Alexandra Blak

Publications (7) View all

• Source

  Available from: Oliver Cooper

  Article: Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.

  Oliver Cooper, Gunnar Hargus, Michela Deleidi, Alexandra Blak, Teresia Osborn, Elizabeth Marlow, Kristen Lee, Adam Levy, Eduardo Perez-Torres, Alyssa Yow, Ole Isacson
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  ABSTRACT: The cardinal motor symptoms of Parkinson's disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/β-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10(-)(8)M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2(+) neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2(+) floor plate-like human neural progenitor cells into FOXA2(+) DA neurons. FOXA2(+) DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.
  Molecular and Cellular Neuroscience 11/2010; 45(3):258-66. · 3.66 Impact Factor
• Source

  Available from: Kambiz Alavian

  Article: CD15, CD24, and CD29 define a surface biomarker code for neural lineage differentiation of stem cells.

  Jan Pruszak, Wesley Ludwig, Alexandra Blak, Kambiz Alavian, Ole Isacson
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  ABSTRACT: Identification and use of cell surface cluster of differentiation (CD) biomarkers have enabled much scientific and clinical progress. We identify a CD surface antigen code for the neural lineage based on combinatorial flow cytometric analysis of three distinct populations derived from human embryonic stem cells: (1) CD15(+)/CD29(HI)/CD24(LO) surface antigen expression defined neural stem cells; (2) CD15(-)/CD29(HI)/CD24(LO) revealed neural crest-like and mesenchymal phenotypes; and (3) CD15(-)/CD29(LO)/CD24(HI) selected neuroblasts and neurons. Fluorescence-activated cell sorting (FACS) for the CD15(-)/CD29(LO)/CD24(HI) profile reduced proliferative cell types in human embryonic stem cell differentiation. This eliminated tumor formation in vivo, resulting in pure neuronal grafts. In conclusion, combinatorial CD15/CD24/CD29 marker profiles define neural lineage development of neural stem cell, neural crest, and neuronal populations from human stem cells. We believe this set of biomarkers enables analysis and selection of neural cell types for developmental studies and pharmacological and therapeutic applications.
  Stem Cells 10/2009; 27(12):2928-40. · 7.78 Impact Factor
• Article: Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.

  Frank Soldner, Dirk Hockemeyer, Caroline Beard, Qing Gao, George W Bell, Elizabeth G Cook, Gunnar Hargus, Alexandra Blak, Oliver Cooper, Maisam Mitalipova, Ole Isacson, Rudolf Jaenisch
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  ABSTRACT: Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.
  Cell 04/2009; 136(5):964-77. · 32.40 Impact Factor
• Article: Fibroblast growth factor receptors cooperate to regulate neural progenitor properties in the developing midbrain and hindbrain.

  Jonna Saarimäki-Vire, Paula Peltopuro, Laura Lahti, Thorsten Naserke, Alexandra A Blak, Daniela M Vogt Weisenhorn, Kai Yu, David M Ornitz, Wolfgang Wurst, Juha Partanen
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  ABSTRACT: Fibroblast growth factors (FGFs) secreted from the midbrain-rhombomere 1 (r1) boundary instruct cell behavior in the surrounding neuroectoderm. For example, a combination of FGF and sonic hedgehog (SHH) can induce the development of the midbrain dopaminergic neurons, but the mechanisms behind the action and integration of these signals are unclear. We studied how FGF receptors (FGFRs) regulate cellular responses by analyzing midbrain-r1 development in mouse embryos, which carry different combinations of mutant Fgfr1, Fgfr2, and Fgfr3 alleles. Our results show that the FGFRs act redundantly to support cell survival in the dorsal neuroectoderm, promote r1 tissue identity, and regulate the production of ventral neuronal populations, including midbrain dopaminergic neurons. The compound Fgfr mutants have apparently normal WNT/SHH signaling and neurogenic gene expression in the ventral midbrain, but the number of proliferative neural progenitors is reduced as a result of precocious neuronal differentiation. Our results suggest a SoxB1 family member, Sox3, as a potential FGF-induced transcription factor promoting progenitor renewal. We propose a model for regulation of progenitor cell self-renewal and neuronal differentiation by combinatorial intercellular signals in the ventral midbrain.
  Journal of Neuroscience 09/2007; 27(32):8581-92. · 7.11 Impact Factor
• Article: Fgfr2 and Fgfr3 are not required for patterning and maintenance of the midbrain and anterior hindbrain.

  Alexandra A Blak, Thorsten Naserke, Jonna Saarimäki-Vire, Paula Peltopuro, Mario Giraldo-Velasquez, Daniela M Vogt Weisenhorn, Nilima Prakash, Michael Sendtner, Juha Partanen, Wolfgang Wurst
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  ABSTRACT: The mid-/hindbrain organizer (MHO) is characterized by the expression of a network of genes, which controls the patterning and development of the prospective midbrain and anterior hindbrain. One key molecule acting at the MHO is the fibroblast growth factor (Fgf) 8. Ectopic expression of Fgf8 induces genes that are normally expressed at the mid-/hindbrain boundary followed by the induction of midbrain and anterior hindbrain structures. Inactivation of the Fgf receptor (Fgfr) 1 gene, which was thought to be the primary transducer of the Fgf8 signal at the MHO, in the mid-/hindbrain region, leads to a deletion of dorsal structures of the mid-/hindbrain region, whereas ventral tissues are less severely affected. This suggests that other Fgfrs might be responsible for ventral mid-/hindbrain region development. Here we report the analysis of Fgfr2 conditional knockout mice, lacking the Fgfr2 in the mid-/hindbrain region and of Fgfr3 knockout mice with respect to the mid-/hindbrain region. In both homozygous mouse mutants, patterning of the mid-/hindbrain region is not altered, neuronal populations develop normal and are maintained into adulthood. This analysis shows that the Fgfr2 and the Fgfr3 on their own are dispensable for the development of the mid-/hindbrain region. We suggest functional redundancy of Fgf receptors in the mid-/hindbrain region.
  Developmental Biology 04/2007; 303(1):231-43. · 4.07 Impact Factor