1.2.1 Stem cell treatment in intracerebral hemorrhage (ICH)

There were four valid articles related to stem cell treatment in ICH. The phase of clinical trials, cell interventions, dosage, infusion site, number of the trials (number of controls), main outcomes, safety, and year were extracted from the manuscript (Table 2).

Stem cell therapy for ICH includes MSCs, olfactory ensheathing cells, neural progenitor cells, umbilical cord mesenchymal cells, and Schwann cells. The doses used vary from 1 × 10⁶ to 5 × 10⁸ injections, and the injection sites are concentrated in the brain (intraventricular, MCA), intrathecally, and venous. The scales used for evaluation included the Barthel index, and so on.¹⁷ The above clinical trials were safe and well tolerated after transplantation. No patients experienced serious adverse reactions or dose-limiting toxicity caused by MSC transplantation, and no adverse outcomes were observed.^{15, 16}

So Yoon Ahn treated three premature infants with severe intraventricular hemorrhage (IVH) with low-dose intraventricular transplantation of MSCs and six infants with high-dose MSCs. The results showed that the transplantation was well tolerated and the cerebrospinal fluid (IL)-6 levels increased after the transplantation. The initial CSF IL-6 and tumor necrosis factor-α levels were significantly correlated with the baseline ventricular index.¹⁵ V Bhati performed ipsilateral MCA arterial infusion of stem cells in 10 patients with acute stroke 8–15 days after stroke onset in the intervention group and no infusion was performed in the control group. It was found that eight patients in the intervention group showed a good clinical outcome, with a modified Rankin scale score of less than 2, and four patients in the control group achieved this outcome.¹⁶ The levels of CD34(+) progenitor cells in peripheral blood of 32 patients with primary ICH within 12 h after onset were detected at admission and 7 ± 1 day after the operation. It was found that the level of CD34(+) progenitor cells on Day 7 was an independent related factor for good prognosis at 3 months after the operation. These results suggest that CD34(+) progenitor cells may be involved in the functional recovery of patients with ICH.¹⁸

1.2.2 Stem cell treatment in brain ischemia

There were 29 articles related to stem cell treatment in brain ischemia. The phase of clinical trials, cell interventions, dosage, infusion site, number of the trials (number of controls), main outcomes, safety, and year are listed as follows (Table 3, Figure 1).

A large number of new clinical trials on cerebral ischemia have been conducted in recent years. In general, the momentum of stem cell therapy for cerebral ischemia into the clinic is strong. There are a variety of intervention methods, among which MSCs, BMMSCs, and NSCs account for the largest part of the cell intervention, followed by umbilical cord mesenchymal cells, CD34+ cells, olfactory ensheathing cells, Schwann cells, and dental pulp stem cells. Intervention methods also include the use of G-CSF, which indirectly regulates the mobilization of stem cells. Overall, the safety of stem cells in the treatment of cerebral ischemia is satisfactory. The adverse events related to the intervention were mild or not significantly different from those in the control group, and no unexpected brain structural abnormalities were observed.^{19, 25} In addition to bone marrow-derived stem cells (such as MSCs and BMMSCs) and CD34+ cells, which are commonly transplanted after autologous cell extraction, the rest of the cells are not easy to extract from themselves, and the treatment mostly involves allogeneic stem cell transplantation, which requires the use of immunosuppressive drugs. The good news, however, is that none of the clinical trials showed oncological or other serious adverse events during follow-up.²⁶ The number of cell injections varied, ranging from 5 × 10⁵ to 3 × 10⁸, and the number of injections was mostly one, with only four trials using multiple repeated cell treatments.^{23, 27, 32, 42} Multiple injections did not show significant safety changes compared to single injections, and patient symptoms also showed some improvement. The route of injection is also relatively diverse. MSCs are mostly injected intravenously, and in some trials, are injections were administered through the ipsilateral putamen of cerebral infarction or intranasally. For other cell types, the MCA route, intrathecal, cerebellomedullary cisterna, and intracerebral infarct area were mostly selected.^{17, 20, 22} The number of injected cells and the volume of solution around the cerebellomedullary cistern and infarct site are relatively small due to the smaller accommodating space than the arterioles and veins. For G-CSF, the injection dose is the standard 10 μg/kg injection for 5 days by a subcutaneous injection.^{39, 40} The modified Rankin scale, the NIHSS scale, the mRS score, the Fugl-Meyer assessment scale, the ARAT test, the European Stroke Scale (ESS), the ESS Motor Subscale (EMS), and the Barthel index (BI) were used to evaluate the function of cerebral ischemia before and after the intervention. Also, imaging and blood biochemical measurements of infarct size and blood biomarkers and other comprehensive assessments were performed.^{22, 34, 40}

