Recapitulation of Transient Abnormal Myelopoiesis Using Patient Derived iPSCs

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4115-4115
Author(s):  
Yoko Nishinaka-Arai ◽  
Akira Niwa ◽  
Mitsujiro Osawa ◽  
Tatsutoshi Nakahata ◽  
Megumu K Saito
Keyword(s):  
Progenitor Cells ◽  
Trisomy 21 ◽  
Hematopoietic Cells ◽  
Chromosome 21 ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Transient Abnormal Myelopoiesis ◽  
Gata1 Mutation

Abstract Down syndrome (DS) is characterized by the trisomy of chromosome 21 and complicated with multi-organ dysfunctions including the hematopoietic system. Among them, myeloproliferative disorder is known as a particular feature of the abnormality in hematopoiesis. At birth, about 10% of DS newborns show an extreme increase in blast cell number of peripheral blood and bone marrow, which is called transient abnormal myelopoiesis (TAM), because the blasts spontaneously disappear within 3 months. Morphologically, blast cells in TAM are similar to those typically found in acute megakaryoblastic leukemia (AMKL). Genetic analysis of blasts in TAM usually shows mutation in GATA1 gene. After spontaneous remission of TAM, 20 to 30% of TAM patients develop AMKL within several years. This type of AMKL is especially called DS related-AMKL (DS-AMKL). This leukemogenic transition from TAM to AMKL is considered to be a typical model of multi-step tumorigenesis. In this model, we deal with the initial part of TAM development in relationship with trisomy 21 and GATA1 mutation, especially focusing on how GATA1 mutation promotes TAM development and why hematopoietic progenitors with GATA1 mutation prevail during embryonic hematopoiesis only in the cells with trisomy 21. In order to address these unsolved issues, we have established a strictly controlled human induced pluripotent stem cell (iPSC) lines derived from DS patients with or without TAM. In this study, to recapitulate the phenotype of TAM and to specify the differentiation stage affected in hematopoietic cells in TAM patients, we differentiated established isogenic iPSC lines into megakaryocytes and erythrocytes in a step-wise manner. For this purpose, we employed two-dimensional differentiation system and compared the frequency of hematopoietic progenitor cells at various stages. For megakaryocytic lineage, we traced their differentiation as follows; hematopoietic progenitor cells committed to megakaryocytic lineage (day 9, CD34+CD41a+CD42b-CD235a-), megakaryoblasts (day 16, CD34-CD41a+CD42b-CD235a-) and promegakaryocytes (day 16, CD34-CD41a+CD42b+CD235a-). For erythrocytic differentiation, CD71+CD235a+ cells were defined as erythroid-committed hematopoietic cells. On nine days after the initiation of hematopoietic differentiation (day 9), the frequency of CD41a+CD235a- cells showed no significant differences irrespective of the status of chromosome 21 and GATA1 genotype. However, on the day 16, while the frequency of promegakaryocytes significantly decreased in GATA1-mutated iPSCs, megakaryoblasts, an earlier stage cells than promegakaryocytes, were increased in GATA1-mutated iPSCs. These data suggest that megakaryocytic maturation is arrested in GATA1-mutated iPSCs at the stage of megakaryoblasts. In GATA1-non-mutated clones, iPSCs with trisomy 21 yielded erythroid-committed CD71+CD235a+ cells more frequently than those with disomy 21. However, in GATA1-mutated clones, either trisomy 21 or disomy 21 iPSC clones never yielded the erythroid-committed cells. Taken these results together, we suspected that these in vitro phenotypes observed in both erythroid and megakaryocytic lineages were caused by the impairment of fate decision in their progenitor cells. In conclusion, we successfully recapitulated the phenotypes of TAM in vitro in regard to the abnormal differentiation into megakaryocytic and erythroid lineages. We noticed that the in vitro phenotype were associated with the GATA1 genotype and the ploidy of chromosome 21. Considering these results, analyses of the megakaryocytic and erythroid progenitor cells, such as CMP, MEP and Mk-p, are important to determine which stage of progenitors is responsible for the impairment of hematopoietic cell maturation and subsequent TAM development. Moreover, we believe that the recapitulated TAM model using iPSCs is helpful for the comprehensive understanding of pathogensis in TAM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2436-2443 ◽  
Author(s):  
MC Yoder ◽  
VE Papaioannou ◽  
PP Breitfeld ◽  
DA Williams
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Yolk Sac ◽  
Hematopoietic Progenitor Cells ◽  
Visceral Endoderm ◽  
Hematopoietic Progenitor

Abstract The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T- antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac- derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.

