽

Human Telomerase

Abstract Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. Recently, it has been reported that telomerase activity level was correlated with the progression and prognosis of hematopoietic malignancies, thus, it is important to analyze telomerase activity. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and unable to isolate specific cell sub-populations. We developed immuno-fluorescence flow cytometry based assay to detect hTERT expression using a monoclonal antibody. This method allows sub-population of cells to be separated according to hTERT expression more easily, combining antibody against cell surface antigen. Expression level of hTERT in T cell leukemia cell line Jurkat and myeloma cell line RPMI8226, KMS12PE, KMS28PE, were very high and well correlated in between this method and real time quantitative PCR. Human mature granulocytes supposed to have no telomerase activity did not show any hTERT expression in this flow-cytometric assay. Immuno-histochemistry demonstrated the specific nuclear expression of hTERT. Bone marrow samples were obtained from 8 MDS patients (5 RA, 1 RAEB, 2 RAEB-t) and analyzed hTERT isolating MDS blast cells. In the blast population, cells expressing hTERT were 33.1% (21.4–40.3) in RA patient sample, while those in RAEB and RAEB-t patients sample were significantly higher at 75.1% (68.2–81.9). When CD34 positive hematopoietic stem cells were analyzed, the cells expressing hTERT were higher in RAEB and RAEB-t than in RA. Mature granulocyte population in bone marrow cells did not show any positivity. Our result suggests that hTERT expression and telomerase activity in the MDS blasts or CD34 positive stem cells were up-regulated during the disease progression and the high expression level of hTERT and telomerase activity were not due to the blast expansion. This technique allows us to easily analyze hTERT expression in hematopoietic malignancy even though the malignant cells were very small in bone marrow sample, e.g. MDS, MM or MGUS of which telomerase activity were supposed to be correlated with the disease progression and prognosis.

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Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽

10.1177/0961203317711013 ◽

2017 ◽

Vol 27 (1) ◽

pp. 49-59 ◽

Cited By ~ 10

Author(s):

X Yang ◽

J Yang ◽

X Li ◽

W Ma ◽

H Zou

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Lupus Nephritis ◽

Bone Marrow Cells ◽

Tfh Cells ◽

Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

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Loss of P-Selectin Promotes the Function of Aged Hematopoietic and Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1577.1577 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1577-1577

Author(s):

Yaoyu Chen ◽

Sullivan Con ◽

Yiguo Hu ◽

Linghong Kong ◽

Cong Peng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Bone Marrow Cells ◽

