MSC-Derived HSCs Can Reconstitute Hematopoietic Function In Patients With Severe Aplastic Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2432-2432 ◽  
Author(s):  
James Q Yin ◽  
Chunji Gao ◽  
Bing Han ◽  
Jianliang Sheng
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Aplastic Anemia ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Severe Aplastic Anemia ◽  
Hematopoietic Stem

Abstract Introduction Naturally-occurring regeneration of cells and tissues is generally involved in four working mechanisms such as directed differentiation, dedifferentiation, trans-differentiation and transdetermination. The better exploring of these mechanisms could be beneficial to develop clinical strategies for regenerative medicine and to reduce the likelihood of immune rejection and relevant complications Recently, “trans-determination” has attracted great controversy, mostly in regards to whether adult stem cells can colonize other tissues after transplantation. More importantly, how to generate large amounts of a particular stem cell type through a transdetermination process remained to be unsolved. Similarly, it is unclear whether mesenchymal stem cells (MSCs) can transdeterminate into hematopoietic stem cells (HSCs). Methods Many technologies were used to validate the transdetermination of adipose-derived mesenchymal stem cells (AD-MSCs) into hematopoietic stem cells (HSCs) from different aspects. They include FACS analysis, PCR tests, immunostaining, expansion and repopulating assays, transplantation analysis and others, showing their in vivo and in vitro potentials for long-term self-renewal and differentiation into multi-lineages of blood cells. Moreover, these AD-HSCs can reconstitute hematopoietic function in six patients. Results We report firstly here that a huge number of human AD-MSCs that are CD44+,CD29+, CD105+, CD166+,CD133-,CD34- could rapidly transdifferentiate into hematopoietic stem cells (CD49f+/CD133+/CD34+) and their descending blood cells in vitro, after transfected with two small RNAs. The sRNAs were high-effectively delivered into MSCs by a novel peptide means. These adipose-derived HSCs (AD-HSCs) could form different types of hematopoietic colonies as nature-occurring HSCs did. Upon the primary and secondary transplantation into sublethally or lethally irradiated mice, these MSC-HSCs engrafted and differentiated into all hematopoietic lineages such as erythrocytes, lymphocytes, myelocytes and thrombocytes. Furthermore, we demonstrated the first evidence that the transdetermination of MSCs was induced by acetylation of histone proteins and activation of many transcriptional factors. More excitingly, these MSC-derived HSCs can reconstitute hematopoietic function in six patients with severe aplastic anemia. Conclusion our findings identify the molecular mechanisms that regulate the directed transdifferentiation of MSCs toward HSCs, create a new source for individual HSC transplantation used for the treatment of blood diseases and cancers, and break the stalemate caused by bone marrow match and graft-versus-host disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Essential Role of Endogenous MOZ in Aberrant HoxA9 and Meis1 Expressions Induced By MOZ Fusion Gene

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2191-2191
Author(s):  
Takuo Katsumoto ◽  
Kazutsune Yamagata ◽  
Yoko Ogawara ◽  
Takuro Nakamura ◽  
Issay Kitabayashi
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Molecular Mechanisms ◽  
Zinc Finger Protein ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Leukemia Cells ◽  
Hematopoietic Stem

