HSC Hierarchy in Primary Myelofibrosis

Abstract Primary Myelofibrosis (PMF) is a Myeloproliferating Neoplasm (MPN) of hematopoietic stem cell origin, characterized by expansion of aberrant myeloid progenitors in the chronic phase that can lead to bone marrow fibrosis development and/or osteosclerosis. In 20-25% of PMF cases transformation to acute myeloid leukemia (AML) is observed. Identification of multiple molecular lesions suggests complex clone dynamics that indicates exceeding sub-clone dominance as PMF progresses. Our aim is to determine the HSC hierarchy orchestrating initiation and development of PMF. In our previous studies we reported a CD133+ HSC population in PMF peripheral blood that represents the aberrant long-term stem cell fraction responsible for PMF reproduction of chronic and acute phases in vivo. Molecular analysis of PMF xenografts indicates sustenance of genetically different HSC clones exhibiting variation in both their engraftment potential and reproduction of PMF parameters in the first mouse model of the disease. To further characterize the succession of molecular lesions determining HSC clone propagation we performed targeted exon sequencing of PMF HSC from 100 PMF patients for 54 genes. Mutations in the epigenetic regulators ASXL1 and EZH2 were detected in 38% and 15% of PMF patients respectively. HSC clonal evolution was determined by single cell molecular and phenotypic analysis in vitro and graft analysis in vivo. Mutations detected in the epigenetic regulators ASXL1 and EZH2 represent founding molecular lesions at the top of PMF HSC hierarchy. ASXL1 mutations precede and are connected with sustenance of clonal hematopoiesis without any significant influence on the HSC differentiation potential. EZH2 mutations are connected with impaired erythropoiesis in vitro and anemia, high engraftment and expansion of pre-leukemic clones in vivo. Occurrence of JAK2 and CALR mutations in the background of mutated epigenetic regulator genes is connected with expansion of HSC subclones and reproduction of chronic phase PMF as atypical myelopoiesis, splenomegaly and induction of fibrosis. Our results indicate genetic heterogeneity of PMF neoplastic HSC is comprised of three different mutational clusters. Mutations in epigenetic regulator genes (Group 1) precede and shape the epigenetic landscape conferring genetic instability to sustain the expansion of pre-leukemic clones (Group 3 mutations). JAK2 and CALR mutations (Group 2) occur later on and are connected with aberrant myelopoiesis of chronic phase disease. HSC clonal dynamics reflect genotype related phenotypes as determinants of chronic and acute phases of PMF. Disclosures No relevant conflicts of interest to declare.

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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 ◽

10.1182/blood.v124.21.4374.4374 ◽

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.

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182 Establishment of expanded potential embryonic stem cell lines from porcine embryos

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab182 ◽

2019 ◽

Vol 31 (1) ◽

pp. 215

Author(s):