Leukemic Stem Cells ◽

Wild Type ◽

Post Transplant ◽

Marrow Cells

Abstract Abstract 1577 Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between the blood cells and their microenvironment. Adhesion molecules like P-selectin are essential to hematopoiesis, and their dysregulation has been implicated in leukemogenesis. We have previously shown a role for P-selectin in chronic myeloid leukemia and demonstrated that in its absence the disease process accelerates. Recently, there has also been speculation that P-selectin may play a role in the aging hematopoietic stem cells (HSCs), as its expression in upregulated as a mouse ages. In this study, we show that the loss of P-selectin function dysregulates the balance of stem cells and progenitors and that these differences become more pronounced with age. We compared the percentages of HSCs, long-term (LT)-HSCs, short-term (ST)-HSCs, multipotent progenitors (MPPs), CMPs, GMPs and MEPs in bone marrow by flow cytometry between wild type (WT) and Selp-/- mice. An age-dependent LT-HSC expansion was observed in WT mice. However, this expansion was prevented by the loss of Selp as observed in Selp-/-mice. Further, we demonstrate that with age LT-HSCs in particular express more elevated levels of P-selectin. LT-HSCs and ST-HSC/MPPs were isolated from the bone marrow of young (2 months old) and old (15 months old) WT mice and examined P-selectin expression by FACS. A significant increase in P-selectin expression was observed in LT-HSCs of old mice, and this increase was not observed in the ST-HSC+MPP subpopulations. We also show that the loss of P-selectin gene has profound effects of stem cell function, altering the capacity of these cells to home. Despite impaired homing capacity, stem cells lacking P-selectin possess a competitive advantage over their wild type counterparts. Using a stem cell competition assay, HSCs derived from Selp-/- mice (CD45.2+) and WT control mice (CD45.2+GFP+) were mixed in 1:1 ratio and transplanted into irradiated WT recipients (CD45.1). The initial findings were potentially indicative of the ability of cells derived from GFP mice to more efficiently home and engraft. Despite this initial advantage, cells derived from Selp-/- eventually exhibited a competitive and statistically significant advantage over the cells derived from GFP mice. At 30 days post-transplant, 49.9±1.4% of the CD45.2 subpopulation was GFP+. At 86 days post-transplant, 25.7±3.3 % of the CD45.2 cells derived from the peripheral blood were GFP+. Similarly, 23.0±3.7% of the CD45.2 cells derived from the bone marrow of these mice were GFP+. Indeed, we demonstrate that recipients of P-selectin deficient bone marrow cells more efficiently repopulate the bone marrow than controls and that this advantage extends and expands in the long-term. Finally, we demonstrate that recipients of leukemic cells lacking P-selectin develop a more accelerated form of leukemia accompanied by significant increases in stem and progenitor cells. Bone marrow cells from donor WT and Selp-/- mice were infected with retrovirus expressing BCR-ABL-GFP, and irradiated WT recipients were transplanted with 2×105 of these transduced donor cells. At 14 days post-transplant, recipient mice from each of the groups were sacrificed, and bone marrow cells were harvested and analyzed by flow cytometry. Recipients of leukemic Selp-/- cells possessed 3.5-fold more LSCs than recipients of wild-type cells. There were 3.1-fold more LT-LSCs and 3.8-fold more ST-LSCs and MPPs in recipients of Selp-/- cells than WT cells. In addition, recipients of leukemic Selp-/- cells possessed significantly more CMP (16.9-fold) and MEP (4.5-fold) cells. Because P-selectin expression increases with age on LT-HSCs, we sought to determine the role that age plays in CML development and progression. Bone marrow cells derived from 15-month-old donor Selp-/- and WT mice were transduced with BCR-ABL, respectively, followed by transplantation of the transduced cells into recipient mice. All recipients of BCR-ABL transduced Selp-/- cells died by 23 days after induction of CML and had a median survival of 19 days, whereas recipients of the transduced WT cells survived significantly longer. This pro-leukemic role for cells lacking P-selectin expression is leukemic stem cell-specific rather than stromal cell-specific and supports an essential role for P-selectin on leukemic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Treatment with 8F4, An Anti-PR1/HLA-A2 T Cell Receptor-Like Antibody, Reduces Established Acute Myeloid Leukemia and Eliminates Leukemia Stem Cells in Mice

Blood ◽

10.1182/blood.v118.21.766.766 ◽

2011 ◽

Vol 118 (21) ◽

pp. 766-766

Author(s):

Anna Sergeeva ◽

Hong He ◽

Kathryn Ruisaard ◽

Karen Clise-Dwyer ◽

Lisa S St. John ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Cell Receptor ◽

