Early Progenitor Cell Populations Research Articles

Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus.

Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.

Soluble Signals and Remodeling in a Synthetic Gelatin-Based Hematopoietic Stem Cell Niche.

Hematopoietic stem cells (HSCs) reside in the bone marrow within niches that provide microenvironmental signals in the form of biophysical cues, bound and diffusible biomolecules, and heterotypic cell-cell interactions that influence HSC fate decisions. This study seeks to inform the development of a synthetic culture platform that promotes ex vivo HSC expansion without exhaustion. A library of methacrylamide-functionalized gelatin (GelMA) hydrogels is used to explore remodeling and crosstalk from mesenchymal stromal cells (MSCs) on the expansion and quiescence of murine HSCs. The use of a degradable GelMA hydrogel enables MSC-mediated remodeling, yielding dynamic shifts in the matrix environment over time. An initially low-diffusivity hydrogel for co-culture of hematopoietic stem and progenitor cells to MSCs facilitates maintenance of an early progenitor cell population over 7 days. Excitingly, this platform promotes retention of a quiescent HSC population compared to HSC monocultures. These studies reveal MSC-density-dependent upregulation of MMP-9 and changes in hydrogel mechanical properties (ΔE = 2.61 ± 0.72) suggesting MSC-mediated matrix remodeling may contribute to a dynamic culture environment. Herein, a 3D hydrogel is reported for ex vivo HSC culture, in which HSC expansion and quiescence is sensitive to hydrogel properties, MSC co-culture, and MSC-mediated hydrogel remodeling.

Conditional Creation and Rescue of Nipbl-Deficiency in Mice Reveals Multiple Determinants of Risk for Congenital Heart Defects.

Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart, which takes place early in development, involves contributions from multiple germ layers, and is controlled by many genes. Here, we use a conditional/invertible genetic strategy to identify the cell lineage(s) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome, in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects (ASDs) at high frequency. Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages, we uncover complex interactions between the cardiac mesoderm, endoderm, and the rest of the embryo, whereby the risk conferred by genetic abnormality in any one lineage is modified, in a surprisingly non-additive way, by the status of others. We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form.

Mass Cytometric Functional Profiling of Acute Myeloid Leukemia Defines Cell-Cycle and Immunophenotypic Properties That Correlate with Known Responses to Therapy.

Acute myeloid leukemia (AML) is characterized by a high relapse rate that has been attributed to the quiescence of leukemia stem cells (LSC), which renders them resistant to chemotherapy. However, this hypothesis is largely supported by indirect evidence and fails to explain the large differences in relapse rates across AML subtypes. To address this, bone marrow aspirates from 41 AML patients and five healthy donors were analyzed by high-dimensional mass cytometry. All patients displayed immunophenotypic and intracellular signaling abnormalities within CD34(+)CD38(lo) populations, and several karyotype- and genotype-specific surface marker patterns were identified. The immunophenotypic stem and early progenitor cell populations from patients with clinically favorable core-binding factor AML demonstrated a 5-fold higher fraction of cells in S-phase compared with other AML samples. Conversely, LSCs in less clinically favorable FLT3-ITD AML exhibited dramatic reductions in S-phase fraction. Mass cytometry also allowed direct observation of the in vivo effects of cytotoxic chemotherapy. The mechanisms underlying differences in relapse rates across AML subtypes are poorly understood. This study suggests that known chemotherapy sensitivities of common AML subsets are mediated by cell-cycle differences among LSCs and provides a basis for using in vivo functional characterization of AML cells to inform therapy selection.

