Microcarrier-Expanded Neural Progenitor Cells Can Survive,Differentiate,and Innervate Host Neurons Better When Transplanted as Aggregates

Abstract

Neuronal progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) are an excellent cell source for transplantation therapy due to their availability and ethical acceptability. However, the traditional method of expansion and differentiation of hESCs into NPCs in monolayer cultures requires a long time, and the cell yield is low. A microcarrier (MC) platform can improve the expansion of hESCs and increase the yield of NPCs. In this study, for the first time, we transplanted microcarrier-expanded hESC-derived NPCs into the striatum of adult NOD-SCID IL2Rgc null mice, either as single cells or as cell aggregates. The recipient mice were perfused, and the in vivo survival, differentiation, and targeted innervation of the transplanted cells were assessed by immunostaining. We found that both the transplanted single NPCs and aggregate NPCs were able to survive 1 month posttransplantation, as revealed by human-specific neural cell adhesion molecule (NCAM) and human nuclear antigen staining. Compared to the single cells, the transplanted cell aggregates showed better survival over a 3-month period. In addition, both the transplanted single NPCs and the aggregate NPCs were able to differentiate into DCX-positive immature neurons and Tuj1-positive neurons in vivo by 1 month posttransplantation. However, only the transplantation of aggregate NPCs was shown to result in mature neurons at 3 months posttransplantation. Furthermore, we found that the cell aggregates were able to send long axons to innervate their targets. Our study provides preclinical evidence that the use of MCs to expand and differentiate hESC-derived NPCs and transplantation of these cells as aggregates produce longer survival in vivo.

Keywords

Neural progenitor cells (NPCs)Transplant Differentiation Aggregate Microcarrier (MC)

Introduction

Cell transplantation therapy is becoming more attractive as a method to treat various neurodegenerative diseases¹. For example, recent clinical studies have reported an effective restorative treatment involving the use of fetal ventral mesencephalic cell transplantation for the clinical treatment of Parkinson's disease (PD)^2–5. However, it has been widely recognized that several critical issues needed to be addressed before cell transplantation therapy could become a routine practice in the clinical treatment of neurodegenerative diseases. These issues include choosing the suitable types of cells^6–11, the accessibility of a viable source of the cells^1,6,9,11, and the optimal differentiation status of the transplantable cells^12–15.

One of the less-recognized issues is that the traditional 2D culture system is laborious, and the cell yield is low and cannot provide large-scale, standardized, transplantable cells for clinical use^1,6. Neural progenitor cells (NPCs) cultured on suspension bioreactors can reach high proliferation rates^16–20, which can speed up and simplify the amplification and differentiation procedure of transplantable cells. In our previous studies, we have developed a microcarrier (MC) platform to improve the expansion of human embryonic stem cells (hESCs) and their subsequent differentiation into NPCs^21,22. Compared with the traditional 2D culture system, this MC platform simplifies the laborious process and produces high cell yields within a short period of time. Nearly 10-fold more NPCs were generated using this MC-based system than by using the traditional 2D culture system²¹. However, the agitation of hESCs in spinner cultures could cause spontaneous differentiation of the hESCs as a result of mechanical stress¹⁶. Thus, it was uncertain whether the NPCs generated from this MC platform could be applied for transplantation therapy in vivo.

Another issue that needs to be addressed in the cell transplantation field is the maintenance of the balance between the survival and the functional maturation of transplanted cells. Transplantation of fewer differentiated precursor cells is usually associated with better survival but weak functional maturation^{6,12,13,23,24}. Additionally, the residual proliferating cells may cause tumorigenesis^13,24. In contrast, well-differentiated neurons usually show low survival rates^6,25,26. The loss of transplanted cells following transplantation can be as much as 90% of the total number of grafted cells^6,25,26. To promote the survival of the transplanted cells, many methods have been developed, such as the addition of supporting cell types that are transplanted along with the neurons²⁷, transplanting cells together with a biodegradable scaffold²⁶, or transplanting cells in aggregate form^28–30. Among these methods, transplanting cells in aggregate form seems to be a promising choice for several reasons^20,30. First, compared to the other methods, no unwanted cells or materials need to be delivered into the brain. Second, preparation of the cell aggregates is simple, and the size of the aggregates can be controlled and standardized.