Cell-to-Cell Contact Is Critical for the Survival of Hematopoietic Progenitor Cells on Osteoblasts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1285-1285
Author(s):  
Jianhua Wang ◽  
Russell Taichman ◽  
Younghun Jung ◽  
Aaron Havens ◽  
Yanxi Sun ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Intracellular Signaling ◽  
Hematopoietic Cells ◽  
Differentially Expressed ◽  
Hematopoietic Progenitor Cells ◽  
Cell Contact ◽  
Type I ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Osteoblasts constitute part of the stromal cell support system in marrow for hematopoiesis, however little is known as to how they interact with hematopoietic stem cells (HSCs). In vitro studies have demonstrated that the survival of HSCs in co-culture with osteoblasts requires intimate cell-to-cell contact. This suggests that the osteoblast-derived factor(s) that supports stem cell activities are either produced in very small quantities, are rapidly turned over, may be membrane-anchored and/or requires the engagement of cell-cell adhesion molecules yet to be determined. In the present report we found that survival of hematopoietic progenitor cells on osteoblasts is dependent upon the engagement of VLA-4 (α4β1) and VLA-5 (α5ß1) receptors using function blocking antibodies. Surprisingly, cell-to-cell contact is not absolutely required to support progenitor activity, but does not require receptor-ligand engagement of the VLA-4 and LFA-1 complexes, which can in part be replaced through the use of recombinant ligands (fibronectin, ICAM-1, VCAM-1). Moreover conditioned once these receptors were engaged, medium derived from HSCs grown on osteoblasts ligands supported significantly greater hematopoietic progenitors in vitro than did osteoblast-conditioned or HSC-conditioned medium alone. As an initial attempt to identify the activity we examined which genes are activated following the establishment of osteoblast-CD34+ cell co-cultures nine separate co-cultures were establsihed and the RNA was pooled and analyized on Affymetrix HG-U133A chips at 24 hours. Initially our analysis revealed that there were 259 genes that are up regulated at 24 hours, and 14 genes that are down regulated. Inspection revealed that 30 of these signals were repeated at least once suggesting that 206 genuine gene candidates were differentially expressed resulting from the co-culture. A significant proportion of the differentially expressed cDNAs represent intracellular signaling ligands 16.5% (n=34) and cell surface receptors 13.5% (n=28). Molecules associated with assembly of the extra cellular matrix or its degradation comprised 7.2% (n=15) of the differentially up regulated molecules. Molecules associated with intracellular signaling, novel sequences and intermediate metabolism comprised the majority of the remaining activities. Amoung the candidates of extra cellular signaling molecules, we noted that IL-6, LIF, MIP-1alpha and SDF-1 were identified in the microarray analysis. This observation was most gratifying as we had previously reported that IL-6, LIF and MIP-1α activities are critical components of an HSC-osteoblast microenvironment. Other notable cytokine messages for BMP-2, CCL7, FGF2b, GRO1α, GRO3, IGF1, IL1ß, IL-8, IL-11, LIF, PDGF-D and the receptors for CCL7 (CCR7). Elevations in mRNA for fibronectin, lysine hydroxylase-like proteins, laminin and Type I collagen suggest that the presence of hematopoietic cells also induces osteoblastic activities. While the identity of those molecules present in the co-cultured medium remain to be identified, the data suggests that hematopoietic cells cooperate with osteoblasts to assemble the various marrow microenvironments by directing the synthesis of osteoblast-derived cytokines to improve HSC survival.

scholarly journals Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2436-2443 ◽  
Author(s):  
MC Yoder ◽  
VE Papaioannou ◽  
PP Breitfeld ◽  
DA Williams
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Yolk Sac ◽  
Hematopoietic Progenitor Cells ◽  
Visceral Endoderm ◽  
Hematopoietic Progenitor

The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T- antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac- derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.

Bone Marrow Endothelial Cell Support of Primitive Hematopoietic Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3573-3573
Author(s):  
Xiaoying Zhou ◽  
Dilani Rosa ◽  
Cynthia Cunningham ◽  
Gregor B. Adams
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Functional Role ◽  
Hematopoietic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Vascular Niche

Abstract Hematopoietic stem cells (HSCs) in the bone marrow (BM) reside in specialized microenvironments known as the stem cell niche. Many reports have found the HSCs to be resident next to the endosteal surface of bone where cells of the osteoblastic lineage are a key component of the so-called endosteal niche. However, HSCs have also been found to reside adjacent to sinusoidal blood vessels. These observations have led to the proposal that HSCs in the adult BM may also reside in a vascular niche. However, the functional role of the vascular niche in hematopoiesis remains to be determined. We wished to evaluate the role that BM endothelial cells (BMECs) play in HSC physiology. To examine this we cultured BMEC-enriched cells in vitro, identified by expression of CD31, Tie-2, VE-cadherin and LDL uptake. We compared these cells to spleen derived ECs and BM stromal cells (BMSCs) in their ability to support primitive hematopoietic cells for extended periods in in vitro culture. We found that BMECs were superior in their ability to support the cobblestone area forming cell activity of VEGF-R1+ HSCs than spleen ECs or BMSCs. We also found that the number of cobblestone area cells was markedly reduced when VEGF-R1− HSCs were cultured on any of the supportive cell layers, however this may be due to an intrinsic difference between these cells as a much higher proportion of VEGF-R1+ HSCs were found to be in the G0 phase of the cell cycle than VEGF-R1− cells. To evaluate the supportive role of BMECs, spleen ECs or BMSCs on hematopoietic progenitor cells (HPC) we cultured purified primitive cells on these supportive layers and the total number of colony-forming unit-culture (CFU-C) cells were examined after 4-days or 7-days co-culture with the feeder cells. The results showed that BMECs or spleen ECs can promote the generation of CFU-C from VEGFR1+ HSCs or VEGFR1− HSCs, yet the tot al number of CFU-C produced from VEGFR1+ HSCs was greater than that from VEGFR1- HSCs. However, both of these cell types were able to support the generation of CFU-Cs to a greater degree than BMSCs. To examine the mechanism of enhanced support of VEGF-R1+ HSCs by the BMECs, we performed real-time PCR analysis for the expression of the VEGF-R1 ligands. Both BMECs and spleen ECs were found to express VEGF-A and –B to similar levels, however the expression of placental growth factor was higher in the BMECs. Whether the increased expression of this factor plays a functional role in the support of the HSCs in currently being evaluated. Our findings suggest that the ECs from BM or spleen can promote the proliferation of hematopoietic progenitor cells, while BMECs can maintain the long-term culture of VEGFR1+ HSCs in vitro. The functional relevance of this in vivo is currently being investigated.