Leukemia Stem Cells ◽

Nsg Mice ◽

Secondary Transfer

Abstract Abstract 766 PR1 (VLQELNVTV) is an HLA-A2-restricted leukemia-associated peptide from proteinase 3 and neutrophil elastase that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of myeloid leukemia. We developed a high affinity T cell receptor (TCR)-like mouse monoclonal antibody (8F4) that binds to a conformational epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes. Moreover, 8F4 mediated complement dependent cytolysis of AML blasts and Lin−CD34+CD38− leukemia stem cells (LSC), but not normal leukocytes. To investigate in vivo biological effects 8F4 on established leukemia, we established xenografts of primary human HLA-A2-positive AML in sublethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Leukemia engraftment was monitored in peripheral blood by flow cytometry. Mice with established PR1/HLA-A2-expressing leukemia were treated with twice-weekly intravenous injections of 200 μg 8F4 or isotype control antibody. Flow cytometry and histology analysis of tissues was used to assess leukemia burden and level of engraftment. After 5 weeks of treatment AML was reduced 300-fold in bone marrow of 8F4-treated mice compared to isotype-treated control animals (0.07 ± 0.06% hCD45+cells versus 20.4 ± 4.1%, n=5 mice per group). Moreover, leukemia stem cells (LSC, CD34+CD38−Lin-) were no longer detected in bone marrow of 8F4-treated mice, compared to 0.88 ± 0.24% in isotype-treated mice. Equally, AML was evident in the liver and spleen of isotype-treated mice (1.1 ± 0.16% and 0.32 ± 0.17%, respectively), but was undetectable in 8F4-treated mice (p<0.001). Similar results were obtained with AML from two additional patients, one with secondary AML (CMML) and one with AML-M7. Bone marrow contained 6.2 ± 3.0% (n=3) AML versus 41 ± 15% (n=2 mice; p=0.06) in the first case and 0.16 (n=1) versus 7.0 ± 4.1 (n=2) in the second case after 2–3 weeks of twice-weekly injection. To confirm 8F4-mediated elimination of LSC, we performed secondary transfer experiment with 1×106 bone marrow cells from 8F4- and isotype-treated mice, transplanted into recipient NSG mice, irradiated with 250 cGy. AML was undetectable in mice that received bone marrow from 8F4-treated animals versus 4.1 ± 2.4% (n=4) in bone marrow of mice that received cells from isotype- treated mice, determined at 16 weeks after secondary transfer. Because PR1/HLA-A2 expression on normal hematopoietic cells (HSC) is similar to LSC in AML patients, we sought to determine whether 8F4 treatment of NSG mice xenografted with CD34-selected umbilical cord blood resulted in elimination of xenograft. Fourteen weeks after transplant stable chimerism (4.1 - 7.7% hCD45+ cells) was established, mice were treated with 50 μg 8F4 intravenously and peripheral blood was monitored weekly for chimerism. Human CD45+ cells decreased to 0.35 – 0.95% by week 1, but increased to 1.9 – 2.1 % hCD45+ cells at week 3. Bone marrow at week three contained myeloid (CD13+CD33+) and lymphoid (CD19+) cells showing that while 8F4 has off- target effects against normal hematopoietic cells, HSC are preserved. This is consistent with our previous studies that showed no 8F4-mediated effect on colony formation of normal bone marrow cells. In conclusion, these results show that anti-PR1/HLA-A2 monoclonal antibody 8F4 is biologically active in vivo and selectively eliminates LSC, but not normal HSC. This justifies continued study of 8F4 as a novel therapy for AML. Disclosures: No relevant conflicts of interest to declare.

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Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.3513.3513 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3513-3513

Author(s):

David B. Sykes ◽

Mark K Haynes ◽

Nicola Tolliday ◽

Anna Waller ◽

Julien M Cobert ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Small Molecules ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Myeloid Differentiation ◽

Prostacyclin Receptor ◽

Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

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A CD123-Targeting Antibody-Drug Conjugate (ADC), IMGN632, Designed to Eradicate Acute Myeloid Leukemia (AML) Cells While Sparing Normal Bone Marrow Cells

Blood ◽

10.1182/blood.v128.22.768.768 ◽

2016 ◽

Vol 128 (22) ◽

pp. 768-768 ◽

Cited By ~ 6

Author(s):