To address the current gaps in knowledge, in this study we systematically assessed the in vivo differentiation abilities of transplanted NPCs that were derived from hESCs and expanded on the MC platform. The NPCs were transplanted into the striatum of adult NOD-SCID IL2Rgc null mice either as single cells or as aggregates. The survival, differentiation potential, and neurite outgrowth of the transplanted cells were assessed by immunostaining. Our results showed that the NPCs transplanted as cell aggregates could survive for a longer period of time (3 months) than the single cells, which could only survive for 1 month. Both the transplanted single NPCs and the aggregate NPCs differentiated into doublecortin (DCX)-positive immature neurons and β-tubulin-III (Tuj1)-positive neurons at 1 month posttransplantation. NeuN-positive mature neurons were found in vivo at 3 months posttransplantation of aggregate NPCs, but not after transplantation of the single cells. We found that the cell aggregates could extend long axons to innervate their targets. These results demonstrated the use of MC-expanded and MC-differentiated hESC-derived NPC aggregates in cell transplantation.

Materials and Methods

Animals

Use of the experimental animals was conducted with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the National Neuroscience Institute (Singapore). The mice were maintained in a pathogen-free facility and exposed to a 12-h light/dark cycle with food and water. Male NOD-SCID IL2Rgc null mice (12 weeks old) were purchased (In Vivos Pte Ltd, Singapore) and used as recipients for the cell transplantation. Efforts were made to minimize animal suffering and the number of animals used. Three recipient mice were analyzed at each time point (1 month or 3 months posttransplantation).

NPC Amplification and Differentiation on the MC

The hESC line HES-3 (46, XX) was obtained from ES Cell International (Singapore). The HES-3 cells were maintained and expanded in mTeSR1 culture media (STEMCELL Technologies, Inc, Vancouver, Canada). Conjugated cell–MC cultures were initiated, expanded, and routinely maintained using a protocol similar to that described previously³¹. In brief, HES-3 monolayer cells were mechanically cut and seeded onto 1 mg/ml Geltrexcoated Cytodex 1 (GE Healthcare, Uppsala, Sweden) MCs with a cell density of 2 × 10⁵ cells/ml. The cells were then allowed to grow for 7 days with daily 80% mTeSR1 media exchange in six-well ultralow attachment (ULA) plates (Corning Inc., Corning, NY, USA) under static conditions. The 7-day-old cell–MC conjugates were routinely passaged by mechanical trituration, and passage 5 (P5) of the exponentially growing cell–MC conjugate cultures was further expanded into 100-ml spinner flasks (Bellco Glass Inc., Vineland, NJ, USA) using a procedure described previously³¹. These cell–MC conjugates were differentiated in suspension spinner flask cultures at an initial cell density of 5 × 10⁵ cells/ml with an additional 1 mg/ml of Geltrexcoated MC. Neural induction media containing reagents from Life Technologies (Waltham, MA, USA), unless other wise stated, 95% DMEM/F12, 1 mM L-glutamine, 1% N2, 20 ng/ml basic fibroblast growth factor (bFGF), 1% (v/v) non-essential amino acids (NEAA) solution, 25 U/ml penicillin, 25 mg/ml streptomycin, 0.1 mM β-mercaptoethanol, 5 μM dorsomorphin (Sigma-Aldrich, St. Louis, MO, USA), and 10 μM SB431542 (Sigma-Aldrich) was used to initiate NPC differentiation for the first 5 days. The NPCs were then maintained in N2B27 medium (95% DMEM/F12, 0.5% L-glut, 1% N2, 2% B27, 25 U/ml penicillin, 25 mg/ml streptomycin, 1% NEAA, and 0.09% β-mercaptoethanol) supplemented with 20 ng/ml of epidermal growth factor (EGF) (PeproTech, Inc., Rocky Hill, NJ, USA) and 20 ng/ml of bFGF (Life Technologies). Nine days after the initial neuronal induction, the NPCs were characterized by flow cytometry and immunocytochemistry prior to transplantation.

Generation of Uniform-Sized NPC Aggregates

NPCs from the MCs were harvested as a single cell suspension using StemPro Accutase (Life Technologies, Grand Island, NY, USA) and filtered through a 40-μm sieve (Greiner Bio-One, Frickenhausen, Germany). Aggrewell 400™ (STEMCELL Technologies) was prepared according to specifications prior to seeding. Neurosphere aggregates were formed on the following day and were dislodged by pipetting. Each neurosphere was comprised of ~1,000 cells and was 100 μm in size. Approximately 250 aggregates were delivered per injection.