Abstract Monocytic leukemia Zinc finger protein (MOZ), a histone acetyltransferase, is involved in chromosome translocations associated with FAB M4/M5 types of acute myeloid leukemia (AML). In normal hematopoiesis, MOZ is essential for self-renewal of hematopoietic stem cells (HSCs) and for expression of HoxA9/Meis1 in hematopoietic stem/progenitor cells (HSPCs). Previously we found that endogenous MOZ is critical for MOZ-TIF2-induced AML. Although MOZ-/- cells expressing the MOZ-fusion serially generated colonies in vitro, they did not induce AML after transplantation into recipient mice. In these cells, up-regulation of Meis1 was impaired, while HoxA9 expression was induced. However, roles of endogenous MOZ in MOZ fusion induced leukemia remained unclear. To elucidate molecular mechanisms, we performed experiments described below. First, to reveal mechanisms in defect of Meis1 expression in MOZ-/- MOZ-fusion leukemia cells, we performed chromatin immune-precipitation assays on Meis1 locus. Coincident with gene expression, active histone marks (H3K9ac, H3K27ac etc.) were disrupted. In contrast, repressive histone modifications (H3K9me2, H3K27me3) were elevated. Next we analyzed requirement of HoxA9 and Meis1 in MOZ fusion induced AML development. When mice were transplanted with MOZ-/- HSPCs simultaneously introduced with MOZ-fusion and Meis1 genes, AML development were induced. On the other hand, when Meis1 was conditionally deleted in MOZ-fusion leukemia cells, AML development was significantly delayed. Mice transplanted with MOZ-/- HSPCs, which were introduced with both HoxA9 and Meis1 genes elicited AML development. Furthermore, we analyzed gene expression profiles of MOZ-/- MOZ fusion leukemia cells. In these cells, expressions of monocyte/macrophage lineage characteristic genes (C/EBPa, Irf8, CD68 etc.) and MLL fusion target genes (Meis1, Mef2c) were decreased. In contract, other hematopoietic lineage characteristic genes (GATA1-3, FOG-1, CD41, Aiolos, Helios, Eag, Epx etc.) were increased. In addition, expression of CDK inhibitor INK4A was also up-regulated. Finally, we tested requirement of endogenous MOZ in various cellular conditions. Previous report showed that AML development was induced by introduction of MOZ-TIF2 not only in hematopoietic stem cells but also in more differentiated Common myeloid progenitors (CMPs) and Granulocyte/Monocyte progenitors (GMPs) (Huntly et al, Cancer Cell 2004). So we introduced MOZ fusion genes in HSCs and CMPs collected from E14.5 MOZ-/- fetal liver. MOZ-/- HSCs, not CMPs, expressing MOZ-TIF2 continuously formed colonies in vitro. In the CMPs expressing MOZ-TIF2, expression of both Meis1 and HoxA9, were abolished. These results suggest that high levels of HoxA9 and Meis1 expressions were respectively required for MOZ-TIF2-induced AML development, and that endogenous MOZ is critical for MOZ-TIF2-induced AML development. Disclosures No relevant conflicts of interest to declare.

Inhibition of PDGFRβ by Imatinib Mesylate Suppresses Proliferation and Alters Differentiation of Human Mesenchymal Stem Cells In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2563-2563
Author(s):  
Fernando Fierro ◽  
Thomas Illmer ◽  
Duhoui Jing ◽  
Philip Le Coutre ◽  
Gerhard Ehninger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Imatinib Mesylate ◽  
Dependent Manner ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Abstract Recent data show that the tyrosine kinase inhibitor Imatinib mesylate (IM) also affects normal hematopoietic stem cells (HSC), T lymphocyte activation and dendritic cell function not relying on the specific inhibition of bcr-abl activity. Mesenchymal stem cells (MSC) have been identified in the bone marrow (BM) as multipotent non-hematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, and cells of visceral mesoderm. MSC interact with HSC, influencing their homing and differentiation through cell-cell contact and the production of factors including chemokines We evaluated possible effects of IM in vitro on human bone marrow-derived MSC. Screening the activity of fourty-two receptor tyrosine kinases by a phospho-receptor tyrosine kinase (RTK)-array revealed an exclusive inhibition of platelet-derived growth factor receptor (PDGFRβ) by IM which consequently affects downstream targets of PDGFRβ as Akt and Erk1/2 signalling pathways in a concentration and time dependent manner. Furthermore, perinuclear multivesicular bodies harbouring PDGFRβ were found within 18–20 hours culture of MSC in the presence of 5 μM IM. Cell proliferation and clonogenicity (evaluated as the capability to form colony forming units - fibroblasts (CFU-F)) of MSC were significantly inhibited by IM in a concentration dependent fashion. IM inhibits significantly the differentiation process of MSC into osteoblasts as evaluated by decreased alkaline phosphatase activity and reduced calcium phosphate precipitates. In contrary, differentiation of MSC into adipocytes was strongly favoured in presence of IM. All these functional deficits described, probably contribute to an observed 50% reduction in the support of clonogenic hematopoietic stem cells, as evaluated by a long term culture-initiating cells (LTC-IC)-based assay. In summary our experiments show that IM inhibits the capacity of human MSC to proliferate and to differentiate into the osteogenic lineage, favouring adipogenesis. This effect is mainly mediated by an inhibition of PDGFRβ autophosphorylation leading to a more pronounced inhibition of PI3K/Akt compared to Erk1/2 signalling. This work confirms the role of PDGFRβ recently described for the proliferation and differentiation potential of MSC and provides a first possible explanation for the altered bone metabolism found in certain patients treated with IM.

