A Single-Arm, Open-Label, Single-Center Clinical Study on Unrelated Umbilical Cord Blood Transplantation for the Treatment of Amyotrophic Lateral Sclerosis (ALS)
Phase: NA
Conditions: ALS (Amyotrophic Lateral Sclerosis)
Sponsor: Institute of Hematology & Blood Diseases Hospital, China

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A Single-Arm, Open-Label, Single-Center Clinical Study on 
Unrelated Umbilical Cord Blood Transplantation for the 
Treatment of Amyotrophic Lateral Sclerosis (ALS) 
 
Study Protocol 
 
 
 
 
 
 
 
NCT number 
：ALS-UCB-2025                    
Version 
Date 
：V2.0                             
：Dec 11. 2025                      
Study Officials 
：Institute of Hematology & Blood 
Diseases Hospital                     
Principal Investigator 
：Zimin Sun                         

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Contents 
1 Confidentiality Statement .................................................................................................... 3 
2 Research Team ...................................................................................................................... 3 
3 Background ........................................................................................................................... 3 
3.1 Current Research Status of ALS .............................................................................. 3 
3.2 Research Progress on HSCT for Neuroinflammatory Diseases ............................ 5 
3.3 Advantages of Unrelated Umbilical Cord Blood Transplantation ........................ 6 
4 Clinical Data of the ALS Cohort ......................................................................................... 8 
4.1 Analysis of Basic Clinical Characteristics Based on Questionnaires from 805 
ALS Patients .................................................................................................................... 8 
4.2 Laboratory Results and In-depth Clinical Assessment of 8 ALS Patients............ 8 
5 Experimental Results ......................................................................................................... 13 
5.1 scRNA-seq ................................................................................................................ 13 
5.2 Flow Cytometry Results .......................................................................................... 14 
5.3 CFC Assay ................................................................................................................ 15 
5.4 Drug Sensitivity Testing .......................................................................................... 16 
6 Animal Experimental Data ................................................................................................ 17 
7 HSCT Protocol and Associated Risks ............................................................................... 18 
7.1 Inclusion Criteria..................................................................................................... 18 
7.2 Exclusion Criteria .................................................................................................... 19 
7.3 Myeloablative Conditioning Regimen and GVHD Prophylaxis Protocol .......... 20 
7.4 Unrelated Umbilical Cord Blood Transplantation Protocol ................................ 22 
7.5 Post-Transplant Monitoring and Management of Potential Complications ...... 23 
8 Evaluation ........................................................................................................................... 25 
8.1 Safety Assessment System ....................................................................................... 26 
8.2 Multidimensional Efficacy Assessment .................................................................. 28 
9 References ........................................................................................................................... 28 
 

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1 Confidentiality Statement 
This protocol is provided to investigators as reference material for the clinical trial 
of unrelated umbilical cord blood transplantation in the treatment of amyotrophic lateral 
sclerosis (ALS). As it contains confidential information and proprietary technology of 
the sponsor, it must not be disclosed to any third party other than the investigators and 
the sponsor without prior authorization. Violators shall be held legally accountable. 
2 Research Team 
Clinical Team: Zimin Sun, Chief Physician and Ph.D. Supervisor, Regenerative 
Medicine Diagnosis and Treatment Center. Specializes in the optimization of clinical 
techniques for umbilical cord blood hematopoietic stem cell transplantation and related 
basic research. (Team members: Erling Chen, Hong Pan, etc.) 
Scientific Research Team: Tao Cheng, Dean, among the first cohort of Tenured 
Professors and Ph.D. Supervisors at Peking Union Medical College. Engaged long-term 
in hematology, stem cell biology research, education, and related administrative work. 
Established the technology system for blood cell reprogramming and gene therapy. 
(Team members: Sha Hao, Linping Hu, etc.) 
Collaborative Team: Yijun Song, Chief Physician and Ph.D. Supervisor, Critical 
Care Medicine Diagnosis and Treatment Center. Long-term involvement in the 
diagnosis, treatment, and research of complex neurological diseases such as epilepsy, 
dementia, and motor neuron diseases. (Team members: Bin Zhang, etc.) 
3 Background 
3.1 Current Research Status of ALS 
ALS, also known as “Lou Gehrig’s Disease”, is a rapidly progressive and fatal 
neurodegenerative disorder with a global prevalence of approximately 0.73-1.89 per 
100,000 individuals [1]. In China, there are an estimated 200,000 ALS patients, with 
about 25,000 new cases diagnosed annually [1]. The exact etiology of ALS remains 

