Pre‐clinical development of a cryopreservable megakaryocytic cell product capable of sustained platelet production in mice

Transfusion ◽  
2019 ◽  
Vol 59 (12) ◽  
pp. 3698-3713 ◽  
Author(s):  
Ami Patel ◽  
Cara Marie Clementelli ◽  
Danuta Jarocha ◽  
Gohar Mosoyan ◽  
Cindy Else ◽  
...  
Keyword(s):  
Clinical Development ◽  
Platelet Production ◽  
Cell Product ◽  
Megakaryocytic Cell

scholarly journals Large, chronic doses of erythropoietin cause thrombocytopenia in mice [see comments]

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 352-358 ◽  
Author(s):  
TP McDonald ◽  
RE Clift ◽  
MB Cottrell
Keyword(s):  
Production Rates ◽  
Short Term ◽  
Cell Competition ◽  
Mean Cell Volume ◽  
Platelet Production ◽  
Platelet Counts ◽  
Chronic Doses ◽  
Blood Volumes ◽  
Megakaryocytic Cell

Abstract Both large, acute doses of erythropoietin (EPO) and short-term hypoxia increase platelet counts in mice, but long-term hypoxia causes thrombocytopenia. Therefore, we tested the hypothesis that EPO injected in large, chronic doses (a total of 80 U of EPO over a 7-day period) might cause thrombocytopenia. EPO caused increased red blood cell (RBC) production, ie, increased hematocrits, RBC counts, mean cell volume (MCV), and reticulocyte counts (from P less than .05 to P less than .0005), and decreased thrombocytopoiesis, ie, decreased platelet counts, percent 35S incorporation into platelets, and total circulating platelet counts (TCPC) (P less than .0005). Femoral marrow megakaryocyte size was unchanged, but megakaryocyte number was significantly (P less than .005) reduced in mice treated with EPO. EPO- injected mice had increased spleen volumes (P less than .0005), but blood volumes (BV) were unchanged. In EPO-treated, splenectomized mice, RBC production was also increased (P less than .05 to P less than .0005) and platelet counts, TCPC, and percent 35S incorporation into platelets were decreased (P less than .05), but BV was not altered. Therefore, the decrease in platelet counts observed in EPO-treated mice was not due to increased BV or to an enlarged spleen. In other experiments, mice were rendered acutely thrombocytopenic to increase thrombocytopoiesis, and platelet and RBC production rates were determined. In mice with elevated thrombocytopoiesis, RBC counts, hematocrits, percent 59Fe RBC incorporation values, and MCV were decreased (P less than .05 to P less than .0005). Because 59Fe RBC incorporation and MCV were not elevated, the decrease in RBC counts and hematocrits does not appear to be due to bleeding. Therefore, we show that large, chronic doses of EPO increase erythropoiesis and decrease thrombocytopoiesis. Conversely, acute thrombocytopenia causes increased thrombocytopoiesis and decreased erythropoiesis. These findings support the hypothesis of competition between precursor cells of the erythrocytic and megakaryocytic cell lines (stem-cell competition) as the cause of thrombocytopenia in EPO-treated mice and the cause of anemia in mice whose platelet production rates were increased.

scholarly journals Large, chronic doses of erythropoietin cause thrombocytopenia in mice [see comments]

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 352-358 ◽  
Author(s):  
TP McDonald ◽  
RE Clift ◽  
MB Cottrell
Keyword(s):  
Production Rates ◽  
Short Term ◽  
Cell Competition ◽  
Mean Cell Volume ◽  
Platelet Production ◽  
Platelet Counts ◽  
Chronic Doses ◽  
Blood Volumes ◽  
Megakaryocytic Cell

