Anne Lindgren

Hydrodynamic stretching of single cells for large population mechanical phenotyping

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.

Directed differentiation of human induced pluripotent stem cells generates active motor neurons

The potential for directed differentiation of human‐induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS‐derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS‐derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS‐derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. STEM CELLS 2009;27:806–811

Female Human iPSCs Retain an Inactive X Chromosome

Generating induced pluripotent stem cells (iPSCs) requires massive epigenome reorganization. It is unclear whether reprogramming of female human cells reactivates the inactive X chromosome (Xi), as in mouse. Here we establish that human (h)iPSCs derived from several female fibroblasts under standard culture conditions carry an Xi. Despite the lack of reactivation, the Xi undergoes defined chromatin changes, and expansion of hiPSCs can lead to partial loss of XIST RNA. These results indicate that hiPSCs are epigenetically dynamic and do not display a pristine state of X inactivation with two active Xs as found in some female human embryonic stem cell lines. Furthermore, whereas fibroblasts are mosaic for the Xi, hiPSCs are clonal. This nonrandom pattern of X chromosome inactivation in female hiPSCs, which is maintained upon differentiation, has critical implications for clinical applications and disease modeling, and could be exploited for a unique form of gene therapy for X-linked diseases.

Derivation of Primordial Germ Cells from Human Embryonic and Induced Pluripotent Stem Cells Is Significantly Improved by Coculture with Human Fetal Gonadal Cells

The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study, we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that codifferentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). To better characterize the iPGCs, we performed Real‐time polymerase chain reaction, microarray, and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure. STEM CELLS 2009;27:783–795

Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells.

Pluripotent stem cells hold significant promise in regenerative medicine due to their unlimited capacity for self‐renewal and potential to differentiate into any cell type of the body. In this study, we demonstrate that proper mitochondrial function is essential for proliferation of undifferentiated ESCs. Attenuating mitochondrial function under self‐renewing conditions makes these cells more glycolytic‐dependent, and it is associated with an increase in the mRNA reserves of Nanog, Oct4, and Sox2. In contrast, attenuating mitochondrial function during the first 7 days of differentiation results in normal repression of Oct4, Nanog, and Sox2. However, differentiation potential is compromised as revealed by abnormal transcription of multiple Hox genes. Furthermore, under differentiating conditions in which mitochondrial function is attenuated, tumorigenic cells continue to persist. Our results, therefore establish the importance of normal mitochondrial function in ESC proliferation, regulating differentiation, and preventing the emergence of tumorigenic cells during the process of differentiation. STEM CELLS 2011;486–495

The roof plate regulates cerebellar cell-type specification and proliferation

During embryogenesis, the isthmic organizer, a well-described signaling center at the junction of the mid-hindbrain, establishes the cerebellar territory along the anterior/posterior axis of the neural tube. Mechanisms specifying distinct populations within the early cerebellar anlage are less defined. Using a newly developed gene expression map of the early cerebellar anlage, we demonstrate that secreted signals from the rhombomere 1 roof plate are both necessary and sufficient for specification of the adjacent cerebellar rhombic lip and its derivative fates. Surprisingly, we show that the roof plate is not absolutely required for initial specification of more distal cerebellar cell fates, but rather regulates progenitor proliferation and cell position within the cerebellar anlage. Thus, in addition to the isthmus, the roof plate represents an important signaling center controlling multiple aspects of cerebellar patterning.

Lmx1a regulates fates and location of cells originating from the cerebellar rhombic lip and telencephalic cortical hem

