The role of growth factors in self-renewal and differentiation of haemopoietic stem cells

1990 ◽  
Vol 327 (1239) ◽  
pp. 85-98 ◽  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Growth Factors ◽  
Stromal Cells ◽  
Cell Lineage ◽  
Self Renewal ◽  
Haemopoietic Stem Cells

Haemopoietic stem cells in vivo proliferate and develop in association with stromal cells of the bone marrow. Proliferation and differentiation of haemopoietic stem cells also occurs in vitro , either in association with stromal cells or in response to soluble growth factors. Many of the growth factors that promote growth and development of haemopoietic cells in vitro have now been molecularly cloned and purified to homogeneity and various techniques have been described that allow enrichment (to near homogeneity) of multipotential stem cells. This in turn, has facilitated studies at the mechanistic level regarding the role of such growth factors in self-renewal and differentiation of stem cells and their relevance in stromal-cell mediated haemopoiesis. Our studies have shown that at least some multipotential cells express receptors for most, if not all, of the haemopoietic cell growth factors already characterized and that to elicit a response, several growth factors often need to be present at the same time. Furthermore, lineage development reflects the stimuli to which the cells are exposed, that is, some stimuli promote differentiation and development of multipotential cells into multiple cell lineages, whereas others promote development of multipotential cells into only one cell lineage. We suggest that, in the bone marrow environment, the stromal cells produce or sequester different types of growth factors, leading to the formation of microenvironments that direct cells along certain lineages. Furthermore, a model system has been used to show the possibility that the self-renewal probability of multipotential cells can also be modulated by the range and concentrations of growth factors present in the environment. This suggests that discrete microenvironments, preferentially promoting self-renewal rather than differentiation of multipotential cells, may also be provided by marrow stromal cells and sequestered growth factors.

Id1 Regulates the Choice of Hematopoietic Stem Cells to Self-Renew or Commit to Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 270-270 ◽  
Author(s):  
Vladimir Jankovic ◽  
Alessia Ciarrocchi ◽  
Piernicola Boccuni ◽  
Robert Benezra ◽  
Stephen D. Nimer
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Self Renewal ◽  
Myeloid Progenitor ◽  
E Protein

Abstract Id proteins belong to the basic helix-loop-helix family of transcription factors and act as dominant negative forms of E-protein transcriptional activators. Id mediated E protein silencing has an essential role in restricting differentiation and maintaining self-renewal in embryonic stem cells. However, the role of Id1 in adult stem cells including HSCs has not been described thus far. Having detected relatively high levels of Id1 mRNA in murine adult HSCs (compared to the committed myeloid progenitor cells) we examined the in vivo HSC function in Id1 deficient mice. We observed a >2 fold reduction in HSC frequency in the bone marrow in 8-10w old Id1−/− mice compared to Id1+/+ animals, detected by both lin-c-kit+Sca-1+ (LKS) cell surface marker profile and Hoechst 33342 dye efflux - “side population” phenotype, as well as a ~25% decrease in total bone marrow cellularity. Although Id1 deficient HSCs show robust long-term competitive repopulating capacity in primary transplant recipients, they have markedly impaired hematopoietic function upon secondary transplantation. Id1 null HSCs show a higher rate of S-phase entry in vivo as measured by 3 day BrdU incorporation (ko: 79.0±3.9% vs. wt: 49.7±7.4) and faster initial doubling times in response to cytokine stimulation in vitro during the first 2 days of culture. This failure to maintain normal HSC numbers and the diminished repopulating capacity, in the presence of enhanced cell cycling, suggests a defect in the regulation of self-renewal in Id1 deficient HSCs. Considering the general function of Id1 as an inhibitor of differentiation, the observed effect of Id1 loss could be explained by the excessive recruitment of LKS cells into the actively proliferating differentiated progenitor pool, at the expense of their self-renewal capacity. Consistent with this, sorted Id1−/− HSCs show accelerated expression of cell surface lineage markers in vitro and an increased ratio of CFU-S8 /CFU-S12 in the in vivo spleen colony forming assay. Global gene expression profiling of Id1+/+ vs. Id1−/− hematopoietic cells (using Affymetrix MOE430 Plus chips) revealed insignificant transcriptional deregulation in the committed myeloid progenitor subsets (CMP, GMP, MEP) in the absence of Id1. Meanwhile, Id1−/− HSCs showed a marked change in gene expression pattern (more than 1500 genes with a ≥2 fold difference in expression levels). Differentially regulated transcripts in Id1+/+ vs. Id1−/− HSCs significantly overlap (~30%) with the observed changes in gene expression that accompany the transition of HSCs to the common myeloid progenitor phenotype. Specifically, genes such as c/EBPα and GATA1 are significantly upregulated in Id1 null immunophenotypic HSCs, consistent with an earlier than normal commitment to myelo-erythroid differentiation. In contrast, several known transcriptional regulators of HSC self-renewal (Bmi1, Gfi1, HoxB4) show no significant change in expression pattern. These data clearly indicate the unique role of Id1 in regulating HSC self-renewal by restricting the rate of HSC commitment to the myeloid progenitor cell fate.

