Differences In Differentiation Capacity Research Articles

Validation of a color deconvolution method to quantify MSC tri-lineage differentiation across species.

Mesenchymal stem cells (MSCs) are a promising candidate for both human and veterinary regenerative medicine applications because of their abundance and ability to differentiate into several lineages. Mesenchymal stem cells are however a heterogeneous cell population and as such, it is imperative that they are unequivocally characterized to acquire reproducible results in clinical trials. Although the tri-lineage differentiation potential of MSCs is reported in most veterinary studies, a qualitative evaluation of representative histological images does not always unambiguously confirm tri-lineage differentiation. Moreover, potential differences in differentiation capacity are not identified. Therefore, quantification of tri-lineage differentiation would greatly enhance proper characterization of MSCs. In this study, a method to quantify the tri-lineage differentiation potential of MSCs is described using digital image analysis, based on the color deconvolution plug-in (ImageJ). Mesenchymal stem cells from three species, i.e., bovine, equine, and porcine, were differentiated toward adipocytes, chondrocytes, and osteocytes. Subsequently, differentiated MSCs were stained with Oil Red O, Alcian Blue, and Alizarin Red S, respectively. Next, a differentiation ratio (DR) was obtained by dividing the area % of the differentiation signal by the area % of the nuclear signal. Although MSCs isolated from all donors in all species were capable of tri-lineage differentiation, differences were demonstrated between donors using this quantitative DR. Our straightforward, simple but robust method represents an elegant approach to determine the degree of MSC tri-lineage differentiation across species. As such, differences in differentiation potential within the heterogeneous MSC population and between different MSC sources can easily be identified, which will support further optimization of regenerative therapies.

Clonal Selection in Acute Myeloid Leukemia Following Intensive Induction Correlates with Differences in Differentiation Capacity of Leukemic Sub-Clones

Abstract Introduction: Leukemic sub-clones which survive chemotherapy during induction period may remain dormant and subsequently lead to relapse in patients with acute myeloid leukemia (AML). No mechanism linking clonal selection to a specific leukemia-associated genetic aberration is known. Clonal selection is a common phenomenon; therefore, any suggested mechanism should be applicable to all kinds of leukemia-specific genetic aberrations. We hypothesized that clonal selection might result from differences in differentiation capacity among sub-clones and that chemo-resistance is not necessarily directly affected by specific mutations. Patients and Methods: We tested this hypothesis in vitro using the kasumi-1 leukemia cell line, which undergoes limited differentiation while growing and is composed of both CD34+ and CD34- sub-populations. Kasumi-1 cells were sorted by fluorescence-activated cell sorting (FACS-Aria IIIu, BD Biosciences) to CD34+CD117+ and CD34-CD117+ sub-populations. Each sub-population was separately exposed to escalating doses of daunorubicin (DNR) and cytarabine (ARA-C) for 66 hours. Cell viability was determined by alamarBlue assay (Bio-Rad, USA); apoptosis was assessed by Annexin-V and propidium iodide (PI) staining. In vivo experiments included BM samples derived from three AML patients, with both NPM1 and FLT3 gene mutations, who received intensive induction and subsequently relapsed. Blast cells were sorted to CD34+CD117+ and CD34-CD117+ sub-populations. Targeted gene sequencing was conducted using Ion Torrent™ Personal Genome Machine® (PGM) System (Life Technologies, USA). Allelic frequency of NPM1 and DNMT3A mutations in CD34+ and CD34-sub-populations was measured. FLT3-ITD was sequenced using theGeneScan method and theallelic ratio (AR) was calculated. Results: Following sorting,cultured kasumi-1 CD34+ cells partly differentiated into CD34- cells and several weeks later, the phenotype became similar to the original kasumi-1 phenotype (a mixture of 34+/- cells). In contrast, the CD34- sub-population maintained its phenotype. Immediately after sorting by CD34 expression, both sub-populations were exposed to increasing doses of DNR or ARA-C. Only the CD34+ sub-population was found to be resistant to ARA-C (at all tested concentrations), as determined by the cell viability assay (fig 1a). The CD34- sub-population exhibited a 4-fold higher apoptosis rate than CD34+ cells after exposure to 0.16 µM ARA-C. DNR (0.01 µM) resulted in 2.5-fold higher apoptosis in the CD34- compared to CD34+ sub-population (fig 1b). The sub-clonal composition of BM blasts obtained from three AML patients at diagnosis and relapse was compared. NPM1 mutation was identified in all patients at diagnosis. FLT3-ITD mutation was identified in one patient at diagnosis and in the other two at relapse. To explore the differentiation capacity of each sub-clone, blasts were sorted to well-defined CD34+ and CD34- subgroups. At diagnosis, most of the leukemic blasts presented with the CD34- phenotype, while at relapse, the CD34+ population grew in all patients. The FLT3-ITD allelic ratio was higher in CD34+ cells. In the first patient, FLT3-ITD was not detected at diagnosis, while at relapse, the wild-type allele of FLT3 was lost and FLT3-ITD was solely present in the CD34+ sub-population. In CD34- cells, the AR was low (0.55) at relapse. In the second patient, the dominant clone at diagnosis was NPM1mutFLT3wtDNMT3Awt and >95% cells were CD34-. At relapse, the FLT3-ITD DNMT3Awt clone became dominant and was mostly located within the rising CD34+ population. Another sub-clone, exhibiting both FLT3-ITD and DNMT3A R882C mutation, was identified and was found to reside only within the CD34+ population. Similar findings were also observed in the third patient. We screened additional eight patients presenting with FLT3-ITD and in 4 of them, while both CD34+/- blasts were observed, the FLT3-ITD carrying clones resided only in one sub-population. Conclusions: Chemo-resistance and survival of a specific AML sub-clone correlate with its phenotype and differentiation level. Even genetic aberrations which have no direct interaction with chemotherapy biological effect, may give rise to clonal selection. Sub-clonal differentiation capacity may affect its sensitivity to chemotherapy. Disclosures No relevant conflicts of interest to declare.