Acute cerebral ischemia is defined as cerebral ischemia within 24 h to 1 week after onset. Stem cell therapy for acute cerebral ischemia is safe, but the efficacy is variable. In a single-arm, phase I clinical trial of bone marrow harvesting and intravenous infusion of autologous BMMNCs (10 million cells/kg) in patients with moderate acute ischemic stroke within 24–72 h after onset, treated patients showed a 1-point reduction in ES in mRS at Day 90, with safe outcomes.²⁸ In another prospective study, Banerjee et al. carried out a nonrandomized, open-label, phase I study, which involved infusion of autologous CD34+ cells through the middle cerebral artery in five patients with severe anterior circulation ischemic stroke who presented within 7 days of symptom onset; improvement in clinical function scores and reduction in lesion volume were observed at 6 months of follow-up in all patients.³¹ G-CSF has also shown the potential to effectively mobilize bone marrow CD34+ stem cells in patients with recent ischemic stroke.³⁹ However, some trials revealed that the treatment effect is not significant. Bone marrow-derived allogeneic Baak LM et al. delivered bone marrow-derived allogeneic MSCs intranasally for the treatment of 10 term neonates with perinatal arterial ischemic stroke; after 4 months, four infants showed asymmetric corticospinal tracts and three had abnormal early movements with no established treatment effect.¹⁹ Another phase II A, randomized, double-blind, placebo-controlled, single-center pilot trial showed similarly modest efficacy.²⁵ Four patients aged ≥60 years with moderate to severe stroke were treated with IV (intravenous) adipose tissue-derived mesenchymal stem cells (Ad-Mscs) within 2 weeks of onset. The median NIHSS score was lower at 24 months of follow-up, but the difference was not significant. There were no differences in the mRS scores between the two groups.

Cerebral ischemia with a duration of 1–3 weeks after onset is called subacute cerebral ischemia. Stem cell therapy for subacute cerebral ischemia has shown reliable safety, and the efficacy is related to the individual differences of patients. A case of a test using IV injection of autologous bone marrow derived MSCs in the treatment of subacute moderately severe ischemic of carotid artery stroke test, a IV therapy using autologous BMMSCs within 7–30 days of the involvement of the patients with ischemic stroke and another case of former cycle ipsilateral MCA arterial infusion of stem cells in treatment of test results shows good feasibility of treatment, and improvement of motor neural plasticity.^{16, 30, 34} Otherwise, treatment with ipsilateral MCA intraarterial infusion of stem cells has also shown favorable clinical outcomes in the majority of patients. However, the efficacy of IV infusions has been questioned in some trials. Kameshwar Prasad conducted a phase II, multicenter, parallel-group, randomized trial using an IV infusion of autologous BMSCs and found no benefit for stroke treatment.³⁰ Another similar trial showed a significant improvement from baseline in terms of the absolute change in the median infarct volume but not in the total infarct volume in the treatment group. There were no statistically significant differences in the median NIHSS, mRS, or BI scores at 12 months compared with the control group.²² Savitz SI, using a carotid infusion of ALD-401 in 100 patients with middle cerebral artery ischemic stroke, showed no difference in the primary efficacy endpoint and a higher incidence of small lesions on MRI in the treatment group compared with the control group.³⁶ Notably, Huns001-01 was injected directly around the subacute infarct area, suggesting that an intratumoral injection may be a more favorable method for delivering cells into the lesion site and improving motor function.⁴³ In a phase II B, single-center, randomized, controlled trial of G-CSF treatment, Timothy J England found a trend toward reduced changes from baseline in MRI ischemic lesion volume, suggesting that subacute administration of G-CSF is safe and that CD34(+) iron labeling in patients with ischemic stroke is feasible.³⁸ Hideo Shichinohe conducted a phase I open-label, uncontrolled dose–response study in which the bone marrow was harvested from each patient's iliac crest 15 days or later after symptom onset, treated as HunS001-01, and injected around the subacute infarct area. The results suggest that intraparenchymal injections may be a more favorable approach for delivering cells to the lesion site and improving motor function.⁴³ The current stage of stem cell therapy has a certain effect and can ensure safety, but the choice of intervention and the condition of the patients will greatly affect the outcome. More clinical trials are needed for comparison of intervention pathways, intervention cells, and the number of intervention cells to obtain more effective and exact effect data.