Mass Cytometric Analysis of AML Stem and Early Progenitor Cells Reveals Karyotype and Genotype-Specific Cell Cycle Properties That Correlate with Known Responses to Chemotherapy

Introduction: Leukemic stem cells (LSCs) are recognized as important mediators of chemotherapy resistance and leukemia relapse. The postulated mechanism for this is the relative quiescence of these cell populations that renders them resistant to cytotoxic agents. This simple hypothesis, however, is supported almost entirely by indirect evidence, and fails to explain the large differences in relapse rates across different AML subtypes. To address this question, we have developed a mass cytometry (MCM) approach to assess the cell cycle of immunophenotypically complex primary samples from patients with AML. By processing samples immediately upon bone marrow harvest, we could determine if AML stem cells were quiescent in vivo and if the cell cycle properties of these cells varied between chemotherapy-responsive versus resistant AML subtypes.Methods: Bone marrow aspirates from 33 AML patients, 3 with APL, 2 with high-risk MDS, 5 with AML who achieved a CR with chemotherapy treatment, and 5 healthy donors (48 total samples) were incubated at 37°C for 15 minutes with 20uM Iodo-deoxyuridine (IdU) immediately after aspiration (<1 min), followed by fixation and storage. Samples were then analyzed with two overlapping 39-antibody MCM panels (50 markers total). Cellular barcoding was utilized to stain and analyze cells in tubes of 20 samples each, enabling direct comparison of samples to each other and to the healthy controls.Results: The high dimensionality of MCM enabled the simultaneous measurement of 25 surface markers and the identification of almost all immunophenotypic populations in human bone marrow. The use of barcoding, and the resultant ability to directly compare samples, enabled the detection of aberrant marker expression at very high resolution (2-3 fold changes). At least one surface marker aberrancy was detected in each AML sample. Unexpectedly, cell cycle analysis revealed that, compared to immunophenotypically similar normal cells, the average fraction of S-phase cells in AML samples was significantly lower. In both AML and healthy samples, the lowest S-phase fraction was found in fully differentiated populations and in hematopoietic stem cells (HSCs) while committed progenitor populations (myelo-monoblasts, promyelocytes, erythroblasts) exhibited the highest S-phase fraction. The HSC and early progenitor cell populations from patients with CBF AML (t(8;21) and inv(16)) demonstrated a significantly higher S-phase fraction than the same cell populations from the other AML samples (7.76% vs. 2.66%; p=0.0014). Furthermore, samples with FLT3-ITD mutations exhibited the lowest S-phase fraction in the HSC and early progenitor cell populations (0.63%), which was significantly lower than the S-phase fraction of the other AML samples (4.37%; p=9.3x10-4).Finally, a subset of patients (n=10) was being treated with hydroxyurea (HU) at the time of their bone marrow aspiration. The effect of HU treatment was manifest as a reduction in the IdU incorporation rate (with no change in S-phase fraction) in the cells of the treated patients. However, neither cell cycle arrest nor apoptosis were observed in these samples. This is in contrast with the commonly observed occurrence of both in leukemic cell lines treated in vitro with HU.Conclusions: By combining fresh sample processing with high-dimensional MCM analysis, we developed an innovative approach for the analysis of hematologic malignancies. Our results suggest that the relative sensitivity of CBF AML to cytotoxic chemotherapy may be the result of the increased fraction of S-phase cells within the HSC and early progenitor cell populations. Conversely, HSC and early progenitor cell populations from patients with FLT3-ITD mutations would be expected to be particularly resistant to cytarabine-based consolidation therapy due to the very low frequency of S-phase cells within these populations. This finding, combined with our observation that the stem and early progenitor cells from the FLT3-ITD samples have high expression of CD33, may provide a mechanistic explanation for the improved disease-free survival recently reported for FLT3-ITD AML patients treated with fractioned gemtuzumab ozogamicin in combination with standard therapy. [Display omitted] [Display omitted] [Display omitted] DisclosuresBehbehani:Fluidigm: Consultancy. Medeiros:Agios: Consulting - Ad board Other. Nolan:Fluidigm, Inc: Consultancy, Equity Ownership.

Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts

LIN28 proteins bind to RNA and regulate developmental timing events in multicellular organisms, in part, by reducing cellular levels of the let-7 family of microRNAs. High-level LIN28 expression in stem cells promotes their self-renewal. Over-expression of the LIN28 proteins causes suppression of let-7 in hematopoietic stem and early progenitor cell populations (CD34+) from adult donors and manifests a more fetal-like phenotype in the erythroid lineage. Here we explore LIN28expression that is restricted to erythroid cells, rather than stem or multi-potential progenitor cells.For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes, as well as an internal ribosomal entry site for puromycin selection (vectors: KLF1-LIN28A-OE and SPTA1-LIN28A-OE). Viral supernatants from these constructs were compared with empty-vector controls in matched transductions of CD34+ cells from three adult human volunteers. The cells were transduced and cultured using a three-phase, serum-free model for ex vivo erythropoiesis. Erythroblast proliferation and differentiation were comparable between control and LIN28-transduced cells assessed by cell counting and flow cytometry with staining for CD71, glycophorin A and thiazole orange. To validate restricted expression of LIN28 in the erythroid lineage, colony formation assays were performed in semisolid methylcellulose containing 1.0 ug/ml puromycin. BFU-E, CFU-GM, CFU-G, CFU-M and GEMM colonies were enumerated 14 days after plating. Puromycin addition to KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions resulted in selection of the erythroid colonies (BFU-E as a percentage of total colonies: Control: 44.6 ± 6.1%; KLF1-LIN28A-OE: 98.4 ± 0.7%, p=0.003; SPTA1-LIN28A-OE: 95.2 ± 1.1%, p=0.005).LIN28A over-expression was confirmed by RT-QPCR (KLF1-LIN28A-OE: 2.1E+05 ± 7.0E+04 copies/ng; SPTA1-LIN28A-OE: 2.2E+05 ± 8.3E+04 copies/ng; Controls: below detection limits) and Western analyses after transduction. Suppression of all let-7 miRNA family members to less than 30% control levels were detected for both vectors resulting in a reduction in total let-7 miRNA (RT-QPCR: Control: 2.0E+07 ± 9.7E+05 copies/ng; KLF1-LIN28A-OE: 5.6E+06 ± 5.6E+05 copies/ng, p=0.003; SPTA1-LIN28A-OE: 4.6E+06 ± 6.2E+05 copies/ng, p=0.003). BCL11A expression levels were also measured by RT-QPCR and Western analyses. While BCL11A showed no significant change at the mRNA level (Control: 1.2E+03 ± 4.5E+02 copies/ng; KLF1-LIN28A-OE: 2.9E+02 ± 7.4E+01 copies/ng, p=0.07; SPTA1-LIN28A-OE: 4.2E+02 ± 3.3E+02 copies/ng, p=0.07), protein analyses of nuclear BCL11A showed moderately reduced levels after KLF1-LIN28A-OE and SPTA1-LIN28A-OE transductions.Globin mRNA and protein levels were investigated and compared with controls. Gamma-globin mRNA was significantly increased in LIN28A-OE samples (Control: 3.6E+06 ± 8.2E+05 copies/ng; KLF1-LIN28A-OE: 1.9E+07 ± 1.7E+06 copies/ng, p=0.007; SPTA1-LIN28A-OE: 1.7E+07 ± 8.9E+05 copies/ng, p=0.003). Fetal hemoglobin (HbF) production was measured at the end of the culture period using High Performance Liquid Chromatography, and was increased in the KLF1-LIN28A-OE and SPTA1-LIN28A-OE samples compared to the control (Control: 7.0 ± 1.4%; KLF1-LIN28A-OE: 31.9 ± 2.7%, p=0.004; SPTA1-LIN28A-OE: 43.0 ± 6.2%, p=0.004). Flow cytometry analyses demonstrated a pan-cellular HbF distribution.In contrast to promoting self-renewal in stem cells, these data suggest that adult erythroblast-restricted LIN28 functions to partially reverse the fetal-to-adult developmental transition in hemoglobin expression. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5164: Lacking clonal relationship in 20% of relapsing diffuse large B-cell lymphomas