Yelena Kovtun ◽

Gregory Jones ◽

Charlene Audette ◽

Lauren Harvey ◽

Baudouin Gerard ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Lines ◽

Bone Marrow Cells ◽

Cell Populations ◽

Normal Bone Marrow ◽

Normal Bone ◽

Healthy Donors ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract Current AML therapies are effective in a subset of patients, but often lead to prolonged myelosuppression. CD123 is an attractive AML target due to its elevated expression on AML compared to normal bone marrow cells. Still, severe myelosuppression and myeloablation have been reported in preclinical studies for some CD123-targeted therapies. Here, we present a novel ADC which selectively kills CD123-positive AML cells over normal bone marrow cells. A novel humanized anti-CD123 antibody with two engineered cysteines for payload conjugation was generated. Indolinobenzodiazepine dimers, termed IGNs, were chosen as payload molecules for the antibody due to their high potency against AML cells. The IGN dimers containing mono-imines alkylate DNA, whereas the di-imine containing IGNs can both alkylate and crosslink DNA. To select an optimal IGN payload, we compared the cytotoxicity of an ADC with a mono-imine IGN (A-ADC) to one with a di-imine IGN (C-ADC) on AML cells, as well as normal bone marrow cells in vitro. Potency of the ADCs was evaluated using AML cell lines that have CD123 levels similar to patient cells and carry markers of poor prognosis (FLT3-ITD , MDR1, EVI1, DNMT3A and TP53), as well as on samples from 11 AML patients. AML cells exposed to either ADC displayed markers of DNA damage, cell cycle arrest and apoptotic cell death by flow cytometry. Both ADCs were highly cytotoxic, generating IC50 values between 0.4 to 60 pM on the cell lines in WST-8 assays and killing 90 percent of progenitors from AML patients between 2 to 46 pM in CFU assays. The C-ADC was, on average, two-fold more active than the A-ADC. The cytotoxicity of both ADCs was CD123 dependent, since masking CD123 with a competing anti-CD123 antibody reduced the potency by more than 100-fold. Toxicity of the ADCs was assessed using bone marrow cells from a healthy human donor. The cells were exposed to the ADCs at 100 pM (a concentration highly potent against all AML samples) for 72 hours, and then markers of apoptosis were detected in different cell populations by flow cytometry. Neither ADC affected the viability of monocytes, lymphocytes and multipotential progenitors, consistent with low CD123 levels in these cell populations. In contrast, an apoptotic signal was detected in myeloid progenitors, the population with the highest CD123 level, following exposure to the C-ADC, but not to the A-ADC. The toxicity of the ADCs was also tested in CFU assays on bone marrow cells from 7 healthy donors, as the assays have been reported to predict clinical myelosuppression. Surprisingly, the C-ADC was, on average, 50-fold more cytotoxic to normal myeloid progenitors than the A-ADC (40 pM vs 2,000 pM IC90 values, respectively) (Figure 1). Finally, we compared CD123 independent toxicity of the ADCs in CD-1 mice. The C-ADC showed significantly reduced tolerability, and unlike the A-ADC, was associated with delayed toxicity manifested by weight loss 30 days after administration. Based on its potent yet highly selective toxicity to AML cells and more favorable tolerability profile, the A-ADC was selected for further study, and renamed as IMGN632. To compare IMGN632 to an ADC previously approved for the treatment of AML, the potency of IMGN632 and gemtuzumab ozogamicin (GO) was tested on bone marrow cells from 11 healthy donors and 17 AML patients, including 4 relapsed/refractory and 8 with strong multidrug resistance (Figure 1). Only 6 of 17 AML samples were sensitive to GO at concentrations that did not impact normal progenitors. In contrast, AML progenitors from all 17 patients were highly sensitive to IMGN632. Importantly, normal progenitors were only affected by IMGN632 at 150-fold higher concentrations. The pronounced difference between AML and normal progenitors in their sensitivity to IMGN632 likely reflects both higher CD123 levels on AML progenitors and the lower sensitivity of normal progenitors to the mono-imine IGN payload we observed in CFU assays. In conclusion, through use of a mono-imine IGN payload, IMGN632 demonstrated potent activity in all tested AML samples at concentrations far below levels that impact normal bone marrow cells, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. These findings together with strong efficacy in multiple AML xenograft models (Kovtun et al., 21st EHA congress, 2016; Adams et al., 58th ASH annual meeting, 2016) support advancing IMGN632 into clinical trials. Disclosures Kovtun: ImmunoGen, Inc.: Employment. Jones:ImmunoGen, Inc.: Employment. Audette:ImmunoGen, Inc.: Employment. Harvey:ImmunoGen, Inc.: Employment. Gerard:ImmunoGen, Inc.: Employment. Wilhelm:ImmunoGen, Inc.: Employment. Bai:ImmunoGen, Inc.: Employment. Adams:ImmunoGen, Inc.: Employment. Goldmacher:ImmunoGen, Inc.: Employment. Chari:ImmunoGen: Employment. Chittenden:ImmunoGen, Inc.: Employment.