Treatment initiated 3 weeks after ischemic stroke was considered to be treatment in the chronic phase. At present, there is no effective treatment for chronic stroke. Fortunately, a considerable number of patients show significant improvement in behavioral endpoints and recovery of neurological and motor function after stem cell treatment.^{17, 20, 37} It is worth noting that using the allogeneic human neural stem cell line CTX0E03, Keith W Muir et al. performed implantation into the ipsilateral putamen of cerebral infarction in 23 adults with significant upper limb motor deficits 2–13 months after ischemic stroke; ARAT improvement was seen only in patients with residual upper limb movement at baseline.²⁶ A single infusion of 2 × 10⁷ UCMscs via a catheter into the M1 segment of the middle cerebral artery by Yongjun Jiang improved muscle strength and modified Rankin scale scores in patients with ischemic stroke, but not in patients with hemorrhagic stroke.³³ Jin Soo Lee conducted an open-label, observer-blinded clinical trial on 85 patients with severe cerebral artery regional infarction. Patients with ischemic stroke received an IV injection of autologous cultured MSCs, while the control group did not receive that individual quarantine. The degree of improvement was related to the level of serum stromal cell-derived factor-1 and the degree of external subventricular region involvement.²³

1.3.1 Stem cell treatment in PD

There are nine valid articles related to stem cell treatment in PD. We read the articles and extract the phase of clinical trials, cell interventions, dosage, infusion site, number of the trials (number of controls), main outcomes, safety, and year as the header list below (Table 4).

The most commonly used interventions are NPC, MSCs, and others including SVF, embryonic dopamine cells, hRPE, and stem cells with superselective arterial catheterization, among which MSCs, SVF, and stem cells with superselective arterial catheterization can be administered by autologous transplantation, while other cells are administered by allogeneic transplantation. The doses of cell grafts were relatively similar, ranging from a minimum of 1 × 10⁶ cells to a maximum of 3 × 10⁷ cells, most of which were concentrated in the order of 106. Most of them were single transplantations. Only one study used two unilateral striatal injections of 3 × 10⁷ NPC each at an interval of 7–57 months, which was the largest dose and the largest number of transplants in all stem cell therapies for PD disease, and the outcome was safe without significant side effects.⁴⁷ The injection sites were the putamen, striatum, lateral ventricles, intra-arterial (intracranial), facial muscles, and the nasal cavity. The scales used included PDQ-39, UPDRS, Hoehn-Yahr and Schwab-England, PSP, Unified Parkinson's Disease Rating Scale, and QOL.^{48, 50, 54}

The intervention can improve the neurological function of patients, improve the symptoms of PD, or maintain the stability of motor function. No serious adverse events or side effects were found in all the trials, and the safety was good, without signs of tumor or stroke.^{46, 49, 54} After transplantation of human neural progenitor cells, six of seven patients showed varying degrees of motor improvement and a trend of enhanced midbrain dopaminergic activity at the 1-year follow-up. At the 4-year follow-up, it was found that undifferentiated NPCS could be safely delivered to the putamen of PD patients using a stereotaxic approach, and midbrain dopaminergic neurotransmission was enhanced.⁴⁶ Two other trials of MSCS transplantation were conducted, although one route was intra-arterial and the other was intraventricular, and both showed significant improvements in symptoms such as facial expression, gait, and freezing episodes.^{48, 49} Autologous implantation of stem cells with superselective arterial catheterization in 50 patients showed a median 51.1% improvement on a unified PD rating scale, and eight patients showed an increased N-acetylaspartate/creatine ratio in the right and left basal ganglia.⁵⁴ One study found that human embryonic dopaminergic-neuron transplantation survised in patients with severe PD and produced some clinical benefit in younger (<60 years) patients, but not in older (>60 years) patients, with fiber growth and increased 18 F-fluorodopamine uptake in transplanted neurons from younger subjects. Yet, after the first year of improvement, 15% of the patients who received the transplant experienced recurrent dystonia and dyskinesia.⁵² Two years later, the authors conducted a second clinical trial of putaminal transfer of embryonic dopamine cells and showed that the symptoms of patients in the cell transplantation treatment group were significantly improved, but 4 out of 34 transplanters had secondary dyskinesia after significant clinical improvement within 1–2 years.⁵¹ We need to be alert to the relationship between this age cutoff and the benefit of treatment. In future trials, we should expand the samples and carefully explore other factors that affect the clinical efficacy to maximize the advantages and avoid the disadvantages, and benefit the public.