Both large, acute doses of erythropoietin (EPO) and short-term hypoxia increase platelet counts in mice, but long-term hypoxia causes thrombocytopenia. Therefore, we tested the hypothesis that EPO injected in large, chronic doses (a total of 80 U of EPO over a 7-day period) might cause thrombocytopenia. EPO caused increased red blood cell (RBC) production, ie, increased hematocrits, RBC counts, mean cell volume (MCV), and reticulocyte counts (from P less than .05 to P less than .0005), and decreased thrombocytopoiesis, ie, decreased platelet counts, percent 35S incorporation into platelets, and total circulating platelet counts (TCPC) (P less than .0005). Femoral marrow megakaryocyte size was unchanged, but megakaryocyte number was significantly (P less than .005) reduced in mice treated with EPO. EPO- injected mice had increased spleen volumes (P less than .0005), but blood volumes (BV) were unchanged. In EPO-treated, splenectomized mice, RBC production was also increased (P less than .05 to P less than .0005) and platelet counts, TCPC, and percent 35S incorporation into platelets were decreased (P less than .05), but BV was not altered. Therefore, the decrease in platelet counts observed in EPO-treated mice was not due to increased BV or to an enlarged spleen. In other experiments, mice were rendered acutely thrombocytopenic to increase thrombocytopoiesis, and platelet and RBC production rates were determined. In mice with elevated thrombocytopoiesis, RBC counts, hematocrits, percent 59Fe RBC incorporation values, and MCV were decreased (P less than .05 to P less than .0005). Because 59Fe RBC incorporation and MCV were not elevated, the decrease in RBC counts and hematocrits does not appear to be due to bleeding. Therefore, we show that large, chronic doses of EPO increase erythropoiesis and decrease thrombocytopoiesis. Conversely, acute thrombocytopenia causes increased thrombocytopoiesis and decreased erythropoiesis. These findings support the hypothesis of competition between precursor cells of the erythrocytic and megakaryocytic cell lines (stem-cell competition) as the cause of thrombocytopenia in EPO-treated mice and the cause of anemia in mice whose platelet production rates were increased.

scholarly journals Infusion of a Cryopreservable Human Megakaryocyte-Biased Cell Product Results in Sustained Platelet Reconstitution In Vivo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 117-117
Author(s):  
Ami Patel ◽  
Manisha Kintali ◽  
Gohar Mosoyan ◽  
Ronald Hoffman ◽  
Camelia Iancu-Rubin
Keyword(s):  
Research Funding ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Cost Effective ◽  
Median Number ◽  
In Vivo Studies ◽  
Platelet Production ◽  
Cell Product ◽  
Cd34 Cells