Mesenchymal Stem Cells from the Wharton’s Jelly of Umbilical Cord Segments Support the Maintenance of Long Term Culture-Initiating Cells from Cord Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3650-3650
Author(s):  
Kent W. Christopherson ◽  
Tiki Bakhshi ◽  
Shamanique Bodie ◽  
Shannon Kidd ◽  
Ryan Zabriskie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Blood Cells ◽  
Hematopoietic Stem ◽  
Cord Blood Cells

Abstract Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton’s Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells.2003; 21: 105–110) (Lee O, et al. Blood.2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton’s Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

Differentially Expressed and Novel Transcripts in Highly Purified Chronic Phase CML Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 193-193
Author(s):  
Yun Zhao ◽  
Allen Delaney ◽  
Afshin Raouf ◽  
Kamini Raghuram ◽  
Haiyan I Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Chronic Phase ◽  
Differentially Expressed ◽  
Pcr Analysis ◽  
Direct Role ◽  
Hematopoietic Stem ◽  
Transcript Levels

Abstract The chronic phase of CML is sustained by rare BCR-ABL+ stem cells. These cells share many properties with normal pluripotent hematopoietic stem cells, but also differ in critical ways that alter their growth, drug responsiveness and genome stability. Understanding the molecular mechanisms underlying the biological differences between normal and CML stem cells is key to the development of more effective CML therapies. To obtain new insights into these mechanisms, we generated Long Serial Analysis of Gene Expression (SAGE) libraries from paired isolates of highly purified lin-CD34+CD45RA-CD36- CD71-CD7-CD38+ and lin-CD34+CD45RA-CD36-CD71-CD7-CD38- cells from 3 chronic phase CML patients (all with predominantly Ph+/BCR-ABL+ cells in both subsets) and from 3 control samples: a pool of 10 normal bone marrows (BMs), a single normal BM and a pool of G-CSF-mobilized blood cells from 9 donors. In vitro bioassays showed the CD34+CD38+ cells were enriched in CFCs (CML: 3–20% pure; normal: 4–19% pure) and the CD34+CD38- cells were enriched in LTC-ICs (CML: 0.2–26% pure; normal: 12–52% pure). Each of the 12 libraries was then sequenced to a depth of ~200,000 tags and tags from libraries prepared from like phenotypes were compared between genotypes using DiscoverySpace software and hierarchical clustering. 1687 (355 with clustering) and 1258 (316 with clustering) transcripts were thus identified as differentially expressed in the CML vs control CD34+CD38− and CD34+CD38+ subsets, respectively. 266 of these transcripts (11 with clustering) were differentially expressed in both subsets. The differential expression of 5 genes (GAS2, IGF2BP2, IL1R1, DUSP1 & SELL) was confirmed by real-time PCR analysis of lin-CD34+ cells isolated from an additional 5 normal BMs and 11 CMLs, and lin-CD34+CD38− cells from an additional 2 normal BMs and 2 CMLs (with dominant Ph+ cells). GAS2 and IL1R1 transcript levels were correlated with BCR-ABL transcript levels in both primitive subsets, and predicted differences in expression of IL1R1 and SELL were apparent within 3 days in CD34+ cord blood cells transduced with a lenti-BCR-ABL-IRES-GFP vs a control lenti-GFP vector (n=3). These findings support a direct role of BCR-ABL in perturbing the expression of these 3 genes. Further comparison of the meta CD34+CD38− and CD34+CD38+ CML cell libraries with most publicly accessible SAGE data revealed 69 novel tags in the CD34+ CML cells that correspond to unique but conserved genomic sequences. Nine of these were recovered by 5′- and 3′- RACE applied to cDNAs pooled from several human leukemic cell lines. These results illustrate the power of SAGE to reveal key components of the transcriptomes of rare human CML stem cell populations including transcripts of genes not previously known to exist. Continuing investigation of their biological roles in primary CML cells and primitive BCR-ABL-transduced human cells offer important strategies for delineating their potential as therapeutic targets.