TMOD-09DISTINCT TUMOR PHENOTYPES FROM NEURAL PROGENITOR CELL-DERIVED TUMORS

Glioblastoma (GBM) is a heterogeneous disease and can exhibit a diversity of cell behaviors and responses to therapy, even when driven by a similar set of oncogenic alterations. Using a murine model for glioma based on the genetic manipulation of neural progenitor cells, we asked whether heterogeneous tumors with different tumor cell behaviors and malignancy could be generated using the same oncogenic driver. We established two tumor progenitor cell (TPC) clones with different survival, an aggressive line with short survival (TPC-S) and a less aggressive line with longer survival (TPC-L). Both TPC clones formed invasive tumors in vivo, however with distinct invasive features. TPC-S cells invaded as cohesive clusters, whereas TPC-L cells invaded diffusely, as single cells. Gene expression analysis, proliferation assays, flow cytometry, and cell differentiation assays, were used to define these two cell populations. Aggressive TPC-S cells expanded at almost twice the rate of TPC-L cells in vitro and were enriched for genes associated with DNA proliferation. TPC-S cells had high expression of neural stem/progenitor cell markers, including Prom1 and Id1 compared to TPC-L. Interestingly, the two lines exhibited differences in differentiation capacity with TPC-S cells unable to differentiate in vitro, while TPC-L cells readily formed both Tuj1 and GFAP expressing cells. In the current project we demonstrate how cells with the same oncogenic driver can give rise to tumors with vastly different properties, reflective of different GBM phenotypes. These lines are being used to examine tumor heterogeneity in both mechanistic and preclinical studies.

Stem Cells, a Reservoir for Life

Stem Cells, a Reservoir for Life

Analysis of Induced Pluripotent Stem Cells from a BRCA1 Mutant Family

SummaryUnderstanding BRCA1 mutant cancers is hampered by difficulties in obtaining primary cells from patients. We therefore generated and characterized 24 induced pluripotent stem cell (iPSC) lines from fibroblasts of eight individuals from a BRCA1 5382insC mutant family. All BRCA1 5382insC heterozygous fibroblasts, iPSCs, and teratomas maintained equivalent expression of both wild-type and mutant BRCA1 transcripts. Although no difference in differentiation capacity was observed between BRCA1 wild-type and mutant iPSCs, there was elevated protein kinase C-theta (PKC-theta) in BRCA1 mutant iPSCs. Cancer cell lines with BRCA1 mutations and hormone-receptor-negative breast cancers also displayed elevated PKC-theta. Genome sequencing of the 24 iPSC lines showed a similar frequency of reprogramming-associated de novo mutations in BRCA1 mutant and wild-type iPSCs. These data indicate that iPSC lines can be derived from BRCA1 mutant fibroblasts to study the effects of the mutation on gene expression and genome stability.

Histology of larval eye-antennal disks and cephalic ganglia of Drosophila cultured in vitro

ABSTRACT A description is given of the histological development of eye-antennal disks and cephalic ganglia of Drosophila melanogaster cultured in vitro for varying lengths of time. The eye-antennal disks do not evaginate but move only to a position normally attained in the late prepupal stage. The relative competence of the various cephalic organs to undergo differentiation is as follows (descending order) : antennal disks, eye disks, brain lobes, ventral ganglion. Possible explanations for this difference in differentiation capacity are given. In general, if a developmental sequence takes place in vivo within 24 h after puparium formation, the same sequence occurs in vitro without a serious time-lag. After this period, however, an interval of from 3 to 9 days is necessary for comparable development.