Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy

Abstract

We developed human mesenchymal stem cell (MSC) lines that could differentiate into various tissue cells including bone, neural cells, bone marrow (BM) stromal cells supporting the growth of hematopoietic stem cell (HSC), and so-called 'tumor stromal cells' mixing with tumor cells. We investigated the applicability of MSC as therapeutic cell transplanting reagents (cytoreagents). Telomerized human BM derived stromal cells exhibited a prolonged lifespan and supported the growth of hematopoietic clonogenic cells. The gene transfer of Indian hedgehog (Ihh) remarkably enhanced the HSC expansion supported by the human BM stromal cells. Gene-modified MSC are useful as therapeutic tools for brain tissue damage (e.g. brain infarction) and malignant brain neoplasms. MSC transplantation protected the brain tissue from acute ischemic damage in the midcerebral artery occlusion (MCAO) animal model. Brain-derived neurotrophic factor (BDNF)-gene transduction further enhanced the protective efficacy against the ischemic damage. MSC possessed excellent migratory ability and exerted inhibitory effects on the proliferation of glioma cells. Gene-modification of MSC with therapeutic cytokines clearly augmented the antitumor effect and prolonged the survival of tumor-bearing animals. Gene therapy employing MSC as a tissue-protecting and targeting cytoreagent would be a promising approach.

Figure 1

Mesenchymal stem cells (MSC) and their differentiation.

Figure 2

Growth, morphology, and cell surface characterization of the primary and the gene‐modified stromal cells. (A) population doubling of primary and gene‐transduced human stromal cells (From Fig. 4 of Kawano et al. ⁽⁴⁾ ) (B) May–Giemsa staining of primary stromal, hTERT‐stromal, large T‐stromal, and ras‐stromal cells, (a) Primary stromal cells (b) hTERT‐stromal cells (c) large T‐stromal cells (d) ras‐stromal cells. Original magnification for all panels is ×100. (From Fig. 3 of Kawano et al. ⁽⁴⁾ ). (C) Flow cytometric analysis of the expression of surface antigens on primary stromal and hTERT‐stromal cells. Stromal cells were immunolabeled with the FITC‐conjugated monoclonal antibody specific for the indicated surface antigen. (From Fig. 5 of Kawano et al. ⁽⁴⁾ ).

Figure 3

Production of primitive hematopoietic cells from cobblestone‐forming cells beneath the primary stromal cell layer or hTERT‐stromal cell layer over 7 weeks. Expanded hematopoietic cells were harvested each week and analyzed. The x‐axis indicates the period of cell expansion, and the y‐axis indicates the number of cells. (A) Total number of cells. (B) Number of CD34 + cells. The number of CD34 + cells was calculated from the percentage of CD34 + cells, which was determined by flow cytometric analysis. (C) Total number of clonogenic cells (CFU‐Cs). (D) Number of CFU‐Mix cells. *P < 0.05 versus primary stromal cells (Student t‐test). (From Fig. 8 of Kawano et al. ⁽⁴⁾ ).

Figure 4

Migration and tumor tropism of implanted MSC. 9 L‐DsR glioma cells were inoculated intracranially into the right basal ganglia and subsequently EGFP (enhanced green fluorescent protein)‐labeled MSC (MSC‐EGFP) were injected directly into either the glioma or the contralateral hemisphere 3 days later. Rats were killed 14 days after tumor inoculation, and brain sections were prepared. As shown in Fig 4 A and C enormous glioma masses staining strongly with hematoxylin were observed in all the rats implanted with 9 L cells, which occupied the right hemisphere and caused the midline to be shifted towards the left hemisphere. Gene‐labeled MSC were mostly to be found at the border between tumor and normal parenchyma, but had also infiltrated into the tumor bed relatively uniformly after intratumoral injection (B). MSC did not migrate out to distal brain parenchyma or into the contralateral hemisphere. Confocal laser microscopy revealed the accumulation of EGFP‐positive MSC, most of which retained their Spindle‐like shape, at the edge of the DsRed‐positive tumor (E). The presence of MSC coincided with glioma cells, which spread out from the main tumor (G). In contrast, MSC inoculated into the contralateral hemisphere migrated away from the initial injection site towards the glioma cells along the corpus callosum (D). These MSC were mostly retained at the corpus callosum and the edge of the tumor adjacent to it (H). Additionally, infiltration of MSC into the tumor was observed. (Modified from Fig. 3 of NakamuraK et al. ⁽¹⁰⁾ ).

Figure 5

Effect of gene‐modified MSC on in vivo tumor growth evaluated by MRI. Representative MRI (T1‐weightened coronal images with Gd‐DTPA enhancing) data are shown. Rat 9 L gliomas were treated with either MSC‐IL2 (rat MSC modified to produce human IL‐2 gene by adenovirus‐mediated gene transduction) or MSC (control unmodified MSC) 3 days after tumor inoculation. MRI was performed every 7 days. For explanation, see the text. (From Fig. 5 of NakamuraK et al.^{( 10 )}).