Human NPCs survived healthy in the cerebro-parenchyma of adult rats without immunosuppression
In the first series of experiments, to directly visualize the survival of grafts, we used 2 reporter human cell lines (H1-CAG-DsRed or H1-CAG-GFP) that were previously generated in our lab with constitutive expression of DsRed or GFP reporter gene, at AAVS1 locus of H1 human embryonic stem cell line cells (hESC) through CRISPR/Cas9 gene targeting28.
Early passaged (P2-P5, Day 26–56 after neural induction onset) human H1-CAG-DsRed derived NPCs were transplanted into the intact striatum of immunocompetent adult rats. Within 4 weeks after transplantation, grafts survived healthy in ALL animals with no need of immunosuppression, and no obvious immune rejection was observed in any time point, from 4 days(dpt) to 4 weeks (wpt) post-transplantation( (0/22 animals). Human cells were identified by co-localization of human specific nuclear antigen (hNA) and DsRed (Fig. 1A). The first case of massive cell death of human grafts (due to the immuno-rejection, see below) happened at 5 wpt, with a rejection rate of 15.4% (2/14) at 5–6 wpt. The rejection rate abruptly increased to more than 50% (9/16) at 8–12 wpt (Fig. 1B-C). Surprisingly, although dead cells (not unequivocally from engrafted cells) might be observed in the injection track or around due to stab injury or secondary inflammation, no brains harbored both live and dead human cell mass within the graft core colony, implicating that the massive graft necrosis was a transient process and took place as an all-or-nothing event.
We repeated the experiment by using another hESC line (HN4, ATCC). All rats (3/3) with intra-striatum HN4 graft had live graft colonies when sacrificed at 4 wpt, whereas overt rejections occurred in 8-wpt rats (4/4), probably due to a high proliferation rate of this batch of HN4 NPCs in a retrospective analysis (Fig.s1).
Engrafted human NPCs had a neuron-restricted cell fate
We used early passaged NPCs to transplant in most of our experiments. After settled down (usually within 2 wpt), these engrafted human NPCs started to expand, and formed rosette-like structure at 3–5 wpt, which disappeared at longer time points. None of the host brains harbored a teratoma within the observation time window (H&E staining, Fig.S2A). It is reassuring that immunostaining of the grafts failed to detect any human cells positive for OCT4, AFP and sox17 as markers for residual pluripotent or non-neural cells (Fig.S2B). Notably, all animals lived healthy without any signs of depression or anxiety before sacrifice.
Although quite a few human cells migrated away along white matters tract or blood vessels(Fig.S2C), the majority of the engrafted cells stayed in the injection sites and dispersed within the vicinity, forming a graft colony (Fig. 1A). In the graft colonies, a large number of cells expressed NESTIN (neural stem cell marker), and/or early neuronal markers, like DCX (neuroblast marker), TUJ1 (early neuron precursor) at 3–8 wpt(Fig. 2A), indicative of an early neuronal differentiation stage. Many graft cells also expressed MAP2 (another neuron marker) during this period, while NeuN expression only manifested by 8–12 wpt (Fig. 2A), supporting the previous notion that prolonged time window is needed for human NPCs to differentiate into mature neurons. We failed to detect GFAP expression in human cells at any time pointed up to 12 wpt (Fig. 2A), indicative of highly pure neuron-restricted human NPCs transplanted.
We further investigated whether the survived human neurons could well incorporate into host neural circuits without immunosuppression by using retrograde monosynaptic tracing method we recently reported 28. We used another genetically modified human embryonic stem cell line(H1-CAG-GTRqp) that constitutionally expresses rabies virus glycoprotein, avian TVA receptor(required for selective infection with EnvA-pseudotyped glycoprotein-deleted rabies virus (ΔGRV)) and EGFP under control of the human CAG promoter. At 11 wpt, rabies virus (ΔGRV) was injected at 0.5 mm-distance above the transplantation site (deep in motor cortex) and rats were perfused 7 days later. The infected human cells co-expressed EGFP and mCherry fluorescence (as the starter neurons, EGFP + mCherry+, Fig. 2B-a), which received the afferent projections from the host neurons that only expressed mCherry (as the traced neurons, EGFP-mCherry+) in both the ipsilateral cortex (Fig. 2B-b) and the ipsilateral thalamus (Fig. 2B-c).