1.3.2 AD and stem cell treatment

We screened and stayed nine valid articles related to stem cell treatment in AD. The phase of clinical trials, cell interventions, dosage, infusion site, number of the trial's cases (number of controls), main outcomes, safety, and year were extracted and are listed as follows (Table 5).

At present, the number of clinical trials of stem cell therapy for human AD is relatively small, and there is a large gap. Hee Jin Kim conducted a phase I clinical trial in nine patients with mild to moderate Alzheimer's dementia.⁵⁵ Four weeks before administration, the Ommaya reservoir was implanted into the right lateral ventricle of the patients. Six patients received a high dose of hucB-Mscs (3.0 × 107 cells/2 mL in all patients in triplicate injections, 4 weeks apart). Follow-up for 3 months showed that there were three serious adverse events thought to be caused by the study products, and other adverse reactions such as fever and headache all resolved within 36 h. At a further 36 months of follow-up, no further serious adverse events were observed. Juan Sanchez-Ramos conducted a double-blind, placebo-controlled, crossover design to administer G-CSF to eight patients with mild to moderate AD for 5 days. No serious adverse events were identified, the most common side effects being transient leukocyte increase, myalgia, and diffuse pain. The results showed that the average paired association learning improved significantly. There was no significant difference in cerebrospinal fluid amyloid-β1–42 levels between 2 weeks of G-CSF treatment and placebo treatment.⁵⁶ In addition, Maler found that early Alzheimer's dementia was significantly associated with age, cerebrospinal fluid β-amyloid (Abeta) 1–42, and the Abeta42/40 ratio, and the obtained data suggested that regenerative hematopoietic support in the central nervous system was insufficient in the early stage of AD.⁵⁷

1.4.1 Immunogenicity of stem cells

Stem cells used in clinical trials are mostly autologous and allogeneic, of which allogeneic stem cells are more commonly used because of their convenience and easy availability. However, stem cells are immunogenic and potentially trigger both adaptive and innate immunity. Most in vitro studies have highlighted the immunosuppressive properties of MSCs, but mismatched MSCs are still immunogenic. Although allogeneic MSCs does not elicit rapid rejection in immunocompetent hosts like mismatched fibroblasts or HSCs, rejection of allogeneic MSCs was slower than that of other allogeneic cell types. However, it is inappropriate to suggest that MSCs are immunologically privileged because they do elicit humoral and cellular immune responses in vivo. The timing and severity of rejection to MSC appear to depend largely on the context and on the balance between immunogenicity and immunosuppressive factor MSC expression. Mismatched allogeneic and allogeneic MSCs can be preferentially present in immunosuppressed in vivo environments, such as tumors, but cannot persist in other tissues within the same animal.^{58, 59} This suggested that local immunosuppression was required to mask MSC immunogenicity.