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Human embryonic stem cell–derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients

Blood ◽

10.1182/blood-2005-05-1922 ◽

2006 ◽

Vol 107 (5) ◽

pp. 2180-2183 ◽

Cited By ~ 93

Author(s):

A. Daisy Narayan ◽

Jessica L. Chase ◽

Rachel L. Lewis ◽

Xinghui Tian ◽

Dan S. Kaufman ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Xenograft Model ◽

Fetal Sheep ◽

Hematopoietic Stem ◽

Hematopoietic Activity

The human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. CD34+/lineage- or CD34+/CD38- cells isolated from human embryonic stem cells (hESCs) differentiated on S17 feeder layer were transplanted by intraperitoneal injections into fetal sheep. Chimerism in primary transplants was established with polymerase chain reaction (PCR) and flow cytometry of bone marrow and peripheral blood samples. Whole bone marrow cells harvested from a primary recipient were transplanted into a secondary recipient. Chimerism was established as described before. This animal was stimulated with human GM-CSF, and an increase in human hematopoietic activity was noted by flow cytometry. Bone marrow aspirations cultured in methylcellulose generated colonies identified by PCR to be of human origin. We therefore conclude that hESCs are capable of generating hematopoietic cells that engraft primary recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the secondary recipient.

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Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains.

Journal of Experimental Medicine ◽

10.1084/jem.178.1.139 ◽

1993 ◽

Vol 178 (1) ◽

pp. 139-149 ◽

Cited By ~ 45

Author(s):

M E Pauza ◽

J A Rehmann ◽

T W LeBien

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Surface ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Gene Rearrangement ◽

Immunoglobulin Gene ◽

Human B Cells ◽

Sds Page

Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow cytometry and were clonal by Southern blotting. Surprisingly, two of the mu/lambda-expressing cell lines contained both kappa alleles in germline configuration, and synthesis/expression of conventional lambda L chains was directly proven by immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in one of them. Thus, human fetal bone marrow B lineage cells harbor the capacity to make functional lambda L chain gene rearrangements without rearranging or deleting either kappa allele. A third unusual cell line, designated 30.30, was observed to coexpress cell surface kappa and lambda L chains associated with mu H chains. The 30.30 cell line had a diploid karyotype, a single H chain rearrangement, both kappa alleles rearranged, and a single lambda rearrangement. Immunoprecipitation/SDS-PAGE confirmed that 30.30 cells synthesized and expressed kappa and lambda L chains. Multiparameter flow cytometry was used to demonstrate the existence of kappa+/lambda+ cells in fetal bone marrow and fetal spleen at frequencies of 2-3% of the total surface Ig+ B cell population. The flow cytometry data was confirmed by two-color immunofluorescence microscopy. The existence of normal human B cells expressing cell surface kappa and lambda refutes the widely accepted concept that expression of a single L chain isotype is immutable. The kappa+/lambda+ cells may represent transients undergoing L chain isotype switching.

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New Monoclonal Antibodies Reactive with Human Bone Marrow-Derived Mesenchymal Stem Cells.

Blood ◽

10.1182/blood.v106.11.4318.4318 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4318-4318

Author(s):

