Grafted Subventricular Zone Neural Stem Cells Display Robust Engraftment and Similar Differentiation Properties and Form New Neurogenic Niches in the Young and Aged Hippocampus

Abstract

: As clinical application of neural stem cell (NSC) grafting into the brain would also encompass aged people, critical evaluation of engraftment of NSC graft-derived cells in the aged hippocampus has significance. We examined the engraftment and differentiation of alkaline phosphatase-positive NSCs expanded from the postnatal subventricular zone (SVZ), 3 months after grafting into the intact young or aged rat hippocampus. Graft-derived cells engrafted robustly into both young and aged hippocampi. Although most graft-derived cells pervasively migrated into different hippocampal layers, the graft cores endured and contained graft-derived neurons expressing neuron-specific nuclear antigen (NeuN) and γ-amino butyric acid in both groups. A fraction of migrated graft-derived cells in the neurogenic subgranular zone-granule cell layer also expressed NeuN. Neuronal differentiation was, however, occasionally seen amid graft-derived cells that had migrated into non-neurogenic regions, where substantial fractions differentiated into S-100β+ astrocytes, NG2+ oligodendrocyte progenitors, or Olig2+ putative oligodendrocytes. In both age groups, graft cores located in non-neurogenic regions displayed many doublecortin-positive (DCX+) immature neurons at 3 months after grafting. Analyses of cells within graft cores using birth dating and putative NSC markers revealed that DCX+ neurons were newly born neurons derived from engrafted cells and that putative NSCs persisted within the graft cores. Thus, both young and aged hippocampi support robust engraftment and similar differentiation of SVZ-NSC graft-derived cells. Furthermore, some grafted NSCs retain the "stemness" feature and produce new neurons even at 3 months after grafting, implying that grafting of SVZ-NSCs into the young or aged hippocampus leads to establishment of new neurogenic niches in non-neurogenic regions.

Significance: The results demonstrate that advanced age of the host at the time of grafting has no major adverse effects on engraftment, migration, and differentiation of grafted subventricular zone-neural stem cells (SVZ-NSCs) in the intact hippocampus, as both young and aged hippocampi promoted excellent engraftment, migration, and differentiation of SVZ-NSC graft-derived cells in the present study. Furthermore, SVZ-NSC grafts showed ability for establishing neurogenic niches in non-neurogenic regions, generating new neurons for extended periods after grafting. This phenomenon will be beneficial if these niches can continuously generate new neurons and glia in the grafted hippocampus, as newly generated neurons and glia are expected to improve, not only the microenvironment, but also the plasticity and function of the aged hippocampus. Overall, these results have significance because the potential application of NSC grafting for treatment of neurodegenerative disorders at early stages of disease progression and age-related impairments would mostly involve aged persons as recipients.

Keywords: Cell transplantation; Cellular therapy; Hippocampal neurogenesis; Neural stem cells; Stem cell grafts; Subventricular zone stem cells.

Figure 1.

Distribution of BrdU-labeled cells derived from subventricular zone-neural stem cell (SVZ-NSC) grafts in the intact young hippocampi (A1, A2) and intact aged hippocampi (B1, B2) at 3 months after grafting. (A3, A4, B3, B4): Magnified views of regions from (A2) and (B2), respectively. (A3, B3): Graft-derived BrdU+ cells within graft cores and in the surrounding regions. (A4, B4): Graft-derived cells that have migrated into the subgranular zone-granule cell layer (SGZ-GCL) of the DG. Scale bars = 500 µm (B1) and 50 µm (B4). Bar charts compare numbers of BrdU+ cells recovered per hippocampus (C1) and volumes of brain tissue containing graft-derived cells (C2) between the intact young hippocampus and the intact aged hippocampus. (D1–E3): The occasional presence of BrdU+ elements inside IBA-1+ cells in the young (D1–D3) and aged (E1–E3) hippocampi, suggesting that only a very small fraction of BrdU+ elements were debris ingested by microglial cells (arrows). (D1–E3, Insets): Magnified views of IBA-1+ microglia containing BrdU+ elements. Scale bar = 25 µm (E1). (F): Bar chart depicting fractions of BrdU+ structures found inside IBA-1+ microglia. Abbreviations: BrdU, 5′-bromodeoxyuridine; DG, dentate gyrus; DH, dentate hilus; GCL, granule cell layer; ML, molecular layer.

Figure 2.

Fractions of cells derived from the SVZ-NSC grafts differentiate into neurons (arrowheads). BrdU+ graft-derived cells (red) differentiate into neurons expressing the NeuN (green) in the intact young hippocampus (A1–A3) and the intact aged hippocampus (B1–B3). (C1–D3): NeuN expression (green) in BrdU+ graft-derived cells (red) that have migrated into the granule cell layer of the intact young hippocampus (C1–C3) and the intact aged hippocampus (D1–D3). (E1–E3): The expression of TuJ-1 (green) among BrdU+ graft-derived cells (red) that have migrated into the non-neurogenic region of the intact young hippocampus. Scale bars = 50 µm (A1–A3, E1, E3) and 25 µm (B1–B3, C1–C3, D1–D3). (F): Bar charts show percentages of graft-derived cells that differentiated into NeuN-positive mature neurons within graft cores and among graft-derived cells that have migrated into the SGZ-GCL of the dentate gyrus in the young and aged hippocampi, The extent of neuronal differentiation is similar in the two age groups for graft cores. However, greater percentages of graft-derived cells differentiated into neurons in the in SGZ-GCL of the young hippocampus (p < .05). Abbreviations: BrdU, 5′-bromodeoxyuridine; NeuN, neuron-specific nuclear antigen; SGZ-GCL, subgranular zone-granule cell layer; SVZ-NSC, subventricular zone-neural stem cell; TuJ-1, β-III tubulin.