M. Nowak-Imialek ◽

X. Gao ◽

P. Liu ◽

H. Niemann

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Differentiation Potential ◽

Germ Layers ◽

Embryonic Tissues

The domestic pig is an excellent large animal in biomedical medicine and holds great potential for testing the clinical safety and efficacy of stem cell therapies. Previously, numerous studies reported the derivation of porcine embryonic stem cell (ESC)-like lines, but none of these lines fulfilled the stringent criteria for true pluripotent germline competent ESC. Here, we report the first establishment of porcine expanded potential stem cells (pEPSC) from parthenogenetic and in vivo-derived blastocysts. A total of 12 cell lines from parthenogenetic blastocysts from Day 7 (12/24) and 26 cell lines from in vivo-derived blastocysts from Day 5 (26/27) were established using defined stem cell culture conditions. These cells closely resembled mouse ESC with regard to morphology, formed compact colonies with high nuclear/cytoplasmic ratios, and could be maintained in vitro for more than 40 passages with a normal karyotype. The pEPSC expressed key pluripotency genes, including OCT4, NANOG, SOX2, and SALL4 at similar levels as porcine blastocysts. Immunostaining analysis confirmed expression of critical cell surface markers SSEA-1 and SSEA-4 in pEPSC. The EPSC differentiated in vitro into tissues expressing markers of the 3 germ layers: SOX7, AFP, T, DES, CRABP2, α-SMA, β-tubulin, PAX6, and, notably, the trophoblast markers HAND1, GATA3, PGF, and KRT7. After injection into immunocompromised mice, the pEPSC formed teratomas with derivatives of the 3 germ layers and placental lactogen-1 (PL-1)-positive trophoblast-like cells. Additionally, pEPSC cultured in vitro under conditions specific for germ cells formed embryoid bodies, which contained ~9% primordial germ cell (PGC)-like cells (PGCLC) that expressed PGC-specific genes, including NANOS3, BLIMP1, TFAP2C, CD38, DND1, KIT, and OCT4 as detected by quantitative RT-PCR and immunostaining. Next, we examined the in vivo differentiation potential of pEPSC and injected pEPSC stably expressing the CAG-H2B-mCherry transgene reporter into porcine embryos. The donor cells proliferated and were localised in both the trophectoderm and inner cell mass of the blastocysts cultured in vitro. After transfer to 3 recipient sows, chimeric embryos implanted and a total of 45 fetuses were recovered on Days 26 to 28. Flow cytometry of single cells collected from embryonic and extraembryonic tissues of the fetuses revealed mCherry+ cells in 7 conceptuses, in both the placenta and embryonic tissues; in 3 chimeric conceptuses, mCherry+ cells were exclusively found in embryonic tissues; and in 2 conceptuses, mCherry+ cells were exclusively localised in the placenta. The contribution of the mCherry+ cells was low (0.4-1.7%), but they were found and co-detected in multiple porcine embryonic tissues using tissue lineage-specific markers, including SOX2, TUJ1, GATA4, SOX17, AFP, α-SMA, and trophoblast markers PL-1 and KRT7 in the placental cells. The successful establishment of pEPSC represents a major step forward in stem cell research and provides cell lines with the unique state of cellular potency useful for genetic engineering and unravelling pluripotency regulation in pigs.

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Chicken embryonic brain: an in vivo model for verifying neural stem cell potency

Journal of Neurosurgery ◽

10.3171/2013.1.jns12698 ◽

2013 ◽

Vol 119 (2) ◽

pp. 512-519 ◽

Cited By ~ 4

Author(s):

Alex Kharazi ◽

Michael L. Levy ◽

Maria Cristina Visperas ◽

Chih-Min Lin

Keyword(s):

Stem Cell ◽

Fetal Brain ◽

Stem Cell Marker ◽

In Vivo Model ◽

Embryonic Brain ◽

In Vitro Differentiation ◽

Differentiation Potential ◽

Marker Expression

Object The multipotency of neural stem cells (NSCs) can be assessed in vitro by detection of stage-specific markers in response to a suitable differentiation signal. This test is frequently used because it is fast and affordable. However, it is not clear how the in vitro potential for multilineage differentiation and stem cell marker expression would reflect the ability of NSCs to engraft into the brain following transplantation. The authors undertook this study to directly compare the in vitro potency and in vivo migration of human NSCs (hNSCs) expanded under conditions of gradually increased concentration of fetal bovine serum (FBS) as a maturation factor. Methods Human NSCs isolated from fetal brain were propagated in serum free media (SF-hNSCs) and in media containing 0.1% and 0.2% serum. At Passage 4 in tissue culture the NSCs were harvested and either differentiated in vitro or transplanted into the lateral ventricle of chicken embryonic brain at the late stage of its development (Hamburger and Hamilton Stage 26). The in vitro differentiation was evaluated by immunostaining with neural or glial specific markers, and the in vivo migration was assessed using immunohistology. Results The authors found that SF-hNSCs successfully engrafted into the chicken embryonic brain, which correlated with their ability to differentiate in vitro. NSCs grown at as low as 0.1% concentration of FBS failed to demonstrate the robust in vivo migration pattern but still preserved the capability to differentiate in vitro. Furthermore, NSCs generated in media containing a higher concentration of FBS (0.2%) lost both the in vivo engraftment and in vitro differentiation potential. Conclusions The present study suggests that marker expression and in vitro differentiation assays might not provide adequate information regarding the behavior of NSCs following their transplantation. The in vivo migration following injection into chicken embryonic brain may provide an important assay of the potency of NSCs.

