Morag H. Stewart

IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro.

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.

Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment.

Human embryonic stem cell (hESC) lines are known to be morphologically and phenotypically heterogeneous. The functional nature and relationship of cells residing within hESC cultures, however, has not been evaluated because isolation of single hESCs is limited to drug or manual selection. Here we provide a quantitative method using flow cytometry to isolate and clonally expand hESCs based on undifferentiated markers, alone or in combination with a fluorescent reporter. This method allowed for isolation of stage-specific embryonic antigen-3–positive (SSEA-3+) and SSEA-3− cells from hESC cultures. Although both SSEA-3+ and SSEA-3− cells could initiate pluripotent hESC cultures, we show that they possess distinct cell-cycle properties, clonogenic capacity and expression of ESC transcription factors. Our study provides formal evidence for heterogeneity among self-renewing pluripotent hESCs, illustrating that this isolation technique will be instrumental in further dissecting the biology of hESC lines.

Prevention of Amino Acid Conversion in SILAC Experiments with Embryonic Stem Cells

Recent studies using stable isotope labeling with amino acids in culture (SILAC) in quantitative proteomics have made mention of the problematic conversion of isotope-coded arginine to proline in cells. The resulting converted proline peptide divides the heavy peptide ion signal causing inaccuracy when compared with the light peptide ion signal. This is of particular concern as it can effect up to half of all peptides in a proteomic experiment. Strategies to both compensate for and limit the inadvertent conversion have been demonstrated, but none have been shown to prevent it. Additionally, these methods combined with SILAC labeling in general have proven problematic in their large scale application to sensitive cell types including embryonic stem cells (ESCs) from the mouse and human. Here, we show that by providing as little as 200 mg/liter l-proline in SILAC media, the conversion of arginine to proline can be rendered completely undetectable. At the same time, there was no compromise in labeling with isotope-coded arginine, indicating there is no observable back conversion from the proline supplement. As a result, when supplemented with proline, correct interpretation of “light” and “heavy” peptide ratios could be achieved even in the worst cases of conversion. By extending these principles to ESC culture protocols and reagents we were able to routinely SILAC label both mouse and human ESCs in the absence of feeder cells and without compromising the pluripotent phenotype. This study provides the simplest protocol to prevent proline artifacts in SILAC labeling experiments with arginine. Moreover, it presents a robust, feeder cell-free, protocol for performing SILAC experiments on ESCs from both the mouse and the human.

An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture

The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. However, the factors responsible for preserving the viability of hESCs in a nascent state remain unknown. We describe a mass spectrometry-based method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. Individual samples were analyzed several times, using successive mass (m/z) and retention time-directed exclusion, without sampling the same peptide ion twice. This iterative exclusion -mass spectrometry (IE-MS) approach more than doubled protein and peptide metrics in comparison to a simple repeat analysis method on the same instrument, even after extensive sample pre-fractionation. Furthermore, implementation of the IE-MS approach was shown to enhance the performance of an older quadrupole time of flight (Q-ToF) MS. The resulting number of identified peptides approached that of a parallel repeat analysis on a newer LTQ-Orbitrap MS. The combination of the results of both instruments proved to be superior to that achieved by a single instrument in the identification of additional proteins. Using the IE-MS strategy, combined with complementary gel- and solution-based fractionation methods, the hESC culture microenvironment was extensively probed. Over 10 to 12 times more extracellular proteins were observed compared with previously published surveys. The detection of previously undetectable growth factors, present at concentrations ranging from 10−9 to 10−11 g/ml, highlights the depth of our profiling. The IE-MS approach provides a simple and reliable technique that greatly enhances instrument performance by increasing the effective depth of MS-based proteomic profiling. This approach should be widely applicable to any LC-MS/MS instrument platform or biological system.

Deconstructing human embryonic stem cell cultures : niche regulation of self-renewal and pluripotency

The factors and signaling pathways controlling pluripotent human cell properties, both embryonic and induced, have not been fully investigated. Failure to account for functional heterogeneity within human embryonic stem cell (hESC) cultures has led to inconclusive results in previous work examining extrinsic influences governing hESC fate (self renewal vs. differentiation vs. death). Here, we attempt to reconcile these inconsistencies with recent reports demonstrating that an autologously produced in vitro niche regulates hESCs. Moreover, we focus on the reciprocal paracrine signals within the in vitro hESC niche allowing for the maintenance and/or expansion of the hESC colony-initiating cell (CIC). Based on this, it is clear that separation of hESC-CICs, apart from their differentiated derivatives, will be essential in future studies involving their molecular regulation. Understanding how extrinsic factors control hESC self-renewal and differentiation will allow us to culture and differentiate these pluripotent cells with higher efficiency. This knowledge will be essential for clinical applications using human pluripotent cells in regenerative medicine.

Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease

Loss-of-function mutations in the mouse ortholog of C9ORF72 cause fatal autoimmunity that is transferable by bone marrow–derived cells, demonstrating a hematopoietic intrinsic function for the protein encoded by this gene. C9orf72, a suppressor of autoimmunity? Mutations in C9ORF72 are a common contributor to amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of this gene is still poorly defined. In new work, Burberry et al. demonstrate that mutations disrupting the normal function of C9orf72 cause mice to develop features of autoimmunity. They further found that transplantation of normal bone marrow into mutant animals ameliorated this phenotype, whereas transplantation of mutant bone marrow into normal animals was sufficient to cause autoimmunity. The authors conclude that C9orf72 acts through hematopoietic cells to maintain normal immune function and suggest that investigations are warranted into whether disruptions in immunity contribute to disease in patients. C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. We show that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neutrophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimately leading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was sufficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and premature mortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their phenotype. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflammation and the development of autoimmunity.

Clonal tracking of hESCs reveals differential contribution to functional assays

Human embryonic stem cells (hESCs) have unique self-renewal and differentiation properties, which are experimentally measured using functional assays. hESC cultures are known to be heterogenous, but whether subsets of cells contribute differently to functional assays has yet to be examined. Here, using clonal tracking by retroviral integration, we analyzed in situ the propensity of individual hESCs to contribute to different functional assays. We observed different clonal distributions in teratomas versus in vitro differentiation assays. Some hESC subsets apparently contributed substantially to lineage-specific embryoid body differentiation and lacked clonogenic capacity, although they had self-renewal ability. In contrast, other subsets of self-renewing hESCs with clonogenic ability contributed to teratoma formation but were less frequently observed after in vitro differentiation. Our study suggests that assays used to measure pluripotency may detect distinct subsets of hESCs. These findings have direct implications for hESC-based therapies that may be optimized based on such functional assays.

Complement targeting of nonhuman sialic acid does not mediate cell death of human embryonic stem cells.

Complement targeting of nonhuman sialic acid does not mediate cell death of human embryonic stem cells

The histone demethylase Jarid1b is required for hematopoietic stem cell self-renewal in mice.

Jarid1b/KDM5b is a histone demethylase that regulates self-renewal and differentiation in stem cells and cancer; however, its function in hematopoiesis is unclear. Here, we find that Jarid1b is highly expressed in primitive hematopoietic compartments and is overexpressed in acute myeloid leukemias. Constitutive genetic deletion of Jarid1b did not impact steady-state hematopoiesis. In contrast, acute deletion of Jarid1b from bone marrow increased peripheral blood T cells and, following secondary transplantation, resulted in loss of bone marrow reconstitution. Our results reveal that deletion of Jarid1b compromises hematopoietic stem cell (HSC) self-renewal capacity and suggest that Jarid1b is a positive regulator of HSC potential.

Human ESC Colony Formation Is Dependent on Interplay Between Self-Renewing hESCs and Unique Precursors Responsible for Niche Generation

Human embryonic stem cell (hESC) cultures are heterogeneous and constituting paracrine signals are required to maintain pluripotency. The cellular interplay and dynamic nature of this heterogeneity is not understood. Here, long‐term hESC imaging and tracking revealed that hESC heterogeneity is dynamic and hESC self‐renewal is dependent on colony‐proximal distributions of paracrine signals. Tracking of hESCs forming colonies revealed that a biologically distinct cell type arises at the colony periphery in the absence of feeders. Higher rates of cell death occur in these hESC‐derived cells, leading to clonal selection of colony reestablishing cells. hESC‐derived feeders co‐transferred during passaging promoted rapid colony recovery and expansion and reduced overall clonal selection of self‐renewing hESCs. Our findings demonstrate that hESC‐derived feeders arise from a distinct subpopulation of hESCs that respond to paracrine cues at the colony periphery that are required to sustain and establish clonal hESC self‐renewal.