Flow Cytometry

Cells were harvested from the MCs and were incubated with mouse primary antibodies against TRA-1-60 (Life Technologies, Frederick, MD, USA) and neural cell adhesion molecule (NCAM) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as well as with an APC-conjugated polysialylated neural cell adhesion molecule (PSA-NCAM) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) antibody at a dilution of 1:100 for 30 min at 4°C in 1% bovine serum albumin (BSA) (Sigma-Aldrich)/phosphate-buffered saline (PBS) (Nacalai Tesque, Kyoto, Japan). The cells were washed with 1% BSA/PBS and then incubated with an Alexa Fluor® 488 goat anti-Mouse IgG (H+L) (Life Technologies, Eugene, OR, USA) and an APC-conjugated isotype control (Miltenyi Biotec GmbH) for 30 min at 4°C in 1% BSA/PBS. The cells were washed with 1% BSA/PBS and analyzed on a Guava easyCyte™ HT Sampling Flow Cytometer (EMD Millipore, Darmstadt, Germany). Gating was set at the point of intersection between the negative and the positive stains, and the percentage of cells from the negative control within the gate was subtracted from the percentage of positive cells.

Cell Transplantation

The mice were anesthetized intraperitoneally (IP) with a mixture of ketamine (Ceva Animal Health Pty Ltd, Glenorie, Australia) and diazepam (Ceva Animal Health Pty Ltd) (100 mg/kg and 20 mg/kg, respectively) and positioned in a stereotactic injection apparatus. Concentrated cell suspensions (1 × 10⁵ cells/μl) were loaded into a Hamilton microliter syringe (7105KH 5.0 μl; Hamilton Company, Bonaduz, Switzerland). Then 2.5 μl of cell suspension was injected into the striatum (AP: +0.5 mm; ML: ±2 mm; DV: –3 mm). The needle was left in place for 5 min after the injection. The single NPCs were injected into the left hemisphere, and the aggregate NPCs were injected into the right hemisphere.

Immunohistochemistry (IHC) and Immunocytochemistry Staining

The mice were transcardially perfused with 4% paraformaldehyde (PFA) (Sigma-Aldrich, Singapore); the brains were then removed, postfixed overnight in 4% PFA, and cryoprotected in 30% sucrose. Coronal brain sections were cut with a frozen sliding microtome. For the immunostaining of brain sections, tissue blocking and antibody incubations were performed with a solution of 1% BSA and 0.1% Triton X-100 (Sigma-Aldrich, Singapore) in PBS. The samples were blocked for 1 h at room temperature (RT), incubated overnight in primary antibody solutions at 4°C, and incubated for 2 h in secondary antibody solutions at RT. After the primary and secondary antibody incubations were complete, the sections were washed four times in PBS. Immunostaining was performed with the following primary antibodies: mouse anti-human nucleus antigen (hNuc) (1:200; Millipore, Singapore), mouse anti-human-specific NCAM (1:50; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-nestin (1:300; Millipore), rabbit anti-NeuN (1:500; Millipore), rabbit anti-doublecortin (DCX) (1:500; Abcam, Singapore), mouse anti-Tuj1 (1:500; Abcam), rabbit anti-glial fibrillary acidic protein (GFAP) (1:1,000; Abcam), rabbit anti-Olig-2 (1:500; Millipore), rabbit anti-tyrosine hydroxylase (TH) (1:1,000; Novus Biologicals, Littleton, CO, USA), rabbit anti-Ki-67 (1:500; Millipore), rabbit anti-DAT (1:1,000; Millipore), and rabbit anti-GABA (1:200; Sigma-Aldrich, Singapore). Alexa Fluor 488/568 goat anti-rabbit/mouse IgG (1:400, Invitrogen, Singapore) was used for fluorescence labeling. A peroxidase-conjugated goat anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, USA) was used for 3,3′-diaminobenzidine (DAB) (Vector Laboratories) labeling, and positive reactions were detected by the avidin–biotin complex (ABC) method (Vector Laboratories). The slices were mounted with fluorescence mounting medium (Dako, Carpinteria, CA, USA), and images were obtained with a confocal microscope (LSM710; Olympus, Tokyo, Japan).