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The Hypoxic Preconditioning Effect On Senescence Cell Process In Cultured Adipose-Derived Mesenchymal Stem Cells (AMSCs)

Indonesian Journal of Cardiology ◽

10.30701/ijc.v39i3.802 ◽

2019 ◽

Vol 39 (3) ◽

Author(s):

Nadiar Dwi Nuarisa ◽

I Gde Rurus Suryawan ◽

Andrianto Andrianto

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Life Span ◽

Hypoxic Preconditioning ◽

Differentiation Potential ◽

Hypoxic Precondition ◽

Significant Difference

Introduction : Stem cell therapy for myocardial regeneration is expected to increase cardiomyocyte proliferation and trigger neovascularization to improve cardiomyocytes. Mesenchymal Stem Cells (MSCs) are ideal candidates for regenerative medicine and immunotherapy. But low viability of MSCs is a major challenge in this alternative therapy. Therefore, a cytoprotective strategy is needed, one of them is hypoxic preconditioning which can significantly increase survival stem cells after being transplanted. MSCs are known to have a limited life span, after experiencing several splits MSC will enter the senescence process. It is known that hypoxia can also increase cell proliferation and differentiation potential in vitro and in vivo through the role of Octamer-4 (Oct-4) as a regulator of the pluripotency gene. Methods : Experimental laboratory studies (in vitro studies) using human-AMSCs which were given hypoxic preconditioning, observed as a immunocytochemistry. Results : The results showed that hypoxic precondition (1% O2) inhibited the senescence process. It can be seen in the lower expression of senescence in hypoxic conditions at P6, P7, P8, P9, P10 compared to normoxic ((p=0,004, p=0,001, p=0,009, p=0,013, p=0,024. There is a significant difference in the senescence expression of each passage in hypoxic and normoxic conditions with the highest expression at P10. In addition, we also observed AMSCs differentiation through the Oct-4 expression. It is showed that Oct-4 expression were higher in hypoxia compared to normoxia on P7, P8, P9, P10 (p=0,009, p=0,009, p=0,030, p=0,0001). Conclusions : Hypoxic preconditioning have the effect of inhibiting the senescence process on Adipose-derived MSCs (AMSCs) or prolonging their life span. The longer life span of AMSCs is also seen by higher cell differentiation potential from increased expression of Oct-4. However, the mechanism of inhibiting the senescence process in hypoxia in stem cells is still remain unknown. Keywords: human-Adipose derived Mesenchymal Stem Cell Cultures (h-AMSCs), Hypoxic Preconditioning, Senescence cell, Oct-4.

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215 COMPARATIVE ANALYSIS OF PORCINE PLURIPOTENT CELL LINES DERIVED FROM SOMATIC CELLS AND VARIOUS EMBRYONIC ORIGINS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab215 ◽

2012 ◽

Vol 24 (1) ◽

pp. 220

Author(s):

J. K. Park ◽

H. S. Kim ◽

K. J. Uh ◽

K. H. Choi ◽

H. M. Kim ◽

...

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Pluripotent Cells ◽

Differentiation Potential

Since pluripotent cells were first derived from the inner cell mass (ICM) of mouse blastocysts, tremendous efforts have been made to establish embryonic stem cell (ESC) lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to derive pluripotent cells of naïve state that represents full pluripotency, due to the frequent occurrence of spontaneous differentiation into an EpiSC-like state during culture in pigs. We have been able to derive EpiSC-like porcine embryonic stem cell (pESC) lines of a differentiated non-ES cell state from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) into porcine fibroblast cells. In this study, we analysed characteristics such as marker expression, pluripotency and the X chromosome inactivation (XCI) status of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential; female XCI activity and a normal karyotype. Here we provide preliminary results that suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. This work was supported by the BioGreen 21 Program (#20070401034031, PJ0081382011), Rural Development Administration, Republic of Korea.