All these data showed that the grafted NPCs were highly neuronal-restricted, could properly migrate, differentiate into neurons as expected, further mature and incorporate into host neural network under no immunosuppression settings.
Late-onset cell death was due to immune rejection
As mention above, at late time points (i.e. 5, 6, 8, 12 wpt), we only saw massive dead human cells deposited in some animals, as identified by dusty DsRed fluorescent signals without cell morphology, where cavitation and cracks were sometimes conspicuous (Fig.S3A and Fig. S3B). The hNA positive immunoreactivity was also found desperately messy and weak, indicative of rapid loss of cellular components of engrafted cells (Fig. 1B). Instead, the graft colony was reoccupied by host cells, many of which with irregular cell body showed robust Iba1 expression, a marker of microglia or monocytes, implicating undergoing destructive phagocytosis of the dead graft cells by host (Fig. 3A).
To figure out whether this seemingly spontaneous late-onset cell death was due to adaptive immuno-rejection, lack of oxygen and glucose supply or other reasons, e.g. spontaneous apoptosis, we further did H&E staining, and CD3 immunostaining, a T-lymphocyte marker. In H&E staining, the rejected grafts were characterized with dense and clustered leucocyte infiltration (Fig.S3B), and many of them were CD3 positive with lymphocyte morphology (Fig. 3B). Perivascular lymphocytic cuffing was present within the graft and at the graft-host interface. We also detected heavy host IgG deposition within rejected graft, suggestive of the precipitation of humoral immunity (Fig. 3C). Moreover, the intense infiltration of host inflammatory cells (microglia and T-lymphocytes) and IgG deposition was circumscribed to necrotic graft remnants, and largely spared the neighboring host structures. In contrast, non-rejected human grafts were completely devoid of any leukocyte infiltrate and IgG deposition, except weak signals detected along the injection track at early time points (Fig. 3B-C).
In another experiment, we transplanted human NPCs into two different parenchymal sites of the same rats with an interval distance of 7 mm (n = 12), and examined at 5 wpt. Human grafts were found dead at both sites in 4 recipients, while others only contained live human graft colony (Fig.S4). This “Both-or-None” phenomenon ruled out the possibility that the elimination of human cells was mainly due to lack of oxygen and glucose supply or spontaneous apoptosis. The reasonable explanation for this is that hosts sensitized by either graft would launch immune attack on both.
Taken together, our results suggested that immuno-rejection might be the major contributing factor to the late-onset death of human grafts. This conclusion also borrowed supports from our another experiment showing that human grafts survived in rat brain up to 6 months when daily cyclosporine immunosuppression was used28.
HLA-ABC expression was NOT the trigger for the late-onset rejection of human NPCs
Studies have demonstrated that MHC expression are of prime importance in allograft rejection and may be the precedent step to xenograft rejection 29,30. Human breast cancer cells (MDA-MB-231), which express high-level HLA-ABC, were quickly rejected after transplanted into the striatum (Fig.S5A). Consistent with previous studies from other labs, human ESC-derived NPCs displayed a low HLA-ABC expression level (Fig.S5B), which might protect them from immune attack at the time of graft when the blood–brain barrier (BBB) was transiently disturbed. So the next question is whether engrafted human NPCs would upregulate the HLA-ABC expression during the survival time and then trigger the late-onset cell rejection. The immunostaining results showed that HLA-ABC expression remained barely detectable in all non-rejecting brains at any time point up to 12 wpt (Fig.S5C), and very few HLA-ABC positive cells could be occasionally identified if given much prolonged survival time(data from another experiment with immune-suppression). We also failed to detect HLA-DR expression (Fig.S5C), which has long been strongly associated to transplant rejection31. Thus, the expressions of HLA-ABC and HLA-DR antigens retained a low level in vivo during our observation time window, and should not be responsible for initiation of the late- onset human cell rejection.