Resistance of Hematopoietic Stem Cells Lacking the KIR Ligand to Autologous NK Cell Attacks in Patients with Acquired Aplastic Anemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2939-2939
Author(s):  
Hiroyuki Maruyama ◽  
Luis J. Espinoza ◽  
Takamasa Katagiri ◽  
Yoshitaka Zaimoku ◽  
Koichi Kashiwase ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Aplastic Anemia ◽  
Nk Cells ◽  
Blood Cells ◽  
Nk Cell ◽  
Gene Repertoire ◽  
Malignant Cells ◽  
Hematopoietic Stem ◽  
Accessory Molecules

Abstract Normal blood cells, including hematopoietic stem cells (HSCs), express KIR ligands (KIR-Ls) to protect themselves from an autologous NK cell attack, and malignant cells lacking KIR-Ls elicit NK cell-mediated killing of themselves. This missing-self mechanism is believed to play an important role in the elimination of malignant cells. However, the mechanisms underlying the killing of KIR-L-lacking malignant cells by NK cells remain unclear due to the heterogeneity of tumor cells in terms of their proliferative capacity, and also because other accessory molecules may be involved in the NK cell attacks, in addition to KIR-Ls. This makes it difficult to clarify the interaction between NK cells and KIR-L-lacking target cells. The lack of class I HLA occurs not only in malignant blood cells, but also in the normal leukocytes of patients with acquired aplastic anemia (AA). These HLA-lacking leukocytes, detectable in 13% of patients with AA, are derived from HSCs that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH), and thereby escape the cytotoxic T-cell (CTL) attack against HSCs. The 6pLOH may involve KIR-L loss in some patients, leading to a change in the susceptibility of the affected HSCs to NK cell-mediated killing. Unlike malignant cells, HLA-lacking leukocytes are essentially the same as the wild-type leukocytes, except for the HLA expression. Studying 6pLOH (+) AA patients with leukocytes lacking KIR-Ls should therefore be useful for clarifying the roles of KIR-Ls and other accessory molecules in the target cell killing by NK cells. Screening of 389 patients with AA using flow cytometry and a SNP array analysis revealed that there were HLA-A allele-lacking leukocytes in 60 (15.4%) patients, which included 36 C1/C2 and 24 Bw4/Bw6 heterozygotes. Unexpectedly, a lack of KIR-Ls as a result of 6pUPD was found in five patients (13.9%, C1 missing in two and C2 missing in three) of the 36 C1/C2 heterozygotes and in five (20.8%) of the 24 Bw4/Bw6 heterozygotes, although the proportion of patients lacking a KIR-L-containing haplotype (20.8%) was significantly lower than that of patients lacking a haplotype that did not contain KIR-Ls (79.2%). Moreover, the median percentage of HLA-A-lacking granulocytes in the 10 patients who lacked a KIR-L-containing haplotype (12.4%, 0.44%-50.3%) was significantly lower than that (55.3%, 1.4%-99.4%) in the 26 patients who lacked a haplotype that did not contain KIR-Ls, suggesting that the HSCs lacking KIR-Ls or their progenies are susceptible to autologous NK cells to some extent, but are not eliminated completely. To clarify the mechanisms underlying the HSC resistance to NK cells, we determined the KIR gene repertoire and the haplotype of seven patients whose 6pLOH(+) leukocytes were lacking a KIR-L-containing haplotype. All patients possessed inhibitory KIR genes responsive to corresponding KIR-Ls, a finding that negates the possibility that NK cells failed to undergo licensing in these patients. Although the frequency of the KIR-B haplotype, a haplotype associated with a higher cytotoxic function of NK cells, in the seven patients was lower (14%) than that in Japanese healthy individuals (40.1%), two patients possessed the KIR-B haplotype. Phenotypic analyses of the NK cell subsets defined by anti-2DL1, anti-2DL2/2DL3 and anti-3DL1 antibodies showed that all seven patients had a similar percentage of the eight different NK cell subsets, which included 0.5 to 8% of effector NK cells capable of killing leukocytes lacking corresponding KIR-Ls. The expression level of HLA-E was comparable between HLA-A-lacking and HLA-A-retaining monocytes. The expression of NKG2A on the effector NK cells was also comparable to that of the other NK cell subsets in the 6pLOH(+) patients. Our study demonstrated, for the first time, that HSCs lacking KIR-Ls can evade autologous NK cell attack through an as yet unknown mechanism(s) and can continue to generate blood cells in patients with AA. Disclosures No relevant conflicts of interest to declare.

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