For the immunostaining of cells prior to transplantation, the cells were plated onto TC plates coated with Geltrex (Thermo Fisher Scientific, Singapore) and fixed in 4% paraformaldehyde; they were then washed with PBS, permeabilized, and blocked with 0.1% Triton X-100/10% goat serum (Sigma-Aldrich, Singapore)/PBS. The following primary antibodies were used: a polyclonal antibody against the human nestin repeat (nestin, 1:200; Covance, Singapore) and a monoclonal antibody against paired box gene 6 (PAX6, 1:500; Developmental Studies Hybridoma Bank, Iowa City, IA, USA). The cells were washed with 1% BSA/PBS and incubated with the following secondary antibodies: Alexa Fluor® 488/594 goat anti-mouse/rabbit IgG (Invitrogen). Nuclei were stained with SlowFade® Gold Antifade Reagent (Thermo Fisher Scientific) with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using an inverted fluorescence microscope (IX70; Olympus).

Stereological Quantification of Grafted NPCs

The total numbers of grafted NPCs that were immunoreactive for hNuc, Ki-67, and NeuN were estimated using stereological, unbiased, and systematic sampling^32,33. Frozen coronal sections (16 μm thick) were cut and continuously collected from the focal plane extending from the area in which the grafted cells started to emerge to the plane where the grafted cells disappeared. Each 15th section was used for stereological quantification. The optical images were obtained by an Olympus microscope using a 40× oil immersion objective. Cells were manually counted using FV10-ASW software (Olympus), and the total number per animal was corrected for the series number. Three animals were analyzed to estimate the average number of surviving NPCs within the graft.

Statistical Analysis

At least three mice were used in each experimental condition, and similar results were obtained. Statistical analyses were carried out by using GraphPad Prism Software (San Diego, CA, USA) and performed using Student's t-test. All data are presented as the means and the standard deviation (mean ± SD). Statistical significance was defined at values of p < 0.001, p < 0.01, and p < 0.05 compared to the control group.

Results

Generation and Characterization of hESC-Derived NPCs for Transplantation

We have previously reported that human-derived induced pluripotent stem cells (hiPSCs) [iPSCs (IMR90) and HES-3] can be expanded and efficiently differentiated into high-density cultures of NPCs on MCs, which eliminates the need for multiple cell dissociation and replating steps that are present in the conventional differentiation procedure²¹. In this study, the expensive recombinant protein Noggin was replaced with two small molecules, dorsomorphin and SB431542, to induce neural differentiation via simultaneous inhibition of the bone morphogenetic protein (BMP) and activin/nodal signaling pathways³⁴, resulting in a more cost-effective bioprocess. After 9 days of cultivation (Fig. 1A), NPCs cultured on MCs showed high expression of NPC markers (96.6% NCAM positive and 97.5% PSA-NCAM positive), and the absence of undifferentiated induced pluripotent stem cells (hiPSCs) (TRA-1-60 negative) via flow cytometry analysis (Fig. 1B). These NPCs were later used to generate NPC aggregates (Fig. 1C) for transplantation into the striatum of NOD-SCID IL2Rgc null mice. An immunocytochemical analysis also demonstrated high expression levels of the neural cell markers PAX6 and nestin (Fig. 1D) in MC-cultured NPCs.

Figure 1.

Generation and characterization of neural progenitor cells (NPCs) for transplantation. (A) Schematic diagram of the generation of neural progenitor cells (NPCs) on a microcarrier (MC) platform for the transplantation studies. (B) Characterization of day 9 NPC-MC conjugates prior to transplantation. Representative flow cytometry analysis of cells for a pluripotent stem cell marker, TRA-1-60, and the NPC markers hNCAM and PSA-hNCAM is shown. The shaded peaks represent the isotype controls. (C) Generation of uniform-sized NPC aggregates using Aggrewell 400™. Scale bar: 100 μm. (D) Immunocytochemistry analysis showing positive staining for PAX6 (green) and nestin (red) in the NPCs differentiated on the MC platform. Cell nuclei are indicated by DAPI (blue). Scale bars: 50 μm.

Short-Term Survival of Single NPCs and NPC Aggregates

Previous work using MC-expanded NPCs showed enhanced proliferation and differentiation in vitro²¹. To determine the differentiation efficiency in vivo, we performed NPC transplants in NOD-SCID IL2Rgc null mice. hESC-derived NPCs were differentiated in vitro for 9 days (D9 NPCs) and then used for transplantation because at this time point expression of human NCAM is observed, but no expression of TRA-1-60 (a stem cell surface marker) is present, suggesting it may be a suitable stage for transplantation (Fig. 1). The NPCs were transplanted either as single cells or as aggregates of equal cell numbers (2.5 × 10⁵ cells per animal).