Xiaoyu Lai ◽

He Huang ◽

Yi Luo ◽

Zhixiong Liao ◽

Jiangen Shen

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Monoclonal Antibodies ◽

Cell Lines ◽

Indirect Immunofluorescence ◽

Fluorescent Antibody ◽

Surface Antigens ◽

Hybridoma Cell Lines

Abstract Human mesenchymal stem cells (hMSCs) could be well isolated and expanded from bone marrow and have been widely studied. In addition to hMSCs have ability to support hematopoiesis, they have generated a great deal of excitement as a potential source of cells for regenerative medicine and tissue engineering owing to their dramatic potential of proliferation and differentiation. However, the precise identity of hMSCs remains a challenge and the homogeneity of hMSCs is still controversial due in part to the lack of useful cell-specific markers. Thus, the objective of our study was to development monoclonal antibodies (MAbs) reactive with hMSCs surface antigens. hMSCs were isolated from bone marrow of volunteers on a density gradient solution by Ficoll-paque and cultured (LG-DMEM, 10%FBS) with their characteristic of adherence. hMSCs phenotype were analyzed by flow cytometry and demonstrated that they were uniformly positive for CD29, CD44, CD166 and CD105, negative for CD34, CD45 and HLA-DR. All hMSCs could be successfully induced to differentiate into osteogenic precursor cells, adipocytes and neuron cells when treated by correctly conditioned medium. There were detected to be no significant differences of phenotypes, growth characteristics and the potential of differentiation after either passage or programmed cryopreservation. Cultured and passaged hMSCs were used to immunized BALB/C mice at 1-week interval for 4 times, and their splenocytes subsequently harvested were fused with mouse SP 2/0 myeloma cells in the presence of PEG followed by culturing in the HAT selective medium. Indirect immunofluorescence assay (fluorescent antibody reacted with IgG and IgM isotypes) was utilized to preliminary screen the hybridoma colonies which synthesized and secreted antibodies having the specificity for cultured hMSCs. Out of 1248 wells initially plated, only 27 wells were tested positive for reactivity with living cultured hMSCs, and finally we got 5 hybridoma cell lines which could secreted MAbs steadily, named ZUB1, ZUB4, ZUC3, ZUE12, ZUF10. In order to identify hybridomas which secreted antibodies were only specific for hMSCs, peripheral blood cells, human malignant hematopoietic cell lines and rat marrow-derived mesenchymal stem cells were detected by indirect immunofluorescence analysis and subsequently a series of mesenchymal and non-mesenchymal derived tissues of human were performed by immunohistochemistry staining. The results demonstrated that all these 5 MAbs were specific for hMSCs except that ZUC3 had cross reaction with rat marrow-derived mesenchymal stem cells, and there were a few positive cells in the bone marrow. The subtype of monoclonal antibody ZUC3 was IgM, while other four MAbs were IgG1 and light chains of all MAbs were kappa. Using hybridoma cell lines, large amount of MAbs in ascites were generated then purified. MAbs ZUB1, ZUB4, ZUC3, ZUE12, ZUF10 were used to detected cultured hMSCs by flow cytometry, and percentage of positive cells are 87.39%, 88.07%, 88.12%, 69.89%, 83.67%, respectively. In conclusion, there 5 new MAbs that developed reactived with hMSCs surface antigens. Our study would further the understanding of hMSCs biological characterization and have potential application of new MAbs for identifying, sorting hMSCs derived from bone marrow. Further studies will focus on the characterization and epitopes of the MAbs to hMSCs.

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Effect of Thrombospondin-1 on Apoptosis of Human Megakaryocytic Leukemia Cells

Blood ◽

10.1182/blood-2021-149054 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4351-4351

Author(s):

Huimin Kong ◽

Qianli Jiang ◽

Weiqing Su ◽

Yi Luo ◽

Hui Ge ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Line ◽

Caspase 3 ◽

Bone Marrow Cells ◽

Leukemia Cell Line ◽

Extracellular Matrix Protein ◽

Mouse Bone Marrow ◽

Thrombospondin 1 ◽

Marrow Cells

Abstract Background: Thrombospondin 1 (TSP-1) is an extracellular matrix protein that interacts with a wide array of ligands including cell receptors, growth factors, cytokines, and proteases to regulate various physiological and pathological processes. TSP-1 induces apoptosis of endothelial and cancer cells via its receptor CD36. This study was to investigate the effect of TSP-1 on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. Methods: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations, to investigate the effect of TSP-1 on the growth of megakaryocytes. The early apoptosis and the activity of the apoptotic protease caspase-3 were detected by flow cytometry. Bone marrow cells of mice were cultured for CFU-MK and stained with acetylcholine to detect the differentiation of megakaryocytes. Results: CD36 antigen was detected on the surface of Meg-01 cells by flow cytometry and immunocytochemistry. TSP-1 (5 μg/mL) inhibited the proliferation of Meg-01 cells, but not M-07e cells (CD36 -). After the addition of CD36 antibody FA6-152 (5, 10 and 25 μg/mL), the inhibitory effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5 and 7.5 μg/mL) exerted a pro-apoptotic effect by increasing the expression of Annexin V (P<0.01) and caspase-3 activation (P<0.01). Addition of FA6-152 (25 μg/mL) can significantly reduce the apoptosis induced by TSP-1 in Meg-01 cells. In addition, TSP-1 (5, 10 and 25 μg/mL) repress the formation of CFU-MK in mouse bone marrow cells, while β-TG did not. This repression was relieved efficiently by FA6-152 (25 μg/mL). Conclusion: TSP-1 could inhibit the proliferation of Megakaryocyte cell line Meg-01 cells, and induce it`s apoptosis. TSP-1 may induce apoptosis of Meg-01 cells via CD36 or caspase-3, which provides a potential new drug choice for clinical treatment of megakaryocytic leukemia. Disclosures No relevant conflicts of interest to declare.

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