Karen M. Helm

De novo generation of white adipocytes from the myeloid lineage via mesenchymal intermediates is age, adipose depot, and gender specific

It is generally assumed that white adipocytes arise from resident adipose tissue mesenchymal progenitor cells. We challenge this paradigm by defining a hematopoietic origin for both the de novo development of a subset of white adipocytes in adults and a previously uncharacterized adipose tissue resident mesenchymal progenitor population. Lineage and cytogenetic analysis revealed that bone marrow progenitor (BMP)-derived adipocytes and adipocyte progenitors arise from hematopoietic cells via the myeloid lineage in the absence of cell fusion. Global gene expression analysis indicated that the BMP-derived fat cells are bona fide adipocytes but differ from conventional white or brown adipocytes in decreased expression of genes involved in mitochondrial biogenesis and lipid oxidation, and increased inflammatory gene expression. The BMP-derived adipocytes accumulate with age, occur in higher numbers in visceral than in subcutaneous fat, and in female versus male mice. BMP-derived adipocytes may, therefore, account in part for adipose depot heterogeneity and detrimental changes in adipose metabolism and inflammation with aging and adiposity.

Adult lung side population cells have mesenchymal stem cell potential

BACKGROUND The development of stem cell therapy for pulmonary diseases remains a challenge. Many diverse cell types reside within the lung and a common stem cell has not yet been identified. A basic understanding of lung stem cell fate during disease may prove important for drug intervention as well as autologous therapies. Niches for resident mesenchymal stem cells (MSC) have been identified in many adult tissues and more recently in the lung. We present data to confirm the observation that non-hematopoietic CD45(neg) lung side population (SP) cells contain MSC, single cells capable of multilineage differentiation. METHODS We carried these observations forward by analyzing the MSC potential of single-cell clones, as well as their chromosomal stability and telomerase activity. RESULTS The expression of MSC markers was characterized in mouse CD45(neg) lung SP by flow cytometry on freshly isolated or cultured clonal populations. The karyotype of these cells was subsequently assayed by banding analysis, and telomerase activity was assessed using quantitative polymerase chain reaction. MSC differentiation potential was confirmed by the characteristic ability of single-cell clones to differentiate into cells of three mesenchymal lineages, chondrocytes, adipocytes and osteocytes. Differentiation was confirmed by histochemical analysis. All analyzed populations of CD45(neg) lung SP expressed mesenchymal markers (CD44, CD90, CD105, CD106, CD73 and Sca-I) and lacked hematopoietic markers (CD45, c-kit, CD11b, CD34 and CD14). The cultured and clonal CD45(neg) lung SP had normal chromosomal structures and expressed high levels of telomerase. After being expanded and cultured in differentiation medium, all populations of CD45(neg) lung SP demonstrated adipogenic, osteogenic and chrondrogenic potential. Adult CD45(neg) lung SP cells are a source of MSC. DISCUSSION In defining this tissue-specific stem cell population in the lung, we are now better able to clarify a potential role for them in lung diseases.

Identification of Novel Resident Pulmonary Stem Cells: Form and Function of the Lung Side Population

Resident lung stem cells function to replace all lineages of pulmonary tissue, including mesenchyme, epithelium, and vasculature. The phenotype of the lung side population (SP) cells is currently under investigation; their function is currently unknown. Recent data suggest lung SP cells are an enriched tissue‐specific source of organ‐specific pulmonary precursors and, therefore, a source of adult stem cells. The adult lung SP cell population has been isolated and characterized for expression of markers indicative of stem cell, epithelial, and mesenchymal lineages. These studies determined that the adult mouse lung SP has epithelial and mesenchymal potential that resides within a CD45− mesenchymal subpopulation, as well as limited hematopoietic ability, which resides in the bone marrow–derived CD45+ subpopulation. The ability to identify these adult lung precursor cells allows us to further study the potential of these cells and their role in the regulation of tissue homeostasis and response to injury. The identification of this target population will potentially allow earlier treatment and, long term, a functional restoration of injured pulmonary tissue and lung health.