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults. Nearly half of the patients can be cured with standard therapeutic regimens. However, a substantial number of patients experience a recurrence. In general, lymphoma recurrences are considered to represent a relapse of the original neoplasm, but this concept has been recently challenged by demonstration of clonally unrelated relapses by immunoglobulin (heavy chain) gene (IGH) rearrangement analysis. Although being the most widely used method for clonality testing, IGH fragment length analysis has a very limited output of information not giving any insight about tumor heterogeneity and evolution in time. Also, the result is based only on a single gene in the whole genome, and therefore it can be error-prone. We applied a genome-wide clonality testing on 17 paired primary and relapsed DLBCL aiming to provide an unambiguous answer about the existence of clonally unrelated recurrences in this entity. Additionally, we profiled 10 cases of non-relapsing DLBCL searching for genetic markers at diagnosis which could predict lymphoma relapse. Methods: Genomic DNA extracted from formalin-fixed paraffin-embedded tissue with at least 70% tumor content was used for the analysis. Array-comparative genomic hybridization was utilized to detect chromosomal copy number aberrations and to determine clonal relationship status of each primary-relapse pair. This result was then verified by IGH gene fragment length analysis. Directed deep-sequencing was performed on a custom hot-spot panel consisting of most frequently mutated genes in DLBCL. Results: Among 17 cases of recurring DLBCL, 3 relapses (17%) were identified which were clonally unrelated to the primary disease by copy number aberration and IGH fragment length analysis. Different modes of tumor progression could be detected within clonally related set of relapses, providing evidence of a common early progenitor cell population in some patients. Moreover, some DLBCL exhibit substantial intra- and inter- tumoral heterogeneity, which is probably a source of variation in patient outcomes and clonal progression. Comparison of copy number aberration and point mutation profiles between relapsing and non-relapsing tumors yielded significant differences, which could be useful for understanding the genetic basis of DLBCL recurrence. Conclusions: We provide a clear evidence for the existence of clonally unrelated relapses in DLBCL. The existence of clonally unrelated relapses might be of clinical importance since relapses are usually treated more aggressively than primary neoplasms with a significant treatment-related morbidity, and this may be by-passed in the former instances. Moreover, the high definition clonal analyses provide new insights into the molecular profiles of tumors in vivo, and therefore might contribute to the development of more personalized approaches for cancer treatment. Citation Format: Darius Juskevicius, Alexander Rufle, Christian Ruiz, Stephan Dirnhofer, Alexandar Tzankov. Lacking clonal relationship in 20% of relapsing diffuse large B-cell lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5164. doi:10.1158/1538-7445.AM2014-5164

A Postnatal Pax7+ Progenitor Gives Rise to Pituitary Adenomas

Pituitary adenomas are classified into functioning and nonfunctioning (silent) tumors on the basis of hormone secretion. However, the mechanism of tumorigenesis and the cell of origin for pituitary adenoma subtypes remain to be elucidated. Employing a tamoxifen-inducible mouse model, we demonstrate that a novel postnatal Pax7(+) progenitor cell population in the pituitary gland gives rise to silent corticotroph macro-adenomas when the retinoblastoma tumor suppressor is conditionally deleted. While Pax transcriptional factors are critical for embryonic patterning as well as postnatal stem cell renewal for many organs, we have discovered that Pax7 marks a restricted cell population in the postnatal pituitary intermediate lobe. This Pax7(+) early progenitor cell population is overlapping but ontologically downstream of the Nestin(+) pituitary stem cell population, yet upstream of another newly discovered Myf6(+) late progenitor cell population. Interestingly, the Pax7(+) progenitor cell population is evolutionarily conserved in primates and humans, and Pax7 expression is maintained not only in murine tumors but also in human functioning and silent corticotropinomas. Taken together, our results strongly suggest that human silent corticotroph adenomas may in fact arise from a Pax7 lineage of the intermediate lobe, a region of the human pituitary bearing closer scientific interest as a reservoir of pituitary progenitor cells.