When the cells were grafted as single cells, the graft volume widths ranged from 210 μm to 230 μm, with an average width of 223.33 ± 11.55 μm (Fig. 2A) at 1 month posttransplantation. In contrast, when the cells were grafted as aggregates, the graft volume size was significantly larger, with an average width of 996.67 ± 73.71 μm (Fig. 2A). As shown by the immunostaining with an antibody against human-specific NCAM, both the transplanted single NPCs and aggregate NPCs exhibited substantial survival at 1 month posttransplantation in the NOD-SCID IL2Rgc null mice (Fig. 2B). We also assessed the number of surviving grafted cells per graft using stereological counting. For single-cell transplantation, approximately 0.337 × 10⁵ hNuc-positive cells survived 1 month posttransplantation (Fig. 3C). These counts reflected 13.48% of the initial transplanted cells. In contrast, approximately 10.732 × 10⁵ hNuc-positive cells survived in the aggregate transplant (Fig. 3C). This suggests that the survival ability of cells is better if they are transplanted as aggregates. In addition, we found that both the transplanted single NPCs and aggregate NPCs showed positive expression of the neural stem cell marker nestin (Fig. 2B), indicating that the transplanted NPCs were still neuronal precursors. We also noticed that Ki-67-positive cells (proliferating cells)³⁵ were present in both the single cell transplants and aggregate transplants 1 month posttransplantation. However, the percentage of the proliferating cells (approximately 8%) was not different between these two types of transplantation (Fig. 3D).

Figure 2.

Short-term survival of single NPCs and NPC aggregates. (A) Comparison of the survival of single NPCs and aggregate NPCs at 1 month posttransplantation. The transplanted cells were labeled with an antibody against human NCAM. Scale bar: 300 μm. The bar chart shows the quantification of the diameter of the single cell grafts and the aggregate grafts. The values represent means ± SD, n = 3, ∗∗p < 0.01. (B) Both the transplanted single cells and cell aggregates expressed the NPC marker nestin at 1 month posttransplantation. The transplanted cells were labeled with human-specific NCAM (red). G, graft; H, host tissue. Scale bar: 50 μm.

Figure 3.

Portion of proliferating cells for both single NPCs and NPC aggregate transplantation. (A) Proliferating cells (Ki-67 positive, green in merged images) were present among the transplanted cells (hNuc positive, red in merged images). Upper panel: single cell transplantation; lower panel: aggregate transplantation. Scale bars: 100 μm. (B) High-magnification images showing that the proliferating cells resided mainly in the neural rosette. Scale bar: 50 μm. (C) Number of hNuc-positive cells, which represent the surviving transplanted cells, in single cell transplants and aggregate transplants, respectively. Aggregate transplantation produced a significantly higher number of surviving transplanted cells than single cell transplantation. (D) The percentages of proliferating cells (Ki-67-positive cells) of the total surviving transplanted cells (hNuc-positive cells) were not significantly different between the single cell transplantation and aggregate transplantation.

Short-Term Neuronal Differentiation of Single NPCs and NPC Aggregates

We subsequently examined the differentiation potential of the transplanted NPCs and their capacity to give rise to mature neurons. To detect differentiation into immature neurons, we immunostained the transplanted cells with an antibody against doublecortin (DCX). As shown in Figure 4A, nearly all of the transplanted cells were positive for DCX (Fig. 4A). Moreover, we found that a significant portion of the transplanted cells gave rise to Tuj1-positive cells in both the single NPC and NPC aggregate grafts (Fig. 4B). However, at this stage, no NeuN-positive cells were found in either the single cell or aggregate grafts (Fig. 4C), suggesting that neuronal differentiation of the transplanted NPCs was still in the initial stage at 1 month posttransplantation. Immunostaining for hNuc was used to identify the transplanted cells (Fig. 4C). Our results indicated that the differentiation potential of the transplanted cells was determined by the intrinsic properties of the transplanted cells and was not affected by the method of cell delivery (i.e., single cell vs. aggregate). We also found no dopaminergic neurons [i.e., TH-positive cells (Fig. 5A)] in either the single NPC or aggregate NPC grafts.

Figure 4.