scholarly journals MicroRNA-28-5p Regulates Liver Cancer Stem Cell Expansion via IGF-1 Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Qing Xia ◽  
Tao Han ◽  
Pinghua Yang ◽  
Ruoyu Wang ◽  
Hengyu Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Liver Cancer ◽  
Critical Role ◽  
Self Renewal ◽  
Sorafenib Treatment ◽  
The Impact

Background. MicroRNAs (miRNAs) play a critical role in the regulation of cancer stem cells (CSCs). However, the role of miRNAs in liver CSCs has not been fully elucidated. Methods. Real-time PCR was used to detect the expression of miR-miR-28-5p in liver cancer stem cells (CSCs). The impact of miR-28-5p on liver CSC expansion was investigated both in vivo and in vitro. The correlation between miR-28-5p expression and sorafenib benefits in HCC was further evaluated in patient-derived xenografts (PDXs). Results. Our data showed that miR-28-5p was downregulated in sorted EpCAM- and CD24-positive liver CSCs. Biofunctional investigations revealed that knockdown miR-28-5p promoted liver CSC self-renewal and tumorigenesis. Consistently, miR-28-5p overexpression inhibited liver CSC’s self-renewal and tumorigenesis. Mechanistically, we found that insulin-like growth factor-1 (IGF-1) was a direct target of miR-28-5p in liver CSCs, and the effects of miR-28-5p on liver CSC’s self-renewal and tumorigenesis were dependent on IGF-1. The correlation between miR-28-5p and IGF-1 was confirmed in human HCC tissues. Furthermore, the miR-28-5p knockdown HCC cells were more sensitive to sorafenib treatment. Analysis of patient-derived xenografts (PDXs) further demonstrated that the miR-28-5p may predict sorafenib benefits in HCC patients. Conclusion. Our findings revealed the crucial role of the miR-28-5p in liver CSC expansion and sorafenib response, rendering miR-28-5p an optimal therapeutic target for HCC.

Donor Origin of Multipotent Adult Progenitor Cells in Radiation Chimeras.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1395-1395
Author(s):  
Morayma Reyes ◽  
Jeffrey S. Chamberlain
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Y Chromosome ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Multipotent Adult Progenitor Cells