Leukemia Inhibitory Factor Secretion is a Predictor and Indicator of Early Progenitor Status in Adult Bone Marrow Stromal Cells

Bone marrow-derived stromal cells (BMSCs) are defined by their ability to self-renew and differentiate into at least three mesenchymal cell types (bone, adipose, and cartilage). The inability to isolate a reliably efficacious and homogeneous population of early progenitor cells has limited efforts to increase their therapeutic potential. In this study, we focused on identifying protein markers that may be employed to predict the efficacy of a cultured BMSC population. Markers of progenitor status were identified by comparing BMSCs at early and late passage, donor-matched skin fibroblasts, and commercially available dermal fibroblast cell lines. Differentiation potential was determined according to in vitro assays of osteogenesis, adipogenesis, and chondrogenesis. Early-passage BMSCs differentiated into all three lineages, whereas late-passage BMSCs and both fibroblast preparations did not. To identify novel markers of early progenitors, microarray transcript analysis between early-passage BMSCs and fibroblasts was performed. Messenger RNA encoding the cytokine leukemia inhibitory factor (LIF) was identified as differentially expressed. Enzyme-linked immunosorbent assay on conditioned media confirmed that LIF secretion was much higher from early progenitor BMSCs than donor-matched or commercial lines of fibroblasts and dropped with extensive expansion or induction of differentiation. In clonally expanded BMSCs, colonies that retained progenitor status expressed significantly higher levels of LIF than those that failed to differentiate. Our results indicate that LIF expression may represent a marker to quantify the differentiation potential of BMSCs and may be especially suited for the rapid, noninvasive quality control of clinical preparations.

Transforming Growth Factor-β Can Suppress Tumorigenesis through Effects on the Putative Cancer Stem or Early Progenitor Cell and Committed Progeny in a Breast Cancer Xenograft Model

The transforming growth factor-beta (TGF-beta) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-beta is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-beta has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. First, TGF-beta reduces the size of the putative cancer stem or early progenitor cell population, and second it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-beta receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-beta involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the antiproliferative effect of TGF-beta. These data suggest new roles for the TGF-beta pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation.

Long-term T-cell reconstitution after hematopoietic stem-cell transplantation in primary T-cell–immunodeficient patients is associated with myeloid chimerism and possibly the primary disease phenotype

AbstractWe studied T-cell reconstitution in 31 primary T-cell–immunodeficient patients who had undergone hematopoietic stem-cell transplantation (HSCT) over 10 years previously. In 19 patients, there was no evidence of myeloid chimerism because little or no myeloablation had been performed. Given this context, we sought factors associated with good long-term T-cell reconstitution. We found that all patients having undergone full myeloablation had donor myeloid cells and persistent thymopoiesis, as evidenced by the presence of naive T cells carrying T-cell receptor excision circles (TRECs). In 9 patients with host myeloid chimerism, sustained thymic output was also observed and appeared to be associated with γc deficiency. It is therefore possible that the complete absence of thymic progenitors characterizing this condition created a more favorable environment for thymic seeding by a population of early progenitor cells with the potential for self-renewal, thus enabling long-term (> 10 years) T-cell production.

Fetal thymocyte potential for T cell receptor V gamma 3-J gamma 1 junctional modification.

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non-germ-line-encoded nucleotides (N-regions) in the V-J gamma junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N-region additions in V-J gamma joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to the V gamma 3-J gamma 1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. V gamma 3-J gamma 1 sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N-region additions within V-J gamma joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical V gamma 3-J gamma 1 join (a frequent junction lacking N-region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from V gamma 3-J gamma 1 joins.