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The Effect of Aging on Hematopoietic Stem Cell Homing and Engraftment.

Blood ◽

10.1182/blood.v104.11.2673.2673 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2673-2673

Author(s):

Ying Liang ◽

Gary Van Zant ◽

Stephen J. Szilvassy

Keyword(s):

Stem Cell ◽

Proliferative Potential ◽

Older Individuals ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Platelet Recovery ◽

Donor Cells ◽

Recipient Age

Abstract The efficiency with which hematopoietic stem (HSC) and progenitor cells (HPC) home to bone marrow (BM) critically impacts their engraftment after transplantation. Little is known about the effects of aging on this parameter. The present study was thus initiated to test the hypothesis that homing efficiency of HSCs and HPCs to the BM will vary with aging of the donor, recipient, or both. Young or old C57BL/6 BM cells were intravenously injected into lethally irradiated old or young Ly-5 congenic recipient mice. Three or 24 hours later, the numbers of HPCs or HSCs that could be recovered from the BM and spleen were assayed using an in vitro colony-forming cell assay or an in vivo limiting-dilution competitive repopulating unit (CRU) assay and compared to the number of such cells that were initially transplanted. The frequency of CRU in old BM was two-fold higher than that in young BM (~1 per 10,000 vs. 24,000 cells). However, old CRU homed less efficiently to young BM after 24 hours than did young HSCs (3% vs. 10%). The proliferative potential of individual HSCs (measured as the overall level of engraftment in mice transplanted with 1 CRU) did not change with donor age, but was reduced by advanced recipient age, and was also reduced by prior transplantation as observed previously. HSC differentiation potential (defined by the proportion of lymphocytes and myeloid cells in mice transplanted with 1 CRU) was also skewed toward myelopoiesis at the expense of lymphopoiesis with both donor and recipient age. Donor and recipient aging exerted similar effects on the 3 hour BM and spleen seeding efficiency of HPCs, both leading to a 40% decrease in BM homing, and a 5-fold decrease in spleen homing compared with young HPCs transplanted into young recipients. In terms of the rate of hematopoietic engraftment, advanced recipient age resulted in slower erythrocyte and platelet recovery, but moderately accelerated leukocyte regeneration compared with young cells transplanted into young recipients. Conversely, old donor cells exhibited faster leukocyte and platelet recovery, but erythrocyte recovery was essentially unchanged compared to young donor cells. In sum, these data suggest that primitive hematopoietic cells undergo intrinsic aging-related changes in homing efficiency, proliferation/differentiation potential, and engraftment capability that are also extrinsically affected by an older BM microenvironment. These findings have important implications for clinical stem cell transplantation where older individuals often serve as donors for elderly patients.

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Low or Undetectable Numbers of Philadelphia Chromosome Positive (Ph+) Leukemia Cells in the Primitive (CD34posCD38neg) Stem Cell Fraction in Chronic Myeloid Leukemia (CML) Patients during Tyrosine Kinase Inhibitor Therapy.