Abstract Multipotent Adult Progenitor Cells (MAPC) are bone marrow derived stem cells that can be extensively expanded in vitro and can differentiate in vivo and in vitro into cells of all three germinal layers: ectoderm, mesoderm, endoderm. The origin of MAPC within bone marrow (BM) is unknown. MAPC are believed to be derived from the BM stroma compartment as they are isolated within the adherent cell component. Numerous studies of bone marrow chimeras in human and mouse point to a host origin of bone marrow stromal cells, including mesenchymal stem cells. We report here that following syngeneic bone marrow transplants into lethally irradiated C57Bl/6 mice, MAPC are of donor origin. When MAPC were isolated from BM chimeras (n=12, 4–12 weeks post-syngeneic BM transplant from a transgenic mouse ubiquitously expressing GFP), a mixture of large and small GFP-positive and GFP-negative cells were seen early in culture. While the large cells stained positive for stroma cell markers (smooth muscle actin), mesenchymal stem cell makers (CD73, CD105, CD44) or macrophages (CD45, CD14), the small cells were negative for all these markers and after 30 cell doublings, these cells displayed the classical phenotype of MAPC (CD45−,CD105−, CD44−, CD73−, FLK-1+(vascular endothelial growth factor receptor 2, VEGFR2), Sca-1+,CD13+). In a second experiment, BM obtained one month post BM transplant (n=3) was harvested and mononuclear cells were sorted as GFP-positive and GFP-negative cells and were cultured in MAPC expansion medium. MAPC grew from the GFP-positive fraction. These GFP positive cells displayed the typical MAPC-like immunophenotypes, displayed a normal diploid karyotype and were expanded for more than 50 cell doublings and differentiated into endothelial cells, hepatocytes and neurons. To rule out the possibility that MAPC are the product of cell fusion between a host and a donor cell either in vivo or in our in vitro culture conditions, we performed sex mismatched transplants of female GFP donor BM cells into a male host. BM from 5 chimeras were harvested 4 weeks after transplant and MAPC cultures were established. MAPC colonies were then sorted as GFP-positive and GFP- negative and analyzed for the presence of Y-chromosome by FISH analysis. As expected all GFP-negative (host cells) contained the Y-chromosome whereas all GFP-positive cells (donor cells) were negative for the Y-chromosome by FISH. This proves that MAPC are not derived from an in vitro or in vivo fusion event. In a third study, BM mononuclear cells from mice that had been previously BM-transplanted with syngeneic GFP-positive donors (n=3) were transplanted into a second set of syngeneic recipients (n=9). Two months after the second transplant, BM was harvested and mononuclear cells were cultured in MAPC medium. The secondary recipients also contained GFP-positive MAPC. This is the first demonstration that BM transplantation leads to the transfer of cells that upon isolation in vitro generate MAPCs and, whatever the identity of this cell may be, is eliminated by irradiation. We believe this is an important observation as MAPC hold great clinical potential for stem cell and/or gene therapy and, thus, BM transplant may serve as a way to deliver and reconstitute the MAPC population. In addition, this study provides insight into the nature of MAPC. The capacity to be transplantable within unfractionated BM transplant renders a functional and physiological distinction between MAPC and BM stromal cells. This study validates the use of unfractionated BM transplants to study the nature and possible in vivo role of MAPC in the BM.

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 405-405
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Jonas Larsson ◽  
Emma Larsson ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Distinct Population ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Molecular Imaging for Comparison of Different Growth Factors on Bone Marrow-Derived Mesenchymal Stromal Cells’ Survival and ProliferationIn Vivo

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Hongyu Qiao ◽  
Ran Zhang ◽  
Lina Gao ◽  
Yanjie Guo ◽  
Jinda Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Bioluminescence Imaging ◽  
Firefly Luciferase ◽  
Control Group ◽  
Induced Apoptosis

Introduction. Bone marrow-derived mesenchymal stromal cells (BMSCs) have emerged as promising cell candidates but with poor survival after transplantation. This study was designed to investigate the efficacy of VEGF, bFGF, and IGF-1 on BMSCs’ viability and proliferation bothin vivoandin vitrousing bioluminescence imaging (BLI).Methods. BMSCs were isolated fromβ-actin-Fluc+transgenic FVB mice, which constitutively express firefly luciferase. Apoptosis was induced by hypoxia preconditioning for up to 24 h followed by flow cytometry and TUNEL assay. 106BMSCs with/without growth factors were injected subcutaneously into wild type FVB mice’s backs. Survival of BMSCs was longitudinally monitored using bioluminescence imaging (BLI) for 5 weeks. Protein expression of Akt, p-Akt, PARP, and caspase-3 was detected by Western blot.Results. Hypoxia-induced apoptosis was significantly attenuated by bFGF and IGF-1 compared with VEGF and control groupin vitro(P<0.05). When combined with matrigel, IGF-1 showed the most beneficial effects in protecting BMSCs from apoptosisin vivo.The phosphorylation of Akt had a higher ratio in the cells from IGF-1 group.Conclusion. IGF-1 could protect BMSCs from hypoxia-induced apoptosis through activation of p-Akt/Akt pathway.