scholarly journals DOT1L methyltransferase regulates the calcium influx in erythroid progenitor cells in response to erythropoietin

2020 ◽  
Author(s):  
Yi Feng ◽  
Shaon Borosha ◽  
Anamika Ratri ◽  
Sami M. Housami ◽  
V. Praveen Chakravarthi ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Transient Receptor Potential ◽  
Calcium Influx ◽  
Receptor Potential ◽  
Proximal Region ◽  
Yolk Sac ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

ABSTRACTErythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of hematopoietic progenitor cells. An important downstream response of EPO signaling is calcium influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in c-KIT positive erythroid progenitor cells is regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development, including early erythropoiesis. We previously reported that Dot1L knockout (Dot1L-KO) hematopoietic progenitors in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we have detected a marked downregulation of Trpc6 gene expression in Dot1L-KO progenitor cells in the yolk sac compared to wildtype. However, the expression of Trpc2, the positive regulator of Ca2+influx, remained unchanged. The promoter and the proximal region of the Trpc6 gene loci exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. As the loss of DOT1L affects the expression of TRPC6, which inhibits Ca2+influx by TRPC2, Dot1L-KO progenitor cells in the yolk sac exhibit accelerated and sustained high levels of Ca2+influx. Such heightened Ca2+ levels might have detrimental effects on the development of hematopoietic progenitor cells in response to erythropoietin.

scholarly journals Effects of recombinant alpha and gamma interferons on the in vitro growth of circulating hematopoietic progenitor cells (CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM) from patients with myelofibrosis with myeloid metaplasia

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1014-1019 ◽  
Author(s):  
C Carlo-Stella ◽  
M Cazzola ◽  
A Gasner ◽  
G Barosi ◽  
L Dezza ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Hematopoietic Progenitor Cells ◽  
Definitive Treatment ◽  
Hematopoietic Progenitor Cell ◽  
In Vitro Growth ◽  
Hematopoietic Progenitor ◽  
Myeloid Metaplasia ◽  
Myelofibrosis With Myeloid Metaplasia

Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder due to clonal expansion of a pluripotent hematopoietic progenitor cell with secondary marrow fibrosis. No definitive treatment has as yet been devised for this condition, which shows a marked variability in clinical course. To evaluate whether excessive hematopoietic progenitor cell proliferation could be controlled by recombinant human interferon alpha (rIFN-alpha) and gamma (rIFN-gamma), we studied the effects of these agents on the in vitro growth of pluripotent and lineage-restricted circulating hematopoietic progenitor cells in 18 patients with MMM. A significant increase in the growth (mean +/- 1 SEM) per milliliter of peripheral blood of CFU-GEMM (594 +/- 253), CFU-Mk (1,033 +/- 410), BFU-E (4,799 +/- 2,020) and CFU- GM (5,438 +/- 2,505) was found in patients as compared with normal controls. Both rIFN-alpha and rIFN-gamma (10 to 10(4) U/mL) produced a significant dose-dependent suppression of CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM growth. Concentrations of rIFN-alpha and rIFN-gamma causing 50% inhibition of colony formation were 37 and 163 U/mL for CFU-GEMM, 16 and 69 U/mL for CFU-Mk, 53 and 146 U/mL for BFU-E, and 36 and 187 U/mL for CFU-GM, respectively. A marked synergistic effect was found between rIFN-alpha and rIFN-gamma: combination of the two agents produced inhibitory effects greater than or equivalent to those of 10- to 100- fold higher concentrations of single agents. These studies (a) confirm that circulating hematopoietic progenitors are markedly increased in MMM, (b) indicate that these presumably abnormal progenitors are normally responsive to rIFNs in vitro, and (c) show that IFNs act in a synergistic manner when used in combination. Because rIFN-gamma can downregulate collagen synthesis in vivo, this lymphokine could be particularly useful in the treatment of patients with MMM.

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