Blood ◽

10.1182/blood.v112.11.1079.1079 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1079-1079

Author(s):

Satu Mustjoki ◽

Peter Rohon ◽

Katrin Rapakko ◽

Sari Hernesniemi ◽

Perttu Koskenvesa ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Myeloid Leukemia ◽

Chronic Phase ◽

Cell Fraction ◽

Cytogenetic Remission ◽

Cell Fractions ◽

Cd38 Antigen

Abstract Targeted tyrosine kinase inhibitors (TKIs) efficiently induce rapid hematologic and cytogenetic remission in most chronic myeloid leukemia (CML) patients. However, in vitro experiments have suggested that the most primitive CML stem cells residing in the CD34posCD38neg fraction are relatively resistant to TKIs. The prevalence of these stem cells in vivo in patients under TKI therapy is unclear. The aim of this project was to analyze the effect of TKI therapy on Ph+ leukemia stem cell pool in patients and to analyze the proportion of Ph+ cells in different stem cell fractions. A total of 26 chronic phase CML patients were included in the study. 18 patients were treated with imatinib, 5 with dasatinib, and 3 with bosutinib. The median time of TKI treatment was 20 months (range 3–72 months). Large volume (median 30 ml, range 5–55 ml) of bone marrow (BM) aspirate was collected and mononuclear cells (MNC) were isolated. CD34pos cells were separated with paramagnetic beads and further sorted into CD34posCD38pos and CD34posCD38neg cell populations with multicolor flow cytometry in order to analyze progenitor cell fractions of different maturation stage. Proportion of Ph+ cells was determined with interphase FISH by counting 1000 cells in each fraction. The median yield of MNCs from 30 ml of BM aspirate was 280x106 cells resulting in a median of 32 000 CD34posCD38neg cells (range 1000–91000). High-sensitivity counting of the proportion of Ph+ cells was feasible with a median number of counted interphase nuclei of 1005. During TKI therapy the CD34pos cells expressing highest CD38 antigen level were already mostly differentiated into B-cell lineage (CD19 positive). The CD34pos cells expressing low CD38 antigen levels expressed markers of more primitive cells such as C-kit (CD117) and CD133. Of 26 patients with CML, 19 were in complete cytogenetic remission (CCyR) when assessed by metaphase FISH of non-fractionated BM cells (1000 cells analyzed). Only 3 patients had single Ph+ cells in CD34pos cell fractions (less than 1%). In remainder of patients, all progenitor cell fractions, including the most primitive CD34posCD38neg cells, were negative for Ph+ cells. 3 patients had 0–1% of Ph+ cells in non-fractionated BM sample. One of them had 0.2% of Ph+ cells in CD34posCD38neg fraction, but the other 2 patients had 0/1000 Ph+ stem cells. 4 patients had a partial cytogenetic response (5–20% of Ph+ cells in non-fractionated BM sample). Again, the proportion of Ph+ cells was not increased in the most primitive CD34posCD38neg cell fraction. Interestingly, patients who had discontinued imatinib treatment had lower level of Ph+ cells in different CD34pos fractions (median 0.1%) when compared to non-fractionated BM (median 9.3%). Based on our data, in chronic phase CML patients, TKI therapy eradicates most Ph+ CD34pos progenitor cells. Unexpectedly, leukemic stem cells were not enriched in the most primitive CD34posCD38neg cell fraction in vivo. These results differ from the in vitro studies, where CD34posCD38neg cells have been shown to be resistant to TKIs. This could be due to non-physiological conditions (growth factor sensitivity, other cytokines) in cell culture assays. In addition, leukemic stem cells in vivo may be located in the subcortical hypoxic stem cell niche in the BM and are less likely to be aspirated. Our data underline the tremendous proliferative potential of very rare stem cells in CML patients in CCyR, as is evident after discontinuation of TKI therapy. Future studies evaluating the kinetics of disappearance of Ph+ cells from stem cell fractions during TKI therapy and the location of residual Ph+ stem cells in the BM are warranted and may give important information on the depth of the therapy response. Furthermore, this knowledge may aid in targeting therapy to these cells and finding curative treatment strategies in CML.