The cerebellar rhombic lip and telencephalic cortical hem are dorsally located germinal zones which contribute substantially to neuronal diversity in the CNS, but the mechanisms that drive neurogenesis within these zones are ill defined. Using genetic fate mapping in wild-type and Lmx1a−/− mice, we demonstrate that Lmx1a is a critical regulator of cell-fate decisions within both these germinal zones. In the developing cerebellum, Lmx1a is expressed in the roof plate, where it is required to segregate the roof plate lineage from neuronal rhombic lip derivatives. In addition, Lmx1a is expressed in a subset of rhombic lip progenitors which produce granule cells that are predominantly restricted to the cerebellar posterior vermis. In the absence of Lmx1a, these cells precociously exit the rhombic lip and overmigrate into the anterior vermis. This overmigration is associated with premature regression of the rhombic lip and posterior vermis hypoplasia in Lmx1a−/− mice. These data reveal molecular organization of the cerebellar rhombic lip and introduce Lmx1a as an important regulator of rhombic lip cell-fate decisions, which are critical for maintenance of the entire rhombic lip and normal cerebellar morphogenesis. In the developing telencephalon Lmx1a is expressed in the cortical hem, and in its absence cortical hem progenitors contribute excessively to the adjacent hippocampus instead of producing Cajal-Retzius neurons. Thus, Lmx1a activity is critical for proper production of cells originating from both the cerebellar rhombic lip and the telencephalic cortical hem.

A Self‐Renewal Program Controls the Expansion of Genetically Unstable Cancer Stem Cells in Pluripotent Stem Cell‐Derived Tumors

Human germ cell tumors are often metastatic, presumably due to distal site tumor growth by cancer stem cells. To determine whether cancer stem cells can be identified in a transplantation model of testicular germ cell tumor, we transplanted murine embryonic germ cells (EGCs) into the testis of adult severe combined immunodeficient mice. Transplantation resulted in a locally invasive solid tumor, with a cellular component that generated secondary tumors upon serial transplantation. The secondary tumors were invariably metastatic, a feature not observed in the primary tumors derived from EGCs. To characterize the differences between EGCs and the tumor‐derived stem cells, we performed karyotype and microarray analysis. Our results show that generation of cancer stem cells is associated with the acquisition of nonclonal genomic rearrangements not found in the originating population. Furthermore, pretreatment of EGCs with a potent inhibitor of self‐renewal, retinoic acid, prevented tumor formation and the emergence of these genetically unstable cancer stem cells. Microarray analysis revealed that EGCs and first‐ and second‐generation cancer stem cells were highly similar; however, approximately 1,000 differentially expressed transcripts could be identified corresponding to alterations in oncogenes and genes associated with motility and development. Combined, the data suggest that the activation of oncogenic pathways in a cellular background of genetic instability, coupled with an inherent ability to self‐renew, is involved in the acquisition of metastatic behavior in the cancer stem cell population of tumors derived from pluripotent cells. STEM CELLS 2009;27:18–28

Overlapping Function of Lmx1a and Lmx1b in Anterior Hindbrain Roof Plate Formation and Cerebellar Growth

The roof plate is an organizing center in the dorsal CNS that controls specification and differentiation of adjacent neurons through secretion of the BMP and WNT signaling molecules. Lmx1a, a member of the LIM-homeodomain (LIM-HD) transcription factor family, is expressed in the roof plate and its progenitors at all axial levels of the CNS and is necessary and sufficient for roof plate formation in the spinal cord. In the anterior CNS, however, a residual roof plate develops in the absence of Lmx1a. Lmx1b, another member of the LIM-HD transcription factor family which is highly related to Lmx1a, is expressed in the roof plate in the anterior CNS. Although Lmx1b-null mice do not show a substantial deficiency in hindbrain roof plate formation, Lmx1a/Lmx1b compound-null mutants fail to generate hindbrain roof plate. This observation indicates that both genes act in concert to direct normal hindbrain roof plate formation. Since the requirement of Lmx1b function for normal isthmic organizer at the mid–hindbrain boundary complicates analysis of a distinct dorsal patterning role of this gene, we also used a conditional knock-out strategy to specifically delete dorsal midline Lmx1b expression. Phenotypic analysis of single and compound conditional mutants confirmed overlapping roles for Lmx1 genes in regulating hindbrain roof plate formation and growth and also revealed roles in regulating adjacent cerebellar morphogenesis. Our data provides the first evidence of overlapping function of the Lmx1 genes during embryonic CNS development.

Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.

Etv2, a master regulator of endothelial cell fate, can induce fast skeletal muscle cells to transdifferentiate into endothelial cells in the zebrafish embryo.