Comparative Analysis of the CD19+ and CD138+ Cell Antibody Repertoires in the Cerebrospinal Fluid of Patients with Multiple Sclerosis

Increased amounts of intrathecally synthesized IgG and oligoclonal bands have long been recognized as a hallmark of multiple sclerosis (MS). B cells and plasma cells are components of the inflammatory infiltrates in both active and chronic MS lesions, and increased numbers of these cells are present in MS cerebrospinal fluid (CSF). Single-cell RT-PCR was used to analyze both the CD19+ B cell and CD138+ plasma cell populations in CSF of two patients with clinically definite MS and of one MS patient whose CSF was obtained after a clinically isolated syndrome, but before the second episode. Sequence analysis of amplified IgG V region sequences identified the rearranged germline segments, extent of somatic mutation, and clonal relationships within and between the two cell populations in the three MS patients. Expanded B cell and plasma cell clones were detected in each MS CSF and in all three patients the CD138+ IgG repertoire was more restricted. However, little if any significant sequence overlap was observed between the CD19+ and CD138+ repertoires of each donor. Detection of plasma cell clones by single-cell PCR will facilitate the in vitro production of recombinant Abs useful in identifying disease-relevant Ags.

A Single Cell Functions as a Tissue-Specific Stem Cell and the In Vitro Niche-Forming Cell

Tissue-specific stem cell (TSC) behavior is determined by the stem cell niche. However, delineation of the TSC-niche interaction requires purification of both entities. We reasoned that the niche could be defined by the location of the TSC. We demonstrate that a single CD49f(bright)/Sca1(+)/ALDH(+) basal cell generates rare label-retaining cells and abundant label-diluting cells. Label-retaining and label-diluting cells were located in the rimmed domain of a unique clone type, the rimmed clone. The TSC property of self-renewal was tested by serial passage at clonal density and analysis of clone-forming cell frequency. A single clone could be passaged up to five times and formed only rimmed clones. Thus, rimmed clone formation was a cell-intrinsic property. Differentiation potential was evaluated in air-liquid interface cultures. Homogenous cultures of rimmed clones were highly mitotic but were refractory to standard differentiation signals. However, rimmed clones that were cocultured with unfractionated tracheal cells generated each of the cell types found in the tracheal epithelium. Thus, the default niche is promitotic: Multipotential differentiation requires adaptation of the niche. Because lung TSCs are typically evaluated after injury, the behavior of CD49f(bright)/Sca1(+)/ALDH(+) cells was tested in normal and naphthalene-treated mice. These cells were mitotically active in the normal and repaired epithelium, their proliferation rate increased in response to injury, and they retained label for 34 days. We conclude that the CD49f(bright)/Sca1(+)/ALDH(+) tracheal basal cell is a TSC, that it generates its own niche in vitro, and that it participates in tracheal epithelial homeostasis and repair.

ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling

Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2(pos) mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2(pos) cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2(pos) MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-β1 (TGF-β1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-β1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-β1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2(pos) lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.

Characterization and phenotypic analysis of differentiating CD34+human bone marrow cells in liquid culture

Abstract:  Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34+ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34+ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34+ bone marrow cells were introduced into suspension culture and monitored over a 6‐d period using 3‐colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine‐, drug‐ or chemical‐induced alterations in haematopoietic progenitor cell differentiation in vitro.

Human Tracheobronchial Basal Cells. Normal versus Remodeling/Repairing Phenotypes In Vivo and In Vitro

