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First published online 10 August 2004
doi: 10.1242/jcs.01307

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Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells

Andreas Hermann^1,*, Regina Gastl^1,*, Stefan Liebau¹, M. Oana Popa², Jörg Fiedler³, Bernhard O. Boehm⁴, Martina Maisel¹, Holger Lerche^1,2, Johannes Schwarz^5,6, Rolf Brenner³ and Alexander Storch^1,

¹ Department of Neurology, University of Ulm, Helmholtzstr. 8/1, 89081 Ulm, Germany
² Department of Applied Physiology, University of Ulm, Helmholtzstr. 8/1, 89081 Ulm, Germany
³ Division for Biochemistry of Joint and Connective Tissue Diseases, Department of Orthopaedics, University of Ulm, Helmholtzstr. 8/1, 89081 Ulm, Germany
⁴ Division of Endocrinology, Department of Internal Medicine, University of Ulm, Helmholtzstr. 8/1, 89081 Ulm, Germany
⁵ Department of Neurology, University of Leipzig, Liebigstr. 22a, 04103 Leipzig, Germany
⁶ Division of Biology, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA

View larger version (57K): [in a new window]   Fig. 1. Characteristics of adult human mesodermal stromal cells (hMSC) and human marrow-derived neural stem cell-like cells (hmNSC) during expansion. (A) Morphology, fibronectin and nestin expression of hMSC (left panels) and hmNSC (right panels). All hMSCs express high levels of fibronectin, but only 4±3% of cells express very low levels of the NSC marker nestin, whereas some hmNSCs express low levels of fibronectin, but nearly all cells express high levels of nestin. Nuclei are counterstained with DAPI (blue). Bar, 100 µm. (B) Representative sequence of phase-contrast photomicrographs of hmNSCs 1, 5, 12 and 21 days after conversion from hMSCs. A growth curve of hmNSCs revealed by enumerating the cells at each time point under a hemocytometer is also shown (n=5; calculated doubling time 2.6 days). Bar, 50 µm.

View larger version (18K): [in a new window]   Fig. 2. Flow cytometry of hMSCs (upper panels) and hmNSCs (lower panels) cultured for 10-50 and 5-30 population doublings, respectively. Cells were labeled with fluorescence-coupled antibodies against CD9, CD15, CD34, CD45, CD90, CD133, CD166 or immunoglobulin isotype control antibodies. Cells were analyzed using a FACSCalibur flow cytometer. Black line, control immunoglobulin; red line, specific antibody.

View larger version (25K): [in a new window]   Fig. 3. Quantitative transcription profile of hMSC, hmNSCs and terminal differentiated hmNSCs into glial and neuronal cell types. (A) Representative real-time RT-PCR analysis using the LightCycler® technique. Plot of the fluorescence versus the cycle number obtained from SYBR Green detection of serially diluted FN1 mRNA (encoding fibronectin) (left). The horizontal line represents the position of the threshold. The standard curve obtained by plotting cycle number of crossing points versus dilution factor is shown (center), in addition to melting curve analysis showing the specificity of the amplified PCR product (right). (B) Quantitative real-time RT-PCR analyses of mesodermal genes (FN1), proneural genes (SOX1, OTX1, NeuroD1, Neurog2, MSl1), NSC marker genes (NES), glial genes (GFAP, MBP) and neuronal genes (TUBB4/III, SNCA, NTRK1, TH) as well as OCT-4 as a marker for pluripotency in hMSC, hmNSCs and differentiated hmNSCs, respectively, by the neuronal induction protocol for 14 days. Expression levels are expressed relative to the housekeeping gene HMBS (hydroxymethylbilane synthase). For primers, complete names of genes and melting curve analyses demonstrating the specificity of amplified PCR products see Table 1. #, P<0.05; ##, P<0.01 when compared to mRNA levels in hMSCs; +, P<0.05; ++, P<0.01 when compared to mRNA levels in hmNSCs.