scholarly journals Are MSCs Stem Cells? Demonstration of in Vitro and in Vivo Stem Cell Properties of Highly Enriched linneg/CD45neg/CD271pos/CD140alow/Neg Mesenchymal Stromal Cells from Adult Human Bone Marrow

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4374-4374
Author(s):  
Roshanak Ghazanfari ◽  
Hongzhe Li ◽  
Dimitra Zacharaki ◽  
Simón Méndez-Ferrer ◽  
Stefan Scheding
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Adult Human

Abstract Human bone marrow contains a rare population of non-hematopoietic mesenchymal stromal cells (BM-MSC) with multilineage differentiation capacity, which are essential constituents of the hematopoietic microenvironment. Self-renewal and differentiation are the two key properties of somatic stem cells, however, stem cell properties of human adult BM-MSC have not been demonstrated conclusively yet. We have previously shown that low/negative expression of PDGFRα on linneg/CD45neg/CD271pos cells identified a highly enriched population of primary BM-MSC in adult human bone marrow (Li et al. Blood, 2013, 122:3699). Based on this work, the current study aimed to investigate the in-vitro and in-vivo stem cell properties of this putative stromal stem cell population. The in-vitro clonogenic potential of freshly sorted human linneg/CD45neg/CD271pos/PDGFRlow/neg cells was evaluated by utilizing the CFU-F assay as well as the recently-developed mesensphere assay, which enables MSC amplification while preserving an immature phenotype (Isern et al, Cell Reports 2013, 30: 1714-24). Comparable colony frequencies were obtained with both assays (19.3 ± 2 and 17.5 ± 2.3 CFU-F and spheres per 100 plated cells, respectively, n=6, p=0.19). In order to test whether both assays identified the same population of clonogenic cells, colonies and spheres were replated under both conditions for up to three generations. The results showed comparable capacities of CFU-F and mesenspheres to form secondary and tertiary CFU-F and spheres. In-vitro self-renewal as indicated by increasing numbers of CFU-F and spheres (416.6 ± 431.7-fold and 49.5 ± 65.7-fold, respectively, n=3) was observed up to the third generation and decreased thereafter. The total number of generations was five (CFU-F) and six (spheres). In-vitro differentiation assays with both, CFU-F- and sphere-derived cells (tested until passage three) demonstrated tri-lineage differentiation potential (adipocytes, osteoblasts, chondrocytes). In addition, CFU-Fs and spheres had comparable surface marker profiles (CD73, CD90, CD105, and HLA-ABC positive; CD31, CD34 and HLA-DR negative), except for CD90, which was higher expressed on CFU-Fs. To investigate in-vivo self-renewal and differentiation potential of the putative stromal stem cells, linneg/CD45neg/CD271pos/PDGFRlow/neg -derived CFU-F and spheres were serially transplanted s.c into NSG mice. After 8 weeks, implants were harvested, human cells were FACS-isolated (CD90 and CD105 expression), and re-assayed under CFU-F and sphere conditions. Whereas in-vivo self-renewal of CFU-F could not be shown (111.5 ± 36 –fold decrease in total CFU-F numbers after primary transplantation, n=3), sphere self-renewal was clearly demonstrated by increased numbers of spheres after primary as well as secondary transplantation (1.13 ± 0.05 and 2.06 ± 0.26 –fold, respectively, n=3), which is remarkable given the fact that the number of recovered human cells is underestimated due to the isolation approach. Here, confirming GFP-marking experiments are ongoing. Finally, preliminary data indicate that linneg/CD45neg/CD271pos/PDGFRlow/neg –derived spheres display full in-vivo differentiation capacity in primary and secondary transplantations. Taken together, our data demonstrate - for the first time - that primary human linneg/CD45neg/CD271pos/PDGFRlow/neg cells meet stringent stem cell criteria, i.e. in-vitro and in-vivo self-renewal and differentiation. These findings answer the long-open question of the potential stem cell properties of adult human MSC and will enable to better understand the properties of native BM-MSC and their biological role in the bone marrow. Disclosures No relevant conflicts of interest to declare.