Abstract The demand for platelet (PTL) transfusions has steadily increased, straining a supply that is limited by its dependency on donors, short lifespan, risk of infections and alloimmunization. This stimulated the search for alternative PTL sources including PTLs generated ex vivo from primary CD34+ cells and immortalized pluripotent stem cells. These approaches, however, are associated with obstacles such as scalability and encounter identical limitations as donor PTLs: short shelf life, storage at ambient temperature and limited lifespan after infusion. These obstacles lead us to focus our efforts on not producing PLTs but rather a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTLs after transfusion into patients. Umbilical cord blood units (CBU) are readily available sources for stem cell for transplantation. We created an efficient and cost effective culture system in which CB-derived CD34+ cells are first expanded then allowed to mature into MKs. Initially, we determined the optimal culture period (10-11 days) resulting in the greatest number of CD41+/CD42b- and CD41+/CD42b+ MKs which are capable of PTL production. Next, we used research and clinical grade CBU to generate clinically relevant doses of MK. The median number of CD34+ cells selected from one CBU was 2.5x106 with a purity of 90% (n=4). Following expansion and MK maturation, these cells generated 5.8x107 viable total nucleated cells (TNC)/CBU. Out of these, 3.3 x 107 were CD41+ MKs which corresponds to a median cell dose of 4.1x105 CD41+ cells/Kg of body weight. 92% of CD41+ MKs were mature CD42b+ cells which we previously showed that are capable of ex vivo platelet production. Finally, we performed clonogenic assays and found that one CBU can generate ~1.5x106 CFU-MK. One half of these MK-biased cultures was characterized and assessed immediately after culture and the other half was cryopreserved. The fresh product was infused into sublethally irradiated NSG mice and the presence of human PTLs in the mouse peripheral blood (mPB) was evaluated weekly for 8 weeks at which time the animals were also analyzed for hMK chimerism in the bone marrow (BM), spleen (SP) and lung. The results demonstrate that 87% of animals (13 out of 15) had a robust hPTLs population in their PB. hPTL were detected as early as week 1 post infusion and their number peaked on week 4 (median, 6x103 hPTL/μl) after which it plateaued. The release of hPTL in the mPB was accompanied by the presence of hCD41+ MKs in the mBM, SP and the lung indicating that the infused cells provided both early hPTL release and a reservoir of MK precursors for continuous hPTL production. We also found that in addition to MKs, these same organs contained hCD34+, CD45+ and myeloid CD15+ cells. These findings underscore the capabilities of this product which might have broader clinical applicability such as utilization during myeloablative or suppressive chemo/radiotherapy to improve the time and duration for both neutrophil and platelet engraftment. Equally important, we provide novel evidence that the lung is a site for hMK engraftment after transplantation, in line with recent reports recognizing the pulmonary bed as site for platelet production in the mouse. The major advantage of developing a MK-based product over ex vivo generated PTLs is the amenability of the former to cryopreservation thus becoming a readily available cellular therapy which would be amenable to stock-piling. Therefore, portions of the same MK products described above were cryopreserved then subjected to ex vivo and in vivo studies identical to these performed on their fresh counterparts. Following thawing, the average recovery rate was 71% for TNC and 74.3% for CD41+ cells. MK phenotype and morphology as well as the number CFU-MK generated ex vivo were identical to that found in the fresh product. Although the number of TNC in the thawed product was lower than that of its fresh counterpart, the number of hPTL detected after its infusion ranged from 0.4 to 20.5x103 hPTL/μl which is comparable to that detected after infusion of the fresh equivalent, 0.7-16x103 hPTL/μl. In summary, we created a potent transfusable MK cell product that provides robust and sustained PTL and hematopoietic engraftment in vivo and maintains this capability after cryopreservation. Clinical development of such product is now being pursued for the treatment of thrombocytopenia in acute leukemia patients undergoing chemotherapy. Disclosures Hoffman: Summer Road: Research Funding; Janssen: Research Funding; Formation Biologics: Research Funding; Merus: Research Funding; Incyte: Research Funding. Iancu-Rubin:Incyte: Research Funding; Merck: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding.

scholarly journals Multiple Malignant Meningiomas Presenting As Thrombocytopenia

2019 ◽  
Vol 4 (5) ◽  
Keyword(s):  
Mass Effect ◽  
Clinical Development ◽  
Neurological Deficits ◽  
Careful Examination ◽  
Malignant Meningioma ◽  
Dynamic Changes ◽  
Who Grade ◽  
Platelet Production ◽  
Who Grade Iii ◽  
Grade Iii

Backgrounds: Malignant meningiomas are CNS tumors arising from the arachnoids cap cells of the meninges, very rare in infants. Clinically, intracranial hypertension or focal neurological deficits are usually seen for mass effect, rather than leukemoid symptoms. Methods: Retrospectively analyze the detailed clinical development, diagnosis and treatment of a 10-month-old boy initially hospitalized due to leukemoid symptoms. After careful examination, malignant meningioma (WHO grade III) was proved by the biopsy and histopathology. Chemotherapy (three cycle of ifosfamide 100 mg/kg for 3 days, plus doxorubicin 1 mg/ kg for 2 days every 21 days) in combination with imatinib. Results: Dura nodules significantly reduced in size, skin bleeding spots, thrombocytopenia and enlarged superficial lymph almost disappeared. Conclusion: This study was conducted to demonstrate dynamic changes after effective individualized treatment. Meanwhile, we proposed that the invasiveness of meningioma induces somatic DNA damage, leading to abnormal platelet production and megakaryocytic morphology.