View larger version (36K): [in a new window]   Fig. 4. In vitro differentiation of marrow-derived neurosphere-like structures into astroglial, oligodendroglial and neuronal cell types. hmNSCs were differentiated after 5-30 population doublings using the glial induction or the neuronal induction protocol on poly-L-lysine coated coverslips for 14 days. (A) hmNSCs differentiated using the neuronal induction medium were stained for markers for astrocytes (GFAP), oligodendrocytes (GalC), neurons (ß-tubulin-III, MAP2ab), or catecholaminergic cells (TH). Nuclei are counterstained with DAPI (blue). Bars, 50 µm. (B) Quantification of 14-day cultures of hmNSCs differentiated with the glial and the neuronal induction protocols. Data shown are mean±s.e.m. from at least three independent hMSC preparations. *, P<0.05; **, P<0.01 when compared to the percentage of positive cells in the glial induction protocol. (C) Clonal expansion from single hmNSCs. Six representative photomicrographs of neurosphere-like structures derived from single hmNSCs (left). Bar, 50 µm. Differentiation capacity of clonally derived neurosphere-like structures (right panel). Progeny of single cell-derived neurosphere-like structures can be differentiated into neurons (ß-tubulin III+, green) and astrocytes (GFAP+, red). Nuclei are counterstained with DAPI (blue). Bar, 30 µm.

View larger version (35K): [in a new window]   Fig. 5. Functional properties of terminal differentiated hmNSC. (A-B) Dopamine production and release was measured in hmNSCs differentiated using the neuronal induction protocol for 14 days. (A) Representative chromatograms of HPLC-ECD determination of dopamine in medium conditioned for 3 days (left) and extracellular buffer with 56 mM KCl conditioned for 45 minutes (right). (B) Quantification of dopamine in medium conditioned for 3 days (left), in extracellular buffer conditioned for 45 minutes (center), and in extracellular buffer with 56 mM KCl conditioned for 45 minutes (right). #, P<0.05 when compared to extracellular buffer dopamine levels (paired t-test). (C,D) Electrophysiological recordings on hmNSCs differentiated using the glial induction protocol. For voltage-clamp measurements, cells were held at –80 mV and hyperpolarized or depolarized in 10 mV steps between –160 and +70 mV. (C) Example of a sustained outward current shown without and with (inset) leak subtraction using a –P/4 protocol (left). Current-voltage relationship of the normalized outward currents recorded with leak subtraction (n=7, right panel). (D) Example of an inward current without and with leak subtraction (inset: only currents for depolarizing steps to –40, –20, 0, 20 and 60 mV are shown) (left). Peak current-voltage relationship for the same cell with leak subtraction (right panel). The line represents a fit to the following equation: I(V)/Imax= g(V–Vrev)/1+exp[(V–V0.5)/kV], where I (Imax) is the (maximum) membrane current; g, the maximum conductance; V, the applied voltage; Vrev, the reversal potential for Na+; V0.5, the potential of half-maximal activation and kV, a slope factor.

View larger version (29K): [in a new window]   Fig. 6. Osteogenic differentiation ability of both hMSCs and hmNSCs. Both cell types were differentiated after 6-10 passages using the osteoblast differentiation protocol for 10 days. (A) Differentiated hMSCs (upper panel) and hmNSCs (lower panel) were stained for the osteogenic marker alkaline phosphatase (AP). Bar, 50 µm. (B) Quantification of 10-day cultures of hMSCs and hmNSCs differentiated into osteoblasts under normal atmospheric oxygen levels routinely used for osteoblast cultures (21%) and reduced atmospheric oxygen levels used in our neural culture system (3%). Data shown are mean±s.e.m. from at least three independent cell preparations. *, P<0.05; **, P<0.01 when compared to the percentage of AP+ cells in hMSCs. (C) Expression of osteogenic and mesodermal marker genes as well as the transcription factor c-fos in hMSCs and hmNSCs after osteogenic differentiation under 21% (lanes 1, 4) and 3% (lanes 2, 3) atmospheric oxygen levels. Semiquantitative RT-PCR analysis of AP, runt-related transcription factor 2 (RUNX2), SOX9 and the transcription factor c-fos, as well as GAPDH (housekeeping gene) was performed in hmNSC (lanes 1 and 2) and hMSC (lanes 3 and 4).