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Genetic HSC Variability Determines the Drivers of Chronic and Acute Phases of Primary Myelofibrosis

Blood ◽

10.1182/blood.v126.23.1629.1629 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1629-1629

Author(s):

Ioanna N Triviai ◽

Silke Zeschke ◽

Marios Spanakis ◽

Carol Stocking ◽

Nicolaus Kroeger

Keyword(s):

Mouse Model ◽

Colony Formation ◽

Chronic Phase ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Monocytic Differentiation ◽

Leukemic Transformation ◽

Differentiation Capacity

Abstract Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by abnormal differentiation of erythroid-megakaryocytic lineages and expansion of the granulo/monocytic lineage. Accumulation of aberrant myeloid precursors dominates the chronic phase of PMF leading to fibrosis development or leukemic transformation. Recent reports describe that mutation order dictates the prevalence of distinct erythroid subclones in MPN, or that clonality of whole blood mononuclear cells is related to worse prognosis and leukemic transformation. The mutational variability of the stem cell pool determining either the expansion of independent clones dominating chronic phase PMF or the propagation of pre-leukemic progenitors has not been resolved. In our previous studies, we characterized a CD133+ HSC population exhibiting multilineage differentiation capacity in vitro that drives PMF disease and leukemic transformation in a xenotransplantation mouse model. Molecular analysis of PMF-patient derived HSC indicated variability in their mutational burden, which was reflected in their engraftment capacity and disease induction in vivo. Our goal is to determine the genetic lesions within the HSC pool in PMF that determine aberrant myeloid differentiation in the chronic phase or are responsible for blast transformation. CD133+ HSCs from 15 PMF patients were molecularly characterized for the known mutations in MPN by whole exon sequencing. Sorted HSC cells were functionally analyzed at a single cell level for variable myeloid colony formation. 2230 colonies were phenotypically characterized and isolated. Analysis of the PMF HSC clonogenic potential indicates that the presence of mutations in the epigenetic regulator EZH2 correlates with granulo/monocytic differentiation but limited erythroid colony formation potential (0-0,05%), as determined in three different patient samples (2 JAK2-V617F+, 1 CALR-fs*+). Transplantation of these patient samples gave the highest engraftment in our mouse model and in one case, EZH2mu JAK2wt leukemic transformation. CD133+ HSC-derived single colony analysis from this patient indicated that there are 6 different genotypic clones of HSC, which exhibit variable granulo/monocytic differentiation capacity in vitro. From a total of 569 formed colonies, 538 were CFU-GM,-G,-M and 31 BFU-E. PCR analysis of colonies for JAK2-V617F and Sanger sequencing for EZH2-D265H indicates that the presence of JAK2-V617F in hetero- or homozygosity can occur in the EZH2-D265H background without influencing the granulo/monocytic commitment of these mutated HSCs. Interestingly, the limited BFU-Es that arose contained only single JAK2-V617F mutations in the same patient. Moreover, the presence of single EZH2-D265H heterozygous clones, single JAK2-V617F hetero- or homozygous clones, as well as double mutated clones indicate two independent mutational events affecting the same locus and nucleotide have occurred in this patient. In view of the overall high frequency of JAK2-V617F mutations, we predict that the EZH2 mutation was the first mutation in double mutant clones in this patient. Taken together, we show for the first time that JAK2-V617F mutation can occur in independent HSC clones in PMF that exhibit distinctive differentiation potential. Taken into account that AML occurred in vivo from a EZH2mu JAK2wt clone, our studies indicate that JAK2 and CALR mutations sustain the progeny of the chronic phase PMF, while EZH2 mutations might precede those of JAK2 and shape the genomic landscape that supports the expansion of pre-leukemic clones. Disclosures No relevant conflicts of interest to declare.

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Adult-Derived Human Liver Mesenchymal-Like Cells as a Potential Progenitor Reservoir of Hepatocytes?

Cell Transplantation ◽

10.3727/000000007783465154 ◽

2007 ◽

Vol 16 (7) ◽

pp. 717-728 ◽

Cited By ~ 127

Author(s):

Mustapha Najimi ◽

Dung Ngoc Khuu ◽

Philippe Antoine Lysy ◽

Nawal Jazouli ◽

Jorge Abarca ◽

...