Short-term neuronal differentiation of single NPCs and NPC aggregates. (A) The transplanted cells (labeled with human-specific NCAM, red) differentiated into immature neurons expressing DCX. Upper panel: single cell transplantation; lower panel: aggregate transplantation. (B) The transplanted cells (labeled with nestin, green) differentiated into neurons expressing Tuj1 (red). Upper panel: single cell transplantation; lower panel: aggregate transplantation. The higher magnification images show that more of the marginal cells are Tuj1 positive, while more of the inner cells are nestin positive. (C) The transplanted cells (labeled with human nuclear antigen, red) did not differentiate into mature neurons expressing NeuN at 1 month posttransplantation. The higher magnification images show that the transplanted cells are NeuN negative. Upper panel: single cell transplantation; lower panel: aggregate transplantation. Scale bars in the low-magnification images: 100 μm; scale bars in the high-magnification images: 50 μm. G, graft; H, host tissue.

Figure 5.

Transplanted NPC aggregates cannot differentiate into dopaminergic neurons. (A) TH expression (green in merged images) was not detected in the transplanted cells (hNCAM positive, red in merged images) at either 1 month (upper panel) or 3 months (lower panel) posttransplantation. Scale bar: 100 μm. (B) DAT expression (green in merged images) was not detected in the transplanted cells (hNuc positive, red in merged images) at 3 months posttransplantation. Scale bar: 50 μm. G, graft; H, host tissue.

Long-Term Neuronal Survival and Differentiation of NPC Aggregates

To further assess the long-term survival and differentiation ability of the MC-expanded and MC-differentiated NPCs that were transplanted as either single cells or aggregates, we performed immunostaining with an hNuc antibody at 3 months posttransplantation (Fig. 6). Surprisingly, the single NPC grafts exhibited no hNuc-positive cells at this time point (data not shown), which indicated that substantial cell death occurred in the single NPC transplantation group after 1 month posttransplantation. In contrast, the NPC aggregate grafts still showed a well-defined graft core composed of hNuc-positive cells (Fig. 6A, upper panel) at 3 months posttransplantation.

Figure 6.

Long-term neuronal differentiation of NPC aggregates. (A) The NPC aggregates (upper panel, labeled with hNuc antibody, red) differentiated toward a neuronal linage (DCX staining in the middle panel, green), while no obvious oligodendrocyte (OligoDC) (Oligo2 staining in the middle panel, green) or astrocyte (GFAP staining in the middle panel, green) differentiation was found at 3 months posttransplantation. Scale bar: 500 μm. (B) Neuronal maturation and differentiation of the transplanted cells. Upper panel: low magnification showing substantial expression of NeuN in the graft region (red). G, graft; H, host tissue. Scale bar: 100 μm. Lower panel: high magnification showing individual transplanted cells (red) that are NeuN positive. The arrows indicate representative NeuN-positive transplanted cells. Scale bar: 50 μm. (C) GABA immunostaining was found in some of the transplanted cells. G, graft; H, host tissue. Scale bar: 50 μm. The arrows indicate representative GABA-positive transplanted cells. (D) Quantitative analyses of the percentages of mature neurons and GABAergic neurons of the surviving grafted cells.

The multiple lineage differentiation potential of the transplanted NPCs was assessed by immunostaining the grafted cells for either a neuronal marker (DCX), an astrocyte marker [glial fibrillary acidic protein (GFAP)], or an oligodendrocyte marker (Oligo2). Consistent with the observation at 1 month posttransplantation, the transplanted aggregates showed strong expression of DCX at 3 months posttransplantation (Fig. 6A, left panel). In contrast, we found no GFAP-expressing cells and very few oligodendrocytes (Oligo2-positive cells) in the grafted sites at 3 months post-NPC aggregate transplantation (Fig. 6A, middle panel and right panel, respectively), indicating that the NPCs used in our study, which were expanded and differentiated on MCs, do not give rise to astrocytes, and that only a very small percentage of the cells were differentiated into oligodendrocytes in vivo.

Compared to the grafted cells examined at 1 month posttransplantation, we found a significant increase in the portion of cells (53.25% of total surviving cells) that were positive for NeuN, a mature neuronal marker, at 3 months posttransplantation (Fig. 6B and C). These data indicated that the neuronal differentiation and maturation of the transplanted NPCs progressed gradually in vivo. However, no significant expression of TH and DAT was observed in the transplanted cells (Fig. 5), indicating that no DA neurons spontaneously differentiated from the NPCs when they were transplanted into the striatum, even after 3 months. In contrast, a portion of the grafted cells (17.53% of the total surviving cells) differentiated into GABAergic neurons 3 months posttransplantation (Fig. 6B and C).