NOTCH1 Signaling Defines a Leukemia Initiating Cell Population in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1507-1507
Author(s):  
Wenxue Ma ◽  
Kristen M. Smith ◽  
Alejandro Gutierrez ◽  
Heather S. Leu ◽  
Qingfei Jiang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Notch Signaling ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Resistance ◽  
Self Renewal ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Notch1 Receptor

Abstract Abstract 1507 Leukemia initiating cells (LIC) contribute to therapeutic resistance as a result of their capacity to accumulate mutations in pathways, such as the NOTCH1 receptor signaling pathway, that promote self-renewal and survival within specific niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in driving therapeutic resistance. However, the role of NOTCH1 activation in human T-ALL LIC propagation and LIC sensitivity to selective NOTCH1 receptor inhibition has not been examined. The difficulties in maintaining primary cultures of leukemia cells have hampered investigations into the biology of T-ALL LIC and underscore the need for a direct transplantation model to characterize human LIC in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL LIC. Pediatric T-ALL serially transplantable LIC were found to be enriched in the CD34+CD4− and CD34+CD7− fractions of newly diagnosed patient samples. More recently, a CD7+CD1a− glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2Rƒn gamma null (NSG) mice, was identified in primary adult T-ALL without an in vitro expansion. In this study, we identified and molecularly characterized potential LIC populations in pediatric T-ALL without preceding in vitro culture and examined the role of NOTCH1 activation in LIC propagation. To further define the T-ALL LIC, CD34+CD2+CD7+ or CD34+CD2+CD7− cells were isolated from T-ALL primary patients' blood by FACS sorting and transplanted into neonatal RAG2−/− gamma chain−/− mice to determine their leukemic engraftment potential. Limiting dilution experiments were performed with cells from six T-ALL patient samples. Mice transplanted with CD34+CD2+CD7+ or CD34+CD2+CD7− cells developed a T-ALL-like disease characterized by pale bone marrow and enlarged spleen, thymus and liver. Hematopoietic organs were analyzed by flow cytometry and showed engraftment of bone marrow, spleen, thymus and liver. Furthermore, the disease could be serially transplanted. LIC were uniquely susceptible to targeted inhibition in vivo with a therapeutic human NOTCH1 negative regulatory region selective monoclonal antibody (mAb) while normal human hematopoietic progenitors were spared thereby highlighting the cell type and context specific effects of NOTCH signaling. Both the NOTCH1 mAb treatment and lentiviral shRNA knockdown of NOTCH1 reduced NOTCH1, HES1 and c-MYC transcript levels, underscoring the selectivity of NOTCH1 mAb inhibition of NOTCH signaling. These results demonstrate that CD34+CD2+CD7+ and CD34+CD2+CD7− subpopulations are enriched for LIC activity in pediatric T-ALL. Moreover, inhibition of NOTCH signaling by either mAb or shRNA-mediated Notch1 knockdown might be another strategy to target the LIC in T-ALL. Disclosures: No relevant conflicts of interest to declare.

The Potential Role of the Six1/Eya1 Pathway in the Establishment of Leukemia Stem Cells in MLL-ENL – Induced Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1362-1362 ◽  
Author(s):  
Sylvia Takacova ◽  
Pavla Luzna ◽  
Viktor Stranecky ◽  
Vladimir Divoky
Keyword(s):  
Bone Marrow ◽  
Cellular Transformation ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Transcription Profile ◽  
Self Renewal ◽  
Kinetics Of