Intra-cerebroventricular human NPC grafts were vulnerable to immune attack more than the intra-striatum grafts
Notably, in some rejecting cases, we noticed that the grafts protruded into the paraventricular area, where human cell antigen might more easily get access to the immune system and trigger the immune reaction. Recent findings have revealed a role for circumventricular organs, such as the choroid plexus, as gateways in the trafficking of peripheral leukocytes to the CNS 32,33. The meningeal lymphatic vessel was found to drain cerebrospinal fluid directly into cervical lymph nodes 34. So, we hypothesized that the rupture of the paraventricular structure should be detrimental for survival of human grafts. If this is true, the intra-cerebroventricular human grafts should not be able to survive long in immunocompetent rats. So we transplanted human NPCs into the lateral ventricles to see whether the brain ventricles are as immune-privileged as parenchyma. Four weeks later, we did not see any live graft in these animals (n = 6). The ventricles were not enlarged, and only trace of dead cells sparsely scattered in the ventricles, indicative of quick depletion of human cells. Given that the ventricle of rat and mouse brain has been shown to be a favorable site for neural grafts in immunodeficient xenogeneic hosts 35, the death of intra-cerebroventricular graft was unlikely due to non-immunologically environmental factors. More importantly, intra-cerebroventricular graft also induced death of human NPCs deposited on the upper and lower banks of the ipsilateral lateral ventricles or in the fimbria of the hippocampus with T-lymphocyte infiltration. In another experiment, human NPCs were injected close to the lateral ventricle (within 0.5 mm to the lateral ependymal wall), and no human grafts survived at 6 wpt (n = 5). All these results suggested that the rupture of the paraventricular structure was detrimental for the graft survival in immunocompetent rats (the rejection rate: 12.5% (intra-cerebro-parenchyma, 1/8 at 6 wpt) vs. 100.0% (intra-/ para-cerebroventricular, 11/11).
Hypoproliferative human NPC grafts further extended their survival
Low immunogenicity of human NPC grafts (with low HLA-ABC and HLA-DR expression level) could protect them from the host immune attack. Another arm of the CNS immune-privilege is the BBB, which protects the CNS from the immune attack to a great extent 5. The late-onset human graft rejection could be also due to the compromise of this barrier. As mentioned above, the early passaged human NPCs started significant expansion after settled down in the rat brain. When these grafts grew too quickly to disperse promptly, they easily formed a large and incongruous hyperplastic core within short time (Fig. 1 and other figures), which might impose a continuous compression on the surrounding tissues and destruct the local vascular system or break into the paraventricular organ, resulting in intermittent perturbations of local BBB or ventricle-meningeal lymphatic system and finally graft rejection as described above. The heavy T-lymphocyte infiltration and IgG deposition within the rejected graft did demonstrate such vascular or paraventricular damages. So we hypothesized that the rapid and sustained enlargement of grafts might account for the human cell rejection in our experiments. We turned to late passaged NPCs (beyond 90 days after neural induction onset), which exhibited a much lower proliferation capability (Fig. 4A-C). Even better than expected, up to our longest survival time (more than 4 mpt), the late human NPCs survived in the overwhelming majority of animals (2/2 at 4-5w, 9/10 at 8 wpt, 5/6 at 12 wpt, 3/4 at more than 4 mpt) without massive cell death. Graft survival analysis showed that late passaged NPCs with low proliferation survived significantly longer than early passaged NPCs (Fig. 4D, Log-rank test, P < 0.01**). The late hypoproliferative NPC grafts formed a much smaller graft core than the early passaged graft at 4 wpt and thereafter (Fig. 4A vs. B). Moreover, the long-term survived human grafts migrated along the white matters into a wide territory of ipsilateral cerebral cortex without a conspicuous graft core (FigS6).