Neurite Outgrowth From the Transplanted NPC Aggregates

One important criterion for evaluating whether transplanted cells are suitable for clinical therapy is to assess whether these cells can extend neurites over long distances and innervate their target brain regions^9,10. In this study, as indicated by hNCAM staining, neurites arising from the transplanted NPC aggregates extended in two directions. One direction was along the corpus callosum (CC) (Fig. 7A). As early as 1 month posttransplantation of the NPC aggregates, fasciculated neurites were observed extending more than 2 mm away from the transplantation core. The other direction extended rostrally to target the frontal cortex (Fig. 7B). As shown by the IHC-DAB staining in Figure 7B, dense hNCAM-stained neurites were observed in the frontal cortex (Fig. 7B, upper panel), which is 1.5 mm rostral to the transplantation site (Fig. 7B, lower panel). The morphology of individual transplanted cells also revealed long neurites extending from the neuronal cell bodies (Fig. 7B, lower panel). These data indicated that the transplanted NPCs were able to extend long neurites to reach their designated targets in the brain.

Figure 7.

Neurite outgrowth of transplanted NPC aggregates. (A) Immunofluorescence image showing fasciculated neurites (stained with an hNCAM antibody, yellow) arising from the transplantation site and extending along the corpus callosum (CC). Scale bar: 200 μm. (B) IHC-DAB staining using an hNCAM antibody showing that neurites arising from the transplantation site (striatum) projected rostrally to frontal cortex, which is 1.5 mm away from the transplantation site. Scale bar: 500 μm. The enlarged images in the upper panel show numerous neurite fragments in the frontal cortex. The enlarged images in the lower panel (striatum plane) show the long cellular processes that extended from the transplanted cell body. Scale bar: 50 μm.

Discussion

By intrastriatally transplanting the MC-expanded and MC-differentiated NPCs into NOD-SCID IL2Rgc null mice, we have demonstrated that these cells can sustain long-term survival and maintain restricted neuronal differentiation potential in vivo. This finding provides insight into the potential clinical utilization of MC-amplified and MC-differentiated NPCs. Furthermore, the current data clearly showed that the NPCs transplanted as aggregates survived much better than the single cells over a period of 3 months. The aggregate NPCs were also able to gradually differentiate into DCX-positive immature neurons, Tuj1-positive neurons, and NeuN-positive mature neurons in vivo by 3 months posttransplantation. Importantly, the transplanted NPC aggregates were able to innervate their forebrain target regions.

Survival of MC-Cultured NPCs After Transplantation

The MC culture system has several advantages for hESC expansion^20–22, including accelerated propagation and higher cell yields and purity. Culturing in a bioreactor also enables a more controlled environment for differentiation than monolayer methods. Thus, it is preferred for use in clinical cell transplantation therapies. It is also encouraging that our previous studies have integrated the expansion of hESCs and their subsequent differentiation into NPCs together in this MC culture system. However, elements in this culture system, such as the feeding regime, the attachment between the cells and the MC, and the agitation caused by stirring, may all result in characteristically different NPCs. Nevertheless, these NPCs express high levels of human NCAM and PSA-NCAM in vitro. Whether the NPCs generated from the MC culture system are usable for in vivo brain transplantation has not been studied. Moreover, whether dissociation of the differentiated NPCs from the MC would affect the survival and differentiation of the NPCs in the subsequent transplantation had not been tested. In the current study, the MC-amplified and MC-differentiated NPCs could survive up to one month when they were transplanted into the striatum as single cells. However, none of the single transplanted cells were found to have survived in the graft site at 3 months posttransplantation. The initial transplanted cell number, 2.5 × 10⁵ per mouse, is a commonly used amount of cells in mouse striatum transplantation studies and has been shown to result in a significant portion of surviving cells if the cells are prepared using the traditional 2D static method. The decreased in vivo survival of the NPCs derived from the MC culture system might have been caused by the dissociation process or the intrinsic properties of the NPCs. It has been reported that NPCs isolated from the mammalian CNS survive better than other cell types due to their lack of major histocompatibility complex I (MHC)³⁶. It might be possible that MC culture can increase the expression of MHC in hESC-derived NPCs and in turn weaken their in vivo survival ability. Thus, one should be cautious when determining the number of transplanted single cells if they are derived from an MC culture system, and methods to improve their survival should be investigated.