Keyword(s):

Stem Cell ◽

Human Liver ◽

Adult Stem Cells ◽

Scid Mice ◽

Differentiation Potential ◽

Adult Human ◽

Cell Source ◽

Intrasplenic Transplantation

It is currently accepted that adult tissues may develop and maintain their own stem cell pools. Because of their higher safety profile, adult stem cells may represent an ideal candidate cell source to be used for liver cell therapies. We therefore evaluated the differentiation potential of mesenchymal-like cells isolated from adult human livers. Mesenchymal-like cells were isolated from enzymatically digested adult human liver and expanded in vitro. Cell characterization was performed using flow cytometry, RT-PCR, and immunofluorescence, whereas the differentiation potential was evaluated both in vitro after incubation with specific media and in vivo after intrasplenic transplantation of uPA+/+-SCID and SCID mice. Adult-derived human liver mesenchymal-like cells expressed both hepatic and mesenchymal markers among which albumin, CYP3A4, vimentin, and α-smooth muscle actin. In vitro differentiation studies demonstrated that these mesenchymal-like cells are preferentially determined to differentiate into hepatocyte-like cells. Ten weeks following intrasplenic transplantation into uPA+/+-SCID mice, recipient livers showed the presence of human hepatocytic cell nodules positive for human albumin, prealbumin, and α-fetoprotein. In SCID transplanted liver mice, human hepatocyte-like cells were mostly found near vascular structures 56 days posttransplantation. In conclusion, the ability of isolated adult-derived liver mesenchymal stem-like cells to proliferate and differentiate into hepatocyte-like cells both in vitro and in vivo leads to propose them as an attractive expandable cell source for stem cell therapy in human liver diseases.

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Characterization of Dental Pulp Stem Cell Responses to Functional Biomaterials Including Mineralized Trioxide Aggregates

Journal of Functional Biomaterials ◽

10.3390/jfb12010015 ◽

2021 ◽

Vol 12 (1) ◽

pp. 15

Author(s):

Sejin Bae ◽

Bueonguk Kang ◽

Hyungbin Lee ◽

Harrison Luu ◽

Eric Mullins ◽

...

Keyword(s):

Stem Cell ◽

Growth Factors ◽

Dental Pulp ◽

Cell Biology ◽

Primary Objective ◽

Extrinsic Factors ◽

Differentiation Potential ◽

Functional Biomaterials

Introduction: Many studies in stem cell biology have demonstrated that dental pulp stem cells (DPSC) may be highly proliferative and capable of pluripotent differentiation into many different tissue types. Recent advances in stem cell research have outlined methods for directing in vitro or in vivo growth, viability, and proliferation, as well as differentiation of DPSC—although much remains to be discovered. Based upon this information, the primary objective of this study was to understand the functional biomaterials needed to more effectively direct DPSC viability, growth, and proliferation. Methods: Using an approved protocol, previously collected and isolated samples of DPSC from an existing repository were used. Previously established stem cell biomarkers (Sox-2, Oct-4, NANOG) from each isolate were correlated with their proliferation rates or doubling times to categorize them into rapid, intermediate, or slow-dividing multipotent DPSC. Growth factors and other functional dental biomaterials were subsequently tested to evaluate DPSC responses in proliferation, viability, and morphology. Results: Differential responses were observed among DPSC isolates to growth factors, including vascular endothelial growth factor (VEGF) and bone morphogenic protein (BMP-2), and functional biomaterials such as mineralized trioxide aggregates (MTA). The responsiveness of DPSC isolates did not correlate with any single factor but rather with a combination of proliferation rate and biomarker expression. Conclusions: These data strongly suggest that some, but not all, DPSC isolates are capable of a robust and significant in vitro response to differentiation stimuli, although this response is not universal. Although some biomarkers and phenotypes that distinguish and characterize these DPSC isolates may facilitate the ability to predict growth, viability, and differentiation potential, more research is needed to determine the other intrinsic and extrinsic factors that may contribute to and modulate these DPSC responses to these functional biomaterials for biotechnology and bioengineering applications.

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