A key example of differential immunogenicity is not only HLA-dependent allorecognition but also the differential expression of high procoagulant tissue factor (TF/CD142) between different cell products. Different immediate blood-mediated inflammatory response (IBMIR) triggeres differently, which is a major safety concern for intravascular use of different stem cell products. Cells that are not in regular contact with blood often express different amounts of highly procoagulable tissue factor (TF/CD142), and if MSCs are considered to be derived from perivascular cells or pericytes, then TF/CD142 expression serves the biological function of stopping bleeding by triggering thrombosis in response to vascular injury.^60-62 In patients who are not properly anticoagulated, potentially fatal adverse events such as thrombosis and embolism can result.^{63, 64} These reports of adverse events and cases highlight the clinical need for new criteria for blood compatibility assessment. MSCs derived from different tissues vary greatly in their blood compatibility.^{65, 66} Infusion of procoagulants expressing the cellular products of tfscs, such as mesenchymal stem cells, hepatocytes, and islets, triggers innate immune attack (IBMIR), which affects cell engraftment, safety, and efficacy. In general, AT- and PT-derived MSC products express higher levels of TF/CD142, thereby triggering a stronger IBMIR with concomitant thromboembolic events, unless carefully antagonized with anticoagulants. Therefore, blood compatibility tests must be performed in clinical work, and CAT and TEG are commonly used in clinical practice to obtain adequate safety estimates.^{67, 68}

1.4.2 Cryopreservation and freeze–thawing

Cryopreservation and its associated freezing and thawing procedures are one of the final steps in the manufacture and clinical application of a variety of common and feasible cell therapies, but the differences in viability and function between frozen stem cells and fresh stem cells cannot be ignored. Although there are some preclinical data suggesting that thawed MSCs are somehow equivalent to fresh MSCs, there are also some studies confirming that fresh low-passage MSCs can provide more effective therapy.^69-72 Cryopreservation below −135°C can effectively stop cell activity, but storage often encounters some warming events that can cause transient warmth, for example, due to frequent cell batches being removed/introduced from storage devices, which can affect or even damage any cells that remain in the cold environment for a long time. This is especially true when the temperature is increased repeatedly to the extent that it may affect/impair the cell mass. Alternatively, a major problem with cryopreservation of HSPCs is reduced viability due to cell damage during freezing and thawing.⁷³ Such damage can be caused by osmotic disturbance, ice crystal formation, and cellular dehydration.⁷⁴ Freezing and thawing, especially the formation of exothermic ice crystals, can puncture and thus damage cell membranes. Thawed cells readily recover from freezing and can show a higher degree of membrane asymmetry, phosphatidylserine exposure, heat shock protein expression, and actin–cytoskeleton disruption. These processes are associated with potential changes in osmolality, pH, and temperature and must be effectively controlled.⁷⁵ In addition, frozen–thawed MSCs also elicited stronger innate and adaptive immune responses and were more likely to trigger an IBMIR.⁷⁶ The complement activity human serum exposure assay showed that thawed MSCs undergo more rapid lysis due to increased complement activation, and that thawed MSCs may undergo more rapid lysis in vivo via IBMR-mediated detrimental processes immediately after infusion.⁷¹ A recent study demonstrated better clinical outcomes with fresh and low passage cells, with patients treated with freshly harvested cells in low passage having response rates twice as high as those in the frozen–thawed cell treatment group, and impaired immunomodulatory and hemomodulatory properties of cryopreserved MSCs after thawing, leading to faster complement-mediated elimination after blood exposure.⁷⁶

It has been suggested that cryopreserved HSPCS should be infused as soon as possible after thawing, including freezing medium, to maintain their optimal cell viability and function, but prior washing/removal of cryoprotectants is also recommended in the literature, especially if the patient weighs less than 20 kg.^{77, 78} Although removal of DMSO may be ideal to prevent undesirable toxicity, DMSO elution carries a risk of cell graft contamination/damage and requires actual cell handling in the field. These factors raise the possibility of inducing apoptosis in the cell graft, which may delay HSPC engraftment in the patient.⁷³ Techniques that minimize the reduction in survival of cryopreserved stem cells are being actively explored. One approach is to use poly-D-lysine (PDL)-coated microgel encapsulation of MSC before cryopreservation, which needs to be robust enough, safe, and allow paracrine interactions and induction of immune regulation and regeneration.⁷⁹ Similarly, incorporation of cells into hydrogels may be an effective way to extend/optimize their paracrine potency, which may also improve cell survival after thawing and in vivo application.⁸⁰ Hornberger et al. summarized the techniques which lead to the greatest survival of stem cells after freezing, including alternative cryoprotectants and preinfusion cryoprotectants, and showed that the number of CD34+ cells after thawing was a good predictor of post-infusion function.⁷³ In conclusion, the study of freeze–thaw techniques may provide key advantages to improve the safety and efficacy of cell therapies.