Human tracheobronchial epithelial (TBE) basal cells (BCs) function as progenitors in normal tissue. However, mechanistic studies are typically performed in vitro and frequently use BCs recovered from patients who die of nonrespiratory disease. It is not known whether the cadaveric epithelium (1) is undergoing homeostatic remodeling and/or repair, or (2) yields BC clones that represent homeostatic processes identified in tissue. We sought to compare the phenotype of TBE-BCs with that of BCs cultured under optimal clone-forming conditions. TBE pathology was evaluated using quantitative histomorphometry. The cultured BC phenotype was determined by fluorescence-activated cell sorter analysis. Clone organization and cell phenotype were determined by immunostaining. The cadaveric TBE is 20% normal. In these regions, BCs are keratin (K)-5(+) and tetraspanin CD151(+), and demonstrate a low mitotic index. In contrast, 80% of the cadaveric TBE exhibits homeostatic remodeling/repair processes. In these regions, BCs are K5(+)/K14(+), and a subset expresses tissue factor (TF). Passage 1 TBE cells are BCs that are K5(+)/TF(+), and half coexpress CD151. Optimal clone formation conditions use an irradiated NIH3T3 fibroblast feeder layer (American Type Culture Collection, Frederick, MD) and serum-supplemented Epicult-B medium (Stemcell Technologies, La Jolla, CA). The TF(+)/CD151(-) BC subpopulation is the most clonogenic BC subtype, and is enriched with K14(+) cells. TF(+)/CD151(-) BCs generate clones containing BCs that are K5(+)/Trp63(+), but K14(-)/CD151(-). TF(+) cells are limited to the clone edge. In conclusion, clonogenic human TBE BCs (1) exhibit a molecular phenotype that is a composite of the normal and remodeling/reparative BC phenotypes observed in tissue, and (2) generate organoid clones that contain phenotypically distinct BC subpopulations.

Publishing flow cytometry data.

Cellular measurements by flow cytometric analysis constitute an important step toward understanding individual attributes within a population of cells. Assessing individual cells within a population by protein expression using fluorescently labeled antibodies and other fluorescent probes can identify cellular patterns. The technology for accurately identifying subtle changes in protein expression within a population of cells using a vast array of technology has resulted in controversy and questions regarding reproducibility, which can be explained at least in part by the absence of standard methods to facilitate comparison of flow cytometric data. The complexity of technological advancements and the need for improvements in biological resolution results in the generation of complex data that demands the use of minimum standards for their publication. Herein we present a summarized view for the inclusion of consistent flow cytometric experimental information as supplemental data. Four major points, experimental and sample information, data acquisition, analysis, and presentation are emphasized. Together, these guidelines will facilitate the review and publication of flow cytometry data that provide an accurate foundation for ongoing studies with this evolving technology.

Suppression of Apoptosis Induced by Growth Factor Withdrawal by an Oncogenic Form of c-Cbl

The v-Cbl oncogene induces myeloid and B-cell leukemia; however, the mechanism by which transformation occurs is not understood. An oncogenic form of c-Cbl (Cbl-ΔY371) was expressed in the interleukin-3 (IL-3)-dependent cell line 32Dcl3 to determine whether it was able to induce growth factor-independent proliferation. We were unable to isolate clones of transfected 32Dcl3 cells expressing Cbl-ΔY371 that proliferated in the absence of IL-3. In contrast, 32Dcl3/Cbl-ΔY371 cells did not undergo apoptosis like parental 32Dcl3 cells when cultured in the absence of IL-3. Both 32Dcl3 and 32D/CblΔY371 cells arrested in G1 when cultured in the absence of IL-3. Approximately 18% of the 32Dcl3 cells cultured in the absence of IL-3 for 24 h were present in a sub-G1 fraction, while only 4% of the 32D/Cbl-ΔY371 and 2% of the 32D/Bcl-2 cells were found in a sub-G1 fraction. There was no difference in the pattern of tyrosine-phosphorylated proteins observed following stimulation of either cell type with IL-3. The phosphorylation of JAK2, STAT5, and endogenous c-Cbl was identical in both cell types. No differences were detected in the activation of Akt, ERK1, or ERK2 in unstimulated or IL-3-stimulated 32D/Cbl-ΔY371 cells compared with parental 32Dcl3 cells. Likewise, there was no difference in the pattern of phosphorylation of JAK2, STAT5, ERK1, ERK2, or Akt when 32Dcl3 and 32D/CblDY371 cells were withdrawn from medium containing IL-3. The protein levels of various Bcl-2 family members were examined in cells grown in the absence or presence of IL-3. We observed a consistent increased amount of Bcl-2 protein in five different clones of 32D/Cbl-ΔY317 cells. These data suggest that the Cbl-ΔY371 mutant may suppress apoptosis by a mechanism that involves the overexpression of Bcl-2. Consistent with this result, activation of caspase-3 was suppressed in 32D/Cbl-ΔY371 cells cultured in the absence of IL-3 compared with 32Dcl3 cells cultured under the same conditions.