Figure 3.

Fractions of cells derived from the subventricular zone-neural stem cell grafts differentiated into GABA+ neurons in the intact young (A1) and aged (A2) hippocampi. (Insets): Orthogonal views of GABA+ neurons indicated by arrows in A1 and A2. Scale bars = 40 µm (A1) and 20 µm (A2). (A3): Bar chart shows percentages of graft-derived cells that differentiated into GABA+ neurons in young and aged hippocampi. Abbreviation: GABA, γ-amino butyric acid.

Figure 4.

Fractions of cells derived from the subventricular zone-neural stem cell grafts differentiated into S-100β+ (A1, A2) and GFAP+ (B1, B2) astrocytes and Olig2+ oligodendrocyte-like cells (C1, C2) in the intact young (A1, B1, C1) and aged (A2, B2, C2) hippocampi. (Insets): Orthogonal views of cells indicated by arrows in A1, A2, B1, and B2. Scale bar = 20 µm (A1, B1, B2) and 10 µm (A2, C1, C2). Bar charts show percentages of graft-derived cells that differentiated into S-100β+ astrocytes (A3), GFAP+ astrocytes (B3), and Olig2+ oligodendrocyte-like cells (C3) in the young and aged hippocampi. Abbreviation: GFAP, glial fibrillary acidic protein.

Figure 5.

Differentiation of cells derived from the subventricular zone-neural stem cell grafts into neurons and glia in the young and aged hippocampi, as revealed through dual immunofluorescence for AP (graft cell marker) and neural antigens (arrows). (A1–A6): AP+ graft-derived cells that have differentiated into NeuN-expressing neurons in a young hippocampus (A1–A3) and an aged hippocampus (A4–A6). (B1–B6): AP+ graft-derived cells that have differentiated into GABA-expressing interneurons in a young hippocampus (B1–B3) and an aged hippocampus (B4–B6). (C1–C6): AP+ graft-derived cells that have differentiated into S-100β-expressing mature astrocytes in a young hippocampus (C1–C3) and an aged hippocampus (C4–C6). Arrowheads in (C4–C6) denote S-100b+ host astrocytes. (D1–D6): AP+ graft-derived cells that have differentiated into NG2-expressing oligodendrocyte precursor cells in a young hippocampus (D1–D3) and an aged hippocampus (D4–D6). Scale bars = 50 µm (A5, B5, C5, D5). Bar charts compare percentages of NeuN+ neurons (A7), GABA+ interneurons (B7), S-100β+ astrocytes (C7), and NG2+ oligodendrocyte precursors (D7) among graft-derived AP+ cells between the two age groups. Abbreviations: AP, alkaline phosphatase; GABA, γ-amino butyric acid; NeuN, neuron-specific nuclear antigen.

Figure 6.

Presence of DCX+ immature neurons in graft cores at 3 months after grafting. (A1, A2): Examples of grafts displaying DCX+ immature neurons in intact young hippocampi. (A3): An example of a graft in the aged hippocampus exhibiting DCX+ immature neurons. (Insets): Magnified views of graft regions showing the morphology of immature DCX+ neurons. (B1–B4): DCX-expressing neurons among graft-derived cells expressing AP. Note that a fraction of AP+ graft-derived cells express DCX (arrowheads). (C1–C6): IdU-positive newly born cells expressing DCX within graft cores (arrowheads) located in a young hippocampus (C1–C3) and an aged hippocampus (C4–C6). Arrows denote DCX+ neurons that lack IdU. Bar charts compare the percentage of DCX+ neurons that express IdU (C7) and the percentage of IdU+ cells that express DCX (C8) in grafts located in the young and aged hippocampi. Scale bars = 200 µm (A1, B1, C1), 50 µm (insets), and 50 µm (B1–C6). Abbreviations: AP, alkaline phosphatase; DAPI, 4′,6-diamidino-2-phenylindole; DCX, doublecortin; GCL, granule cell layer; HF, hippocampal fissure; IdU, iododeoxyuridine; ML, molecular layer; SGZ, subgranular zone.

Figure 7.

DCX-expressing neurons among graft-derived cells expressing CldU and IdU. (A1–B4): Generation of new DCX+ neurons from graft-derived cells in the young (A1–A4) and aged (B1–B4) hippocampi. Note that a fraction of proliferating cells (IdU+ cells; arrows in A2 and B2) among CldU+ graft cell population (arrows in A1 and B1) express DCX (arrows in A3 and B3), implying that these DCX+ neurons were generated from graft-derived cells during IdU administration (i.e., 1 month after grafting). (A1–A4): CldU+ graft-derived cells that lack IdU indicated by arrowheads (i.e., nonproliferating graft-derived cells). (B1–B4): CldU and IdU+ cells that lack DCX indicated by arrowheads (i.e., newly born cells that did not differentiate into DCX+ neurons). (C1–C4): Graft cores contain putative neural stem cells (NSCs). Note the presence of putative NSCs (triple-labeled cells indicated by arrows in C1–D4) expressing AP (graft cell marker; C1, D1), GFAP (NSC marker; C2, D2), and Sox-2 (another NSC marker; C3, D3). (C1–C4): Host cells expressing GFAP and Sox-2 (arrowheads). Asterisks indicate examples of a host astrocyte that expresses GFAP but not Sox-2. Scale bars = 50 µm (A1–B4) and 20 µm (C1–C4). Abbreviations: AP, alkaline phosphatase; CldU, chlorodeoxyuridine; DCX, doublecortin; GFAP, glial fibrillary acidic protein; IdU, iododeoxyuridine.