Pre-clinical development and molecular characterization of an engineered type 1 regulatory T-cell product suitable for immunotherapy

Cytotherapy ◽  
2021 ◽  
Author(s):  
Jeffrey Mao-Hwa Liu ◽  
Ping Chen ◽  
Molly Javier Uyeda ◽  
Brandon Cieniewicz ◽  
Ece Canan Sayitoglu ◽  
...  
Keyword(s):  
T Cell ◽  
Molecular Characterization ◽  
Regulatory T Cell ◽  
Clinical Development ◽  
Cell Product

Potential role of APRIL as autocrine growth factor for megakaryocytopoiesis

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3169-3172 ◽  
Author(s):  
Désirée Bonci ◽  
Michael Hahne ◽  
Nadia Felli ◽  
Cesare Peschle ◽  
Ruggero De Maria
Keyword(s):  
Cell Differentiation ◽  
Growth Factor ◽  
Potential Role ◽  
Autocrine Growth ◽  
Platelet Production ◽  
Healthy Donors ◽  
Necrosis Factor ◽  
Megakaryocytic Cell ◽  
Platelet Formation

Abstract A proliferation-inducing ligand (APRIL) is a new tumor necrosis factor family member implicated in tumor cell proliferation. We investigated the role of APRIL in megakaryocytopoiesis, a developmental hematopoietic process responsible for progenitor cell differentiation to megakaryoblasts and megakaryocytes, leading to platelet formation. APRIL is not expressed in CD34+ progenitor cells from healthy donors, but it is massively up-regulated during the proliferative phase of megakaryocytic cell differentiation. Exogenous APRIL expression in primary cells increases megakaryocytic cell growth, suggesting that APRIL acts as a proliferative factor in megakaryocytopoiesis. More importantly, neutralization of endogenous APRIL was able to dramatically reduce megakaryocyte expansion and platelet production. Thus, our data provide evidence that APRIL acts as a growth factor for terminal megakaryocytopoiesis and may promote physiologic platelet production.

Interleukin 10-induced thrombocytopenia in normal healthy adult volunteers: evidence for decreased platelet production

2000 ◽  
Vol 111 (1) ◽  
pp. 104-111 ◽  
Author(s):  
Jeffrey A. Sosman ◽  
Amit Verma ◽  
Steven Moss ◽  
Patricia Sorokin ◽  
Michael Blend ◽  
...  
Keyword(s):  
Healthy Adult ◽  
Interleukin 10 ◽  
Platelet Production ◽  
Adult Volunteers

COVID-19 must catalyse changes to clinical development

2020 ◽  
Vol 19 (10) ◽  
pp. 653-654
Author(s):  
Rod MacKenzie ◽  
Peter Honig ◽  
Judy Sewards ◽  
Robert Goodwin ◽  
Marie-Pierre Hellio
Keyword(s):  
Clinical Development

Curcumin: New trends in the formulative and clinical development

Planta Medica ◽  
2014 ◽  
Vol 80 (10) ◽  
Author(s):  
P Morazzoni ◽  
A Riva ◽  
M Ronchi ◽  
G Petrangolini ◽  
W Cabri
Keyword(s):  
Clinical Development

Expression of Platelet Membrane Glycoprotein V in Human Megakaryocytes and Megakaryocytic Cell Lines: A Study Using a Novel Monoclonal Antibody against GPV

1994 ◽  
Vol 72 (05) ◽  
pp. 762-769 ◽  
Author(s):  
Toshiro Takafuta ◽  
Kingo Fujirmura ◽  
Hironori Kawano ◽  
Masaaki Noda ◽  
Tetsuro Fujimoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Immunohistochemical Analysis ◽  
Specific Protein ◽  
Platelet Membrane ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Flow Cytometric ◽  
Polymerase Chain ◽  
Megakaryocytic Cell

SummaryGlycoprotein V (GPV) is a platelet membrane protein with a molecular weight of 82 kD, and one of the leucine rich glycoproteins (LRG). By reverse transcription-polymerase chain reaction (RT-PCR), GPV cDNA was amplified from mRNA of platelets and megakaryocytic cell lines. However, since there are few reports indicating whether GPV protein is expressed in megakaryocytes as a lineage and maturation specific protein, we studied the GPV expression at the protein level by using a novel monoclonal antibody (1D9) recognizing GPV. Flow cytometric and immunohistochemical analysis indicated that GPV was detected on the surface and in the cytoplasm of only the megakaryocytes in bone marrow aspirates. In a megakaryocytic cell line UT-7, GPV antigen increased after treatment with phorbol-12-myri-state-13-acetate (PMA). These data indicate that only megakaryocytes specifically express the GPV protein among hematopoietic cells and that the expression of GPV increases with differentiation of the megakaryocyte as GPIb-IX complex.

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