Cell Delivery Methods Affect the Survival of the Transplanted Cells

As the human brain is thousands of times larger than the brain of the rodent, a sufficient number of transplanted cells must survive over a prolonged period². Unfortunately, this issue cannot be solved by increasing the starting number of the transplanted cells because the cells are much larger than the average pore size of CNS tissue; therefore, they cannot penetrate into the surrounding host tissue^1,6,26,37. A large number of cells can increase mechanical damage to the surrounding host CNS tissue and can also lead to increased apoptosis of the transplanted cells due to lack of oxygen^6,30,37. Thus far, several strategies have been explored to attempt to increase the percentage of viable cell posttransplantation, including, but not limited to, the genetic modification of cells to promote their survival, differentiation, and migration^25,27,38,39. However, few reports have examined the importance of biophysical properties, such as cell–cell contact and the interaction between the transplanted cells and their surrounding matrix proteins^28–30. Our study has demonstrated that the NPCs delivered in aggregate form showed superior survival to those delivered in single-cell form. This likely occurred because the aggregates maintain cell–cell contact, which can promote the survival of the implanted cells. It has been shown that the establishment of cell–cell contact is beneficial for promoting cell survival, and in the absence of matrix attachment cells have been shown to undergo apoptosis^{17,26,29,30,40}. It has also consistently been observed that cells transplanted in aggregate form survive better than cells transplanted as single cells in cell transplantation-based diabetic therapy⁴¹. However, currently, some animal studies on cell transplantation for PD therapy have been conducted as single-cell deliveries. It should be noted that the optimized parameters used in single-cell transplantation may not be applicable for aggregate transplantation. Thus, in the future, more effort should be directed toward studying how to optimize cell aggregate transplantation in PD therapy.

Optimizing the Stage of Differentiation for Transplantation

Defining the appropriate cell differentiation state before grafting (naive vs. predifferentiated) is a key area that needs to be better understood prior to clinical translation of stem cell therapy^{6,7,12,13,15,42}. The optimal differentiation status of neuronal cells varies according to the specific neurodegenerative diseases treated. NPCs are preferred not only because they are capable of differentiating into the same cell type as the surrounding host tissue but also because these cells can provide neurotrophic benefits to the host tissue. For example, it has been shown that intrahippocampal transplantation of neural stem cells (NSCs) can improve cognitive functions in an Alzheimer's disease (AD) mouse model²³. This beneficial effect was shown to be mediated by brain-derived neurotrophic factor (BDNF) secreted from transplanted NSCs. Moreover, it has been reported that NSC transplantation is also effective for schizophrenia treatment⁴³. In our study, we chose to use MC-amplified, hESC-derived, polysialylated neuronal cell adhesion molecule (PSA-NCAM)-positive D9 NPCs as the transplanted cell source because NPCs are a good starting material and can be applied to various neurological diseases. Additionally, it was important to test the survival and the differentiation potential of the NPCs before we further directed them into specific lineages. Although embryonic NPCs are multipotent and capable of sequentially generating neurons and then glia⁴⁴, in our current study the MC-amplified, hESC-derived D9 NPCs were restricted to neuronal differentiation even after a prolonged period in vivo (Fig. 4A). However, maturation of the transplanted NPCs was slow because the NeuN-positive neurons only appeared at 3 months posttransplantation. This indicates that although the microenvironmental cues of the graft can help the transplanted precursor cells to continue to differentiate along the neuronal linage, the intrinsic properties of the transplanted cells greatly determine the differentiation status of the implanted cells⁴⁵. Thus, predifferentiation and patterning is indeed necessary if a particular neuronal type is needed, such as DA neuron patterning in PD cell therapy⁴⁶. It has been reported that only DA cells expressing the A9 substantia nigra phenotype, not the A10 phenotype, can alleviate functional deficits in a rat model of PD⁹.

In conclusion, our study demonstrated that MC-expanded NPCs can survive and differentiate after transplantation into mice over a 3-month period of time. The transplanted cell aggregates exhibited better survival and differentiation ability than the single cells. In addition, the transplanted cell aggregates were able to extend long axons to innervate their targets. Further studies will be required to better optimize cell aggregate transplantation for PD therapy.

Footnotes

Acknowledgments

We thank SingHealth Advanced Bioimaging Core for technical support for image acquisition. Additionally, we thank Dr. Zhang Wei and Miss Christina Lim for their technical advice. This research is supported by the Singapore National Research Foundation under its Translational and Clinical Research Flagship Program and administered by the Singapore Ministry of Health's National Medical Research Council. We also acknowledge the Agency for Science Technology and Research (A ∗STAR) for generous funding of this research. The authors declare no conflicts of interest.

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