Abstract Abstract 1362 During the multistep pathogenesis of acute leukemia (AL), a pool of leukemia stem cells (LSCs) emerges that is capable of limitless self-renewal and ensuring disease maintenance. The molecular mechanism that controls the kinetics of cellular transformation and development of LSCs is largely unknown. Using our MLL-ENL-ERtm mouse model, we have previously shown (Takacova et al., Blood 2009, 114 (22): 947–947, ASH abstract) activation of the ATR/ATM-Chk1/Chk2-p53/p21 checkpoint leading to senescence at early stages of cellular transformation (myeloproliferation), thereby preventing AL development in vivo. Experimental ATM/ATR inhibition accelerated the transition to immature cell states, acquisition of LSC properties and AL development in these mice. The MLL-ENL-ERtm mouse model allows us to study the kinetics of MLL-ENL-ERtm LSC development. We raised the questions how the transformation process progresses from the pre-LSC to the LSC state, and how DNA damage response (DDR) - mediated senescence affects the transition in gene expression. Given that the threshold of DDR signaling events is rate-limiting, we determined the transcription profile of the pre- LSC–enriched cell states derived from bone marrow and spleen of the MLL-ENL-ERtm mice at the early disease stage, and we correlated this transcription profile with the level of DDR, proliferation rate and induction of senescence. Pair-wise comparisons revealed up-regulation of the Six1 transcription factor gene and its cofactor Eya1 in the MLL-ENL-ERtm pre-LSCs in association with aberrant proliferation in both tissues. The notable difference between the two tissues concerning the barrier induction was the higher threshold of DDR and senescence in the bone marrow due to cooperation with inflammatory cytokines that fine-tune the DDR level. Interestingly, the expression of Six1 and Eya1 genes was down-regulated in senescence exclusively in the bone marrow. Consistent with these in vivo data, we found Six1 expression decreased in response to inflammation/DDR-induced senescence in the MLL-ENL-ERtm bone marrow cells cultured in vitro and correlated with SA-beta-gal positivity and p16 up-regulation. Six1 mRNA level was decreased only transiently after ionizing radiation (4 Gy)-induced DDR in the same cell line. These data suggest that Six1 expression is down-regulated in response to high DDR and permanent cell-cycle arrest in the MLL-ENL-ERtm pre-LSCs. Furthermore, we identified the transcription profile of the LSC-enriched cell state after inhibition of DDR in caffeine-treated MLL-ENL-ERtm mice in vivo. Interestingly, the expression level of Six1 and Eya1 was significantly increased in the bone marrow and spleen of the MLL-ENL-ERtm AML mice compared to the early (preleukemia) stage. High expression of Six1 and Eya1 and higher cell number expressing these genes was further confirmed by immunohistochemical staining on tissue sections. The MLL-ENL-ERtm LSC-enriched spleen cells showed increased colony forming ability in vitro and leukemia-initiating potential in serial transplantation experiments compared to pre-LSCs. Moreover, we detected Six1 and Eya1 expression in the infiltrating leukemia cells in tissues of the caffeine-treated MLL-ENL-ERtm AML mice and in a subset of leukemia cells in transplanted mice. Based on these findings and correlations, we hypothesized that the Six1/Eya1 pathway might be involved in regulation of some of the aspects of LSC development as well as invasion and maintenance of leukemia in our MLL-ENL-ERtm mice. Notably, our data indicate that senescence represses a subset of the MLL-ENL-downstream transcription response and prevents full activation of self-renewal. Experiments leading to more detailed understanding of the role of the Six1/Eya1 pathway in the MLL-ENL-induced cellular transformation are ongoing. Disclosures: No relevant conflicts of interest to declare.

In Vivo Generation of Engraftable Murine Hematopoietic Stem Cells from Induced Pluripotent Stem Cells through Hemogenic Endothelium

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3866-3866
Author(s):  
Masao Tsukada ◽  
Satoshi Yamazaki ◽  
Yasunori Ota ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Teratoma Formation