However, further studies are needed to demonstrate that such a rupture of BBB or paraventricular structure occurs prior to the cell rejection, and to determine what antigens on the human cells would trigger this immune reaction in that the expression level of HLA-ABC and HLA-DR antigen remained barely detectable.
The host microglia responded moderately to human NPC grafts
Previous studies have suggested important roles played by microglia in both the antigen presenting and immune attack phases of intracerebral allograft rejection36,37. In the absence of CNS inflammation, microglia showed resting ramified morphology typically of non-overlapping long branching processes and a small cellular body (Fig.S7A). One of the characteristics of microglia in vivo is their ability to survey the parenchyma from their static tiling position38 and to react quickly to even small pathological changes. In response to the human grafts, the host microglia proliferated, migrated into and recolonized the graft area, where host cells including microglia had been squeezed away by the enlarged human graft (Fig. 5). Although individual phagocytic microglia were occasionally observed within the graft, most of the invading microglia showed a migrating rod-like or “cup” shape, or mildly activated with couple thick processes (Fig. 5). This quite differed from their resting ramified morphology in the intact host brain region (Fig.S7A), and also sharply contrasted to the massive end-stage phagocytic microglia in the rejecting region, which showed intensive activation typically with hypertrophic cell body, ring or irregular shape and severely retracted branches (Fig. 3B, Fig.S7B).
We further examined the host MHC-II expression in the non-rejecting animals to seek any early cues underlying the late-onset immuno-rejection. We failed to find any MHC-II expression in astrocytes and endothelial cells (or below the detectable level of the present method) in brain parenchyma, but a subpopulation of microglia within healthy graft area upregulated MHC-II expression (Fig. 5A). Nearly all MHC-II positive microglia were mildly activated except individuals with phagocytic profiling, while resting microglia never showed any MHC-II expression. Notably, the host MHC-II expression level remained relatively low and showed no significant increase over the long-term surviving period (Fig. 5C), implying that the MHC-II positive microglia failed to present antigens to the host immune system and trigger downstream responses, suggestive of an immune-isolation role by the intact BBB and ependymal wall and the “immune privilege” of CNS. Similarly, as mentioned below, although lipopolysaccharide (LPS) challenge increased MHC-II expression within the graft (Fig. 5B), this did not significantly increase the rejection rate of human cells. LPS also induced a sparse MHC-II upregulation in the microglia in a brain-wide manner, indicative of undergoing inflammation (Fig.S8).
All these results proposed that the mild elevation of MHC-II expression in the microglia in response to live human grafts was a sign of exogenous graft-induced inflammation (and/or just small environment changes) or the initial immuno-recognition without downstream immune reaction. The chronological quantification of MHC-II expression showed no gradual signs indicating initiation of rejection in all non-rejecting rats, again supporting the notion that the immuno-rejection was an unexpected and sudden event.
LPS-induced peripheral inflammations did not increase the rejection of human NPC grafts
To test whether or not peripheral inflammation would induce the rejection of the stable human NPC grafts, we used a well-established LPS challenge paradigm 39. Rats were injected intraperitoneally with 2.0 mg/kg LPS for 2 consecutive days at 4.5 wpt when human grafts had settled down. LPS injections induced a brief sickness (e.g. fever, loss of body weight, anorexia and hypokinesia) that lasted for about 3 days except the body weight changes. At 6 wpt (10 days after the second LPS injection), trace of microglia activation could still be discerned from the Iba1 expression level and profiling in a brain-wide manner (Fig.S8). LPS also further increased phagocytic microglia within the graft colony, in circle or ring shape with apparent MHC-II expression (Fig. 5B). Nevertheless, this LPS-induced peripheral inflammation and microglia activation in CNS did not significantly increase the rejection rate of human cells (1/5 in LPS-challenged VS. 1/6 in non-LPS). In consistent with this, we also did not see upregulated lymphocyte infiltration in the LPS-challenged non-rejecting brains.