Abstract Introduction Generation of engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has long been thought an ultimate goal in the field of hematology. Numerous in vitro differentiation protocols, including trans-differentiation and forward programming approaches, have been reported but have so far failed to generate fully functional HSCs. We have previously demonstrated proof-of-concept for the in vivo generation of fully functional HSCs from induced PSCs (iPSCs) through teratoma formation (Suzuki et al., 2013). However, this method is time-consuming (taking over two months), HSCs are generated at low frequencies, and additionally require co-injection on OP9 stromal cells and SCF/TPO cytokines. Here, we present optimization of in vivo HSC generation via teratoma formation for faster, higher-efficiency HSC generation and without co-injection of stromal cells or cytokines. Results First, we screened reported in vitro trans-differentiation and forward programming strategies for their ability to generate HSCs in vivo within the teratoma assay. We tested iPSCs transduced with the following dox-inducible TF overexpression vectors: (1) Gfi1b, cFOS and Gata2 (GFG), which induce hemogenic endothelial-like cells from fibroblast (Pereira et al.,2013); (2) Erg, HoxA9 and Rora (EAR), which induce short-term hematopoietic stem/progenitor cell (HSPC) formation during embryoid body differentiation (Doulatov et,al., 2013); and (3) Foxc1, which is highly expressed the CAR cells, a critical cell type for HSC maintenance (Oomatsu et al.,2014). We injected iPSCs into recipient mice, without co-injection of stromal cells or cytokines, and induced TF expression after teratoma formation by dox administration. After four weeks, GFG-derived teratomas contained large numbers of endothelial-like and epithelial-like cells, and importantly GFG-derived hematopoietic cells could also be detected. EAR-teratomas also generated hematopoietic cells, although at lower frequencies. By contrast, hematopoietic cells were not detected in control teratomas or Foxc1-teratomas. Through use of iPSCs generated from Runx1-EGFP mice (Ng et al. 2010), and CUBIC 3D imaging technology (Susaki et al. 2014), we were further able to demonstrate that GFG-derived hematopoietic cells were generated through a haemogenic endothelium precursor. Next, we assessed whether HSPC-deficient recipient mice would allow greater expansion of teratoma-derived HSCs. This was achieved by inducing c-kit deletion within the hematopoietic compartment of recipient mice (Kimura et al., 2011) and resulted in a ten-fold increase in the peripheral blood frequency of iPSC-derived hematopoietic cells. We further confirmed similar increases in iPSC-derived bone marrow cells, and in vivo HSC expansion, through bone marrow transplantation assays. Finally, we have been able to shorten the HSC generation time in this assay by five weeks through use of transplantable teratomas, rather than iPSCs. Conclusions We have demonstrated that GFG-iPSCs induce HSC generation within teratomas, via a hemogenic endothelium precursor, and that use of HSPC-deficient recipient mice further promotes expansion of teratoma-derived HSCs. These modifications now allow us to generate engraftable HSCs without co-injection of stromal cells or cytokines. Additionally, use of transplantable teratomas reduced HSC generation times as compared with the conventional assay. These findings suggest that our in vivo system provides a promising strategy to generate engraftable HSCs from iPSCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of White Blood Cells in Blood- and Bone Marrow-Based Autologous Therapies

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
William King ◽  
Krista Toler ◽  
Jennifer Woodell-May
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Growth Factors ◽  
Blood Cells ◽  
Platelet Rich Plasma ◽  
White Blood Cells ◽  
Complex Mixture ◽  
Negative Results

There has been significant debate over the role of white blood cells (WBCs) in autologous therapies, with several groups suggesting that WBCs are purely inflammatory. Misconceptions in the practice of biologic orthopedics result in the simplified principle that platelets deliver growth factors, WBCs cause inflammation, and the singular value of bone marrow is the stem cells. The aim of this review is to address these common misconceptions which will enable better development of future orthopedic medical devices. WBC behavior is adaptive in nature and, depending on their environment, WBCs can hinder or induce healing. Successful tissue repair occurs when platelets arrive at a wound site, degranulate, and release growth factors and cytokines which, in turn, recruit WBCs to the damaged tissue. Therefore, a key role of even pure platelet-rich plasma is to recruit WBCs to a wound. Bone marrow contains a complex mixture of vascular cells, white blood cells present at much greater concentrations than in blood, and a small number of progenitor cells and stem cells. The negative results observed for WBC-containing autologous therapies in vitro have not translated to human clinical studies. With an enhanced understanding of the complex WBC biology, the next generation of biologics will be more specific, likely resulting in improved effectiveness.

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