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unknown and is currently believed to result from an interplay between genetic and 
environmental factors. Identified pathogenic genes include C9orf72, SOD1, TARDBP, 
and FUS. The pathogenesis of ALS is complex, with the core feature being the selective 
loss of motor neurons, involving multiple intertwined pathological processes. Major 
hypotheses include glutamate excitotoxicity, oxidative stress, protein misfolding and 
aggregation, and neuroinflammation [2]. 
Early pathological changes in ALS involve dysfunction of both upper and lower 
motor neurons and the persistent activation of neuroinflammatory responses. Microglia, 
the resident immune cells of the central nervous system (CNS), rapidly transition from 
a resting state to a pro-inflammatory (M1) phenotype. This activation leads to the 
release of a large number of inflammatory factors (such as TNF-α, IL-1β, IL-6, NO, 
ROS) and chemokines (such as MCP-1/CCL2), and triggers the NLRP3 inflammasome, 
directly or indirectly damaging motor neurons. Furthermore, systemic immune 
dysregulation plays a significant role in ALS pathogenesis. ALS patients exhibit 
reduced numbers of regulatory T cells (Tregs) in both peripheral blood and the CNS, 
alterations in the number of activated CD8+ T cell infiltrates, and a shift in the T helper 
cell (Th1/Th2) balance towards the pro-inflammatory Th1 phenotype, collectively 
driving the inflammatory microenvironment of the disease [3, 4]. Clinically, elevated 
inflammatory biomarkers (such as pNfL, sTREM2) are often detected in the 
cerebrospinal fluid (CSF) of ALS patients, further corroborating the close association 
between neuroinflammation and ALS disease activity. 
Clinically, ALS patients primarily present with progressively worsening muscle 
weakness, atrophy, fasciculations, and spasticity, ultimately succumbing to dysphagia 
and respiratory muscle paralysis. Currently, therapeutic options for ALS are extremely 
limited. In China, only Riluzole and Edaravone are approved for clinical use. Riluzole 
merely extends patient survival by 2 to 3 months, offering a very modest therapeutic 
benefit [5]. In recent years, exploratory treatment strategies targeting novel pathways 
such as neuroinflammation, immune dysregulation, and energy metabolism have 

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emerged, including the infusion of Tregs, mesenchymal stem cells, and neural stem 
cells. However, these approaches have still failed to halt disease progression 
[NCT05695521, NCT03280056, NCT06973629, NCT02290886]. Given the high 
heterogeneity in ALS pathogenesis and its multisystem involvement, single-target 
intervention strategies are inadequate to comprehensively correct its widespread 
functional abnormalities. Therefore, there is an urgent clinical need for novel therapies 
capable of multi-targeted, systemic intervention. 
3.2 Research Progress on Hematopoietic Stem Cell Transplantation (HSCT) for 
Neuroinflammatory Diseases 
Studies have shown that classic chronic neurodegenerative diseases primarily 
caused by immune dysregulation, such as multiple sclerosis (MS), Alzheimer’s disease 
(AD), and Parkinson’s disease (PD), all exhibit neuroinflammation. CNS is not an 
immune-privileged site but rather undergoes immune surveillance under homeostasis. 
Following injury, various immune cell subsets infiltrate, each exerting distinct and 
interactive effects on the neural parenchyma [6, 7]. Direct channels exist between the 
skull bone marrow and the cerebral meninges. After events like stroke or meningitis, 
these channels can provide a direct pathway, allowing immune cells to be rapidly 
mobilized from the skull bone marrow into the brain [8]. Professor Qiang Liu’s team 
from the Department of Neurology at Tianjin Medical University General Hospital 
systematically analyzed the characteristics of bone marrow hematopoiesis in MS 
patients during the active phase of the disease using techniques such as single-cell 
sequencing, lineage tracing, and flow cytometry. They discovered that hematopoietic 
stem cells (HSCs) in MS patients exhibit a myeloid bias. Autoreactive T cells in MS 
patients home to the bone marrow guided by chemokines like CXCL12. Within the 
bone marrow, these autoreactive T cells highly express CCL5, leading to the activation 
and proliferation of HSCs and their downstream myeloid progenitors. This results in 
abnormal myeloid hyperplasia in the bone marrow, generating a large number of 
monocytes and neutrophils. These cells can drive T cell-mediated autoimmunity and 

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migrate to the CNS, causing inflammatory damage [9]. 
HSCs are among the most widely used and clinically well-established adult stem 
cell types, serving as the source of all blood cells throughout an organism’s lifetime 
[10]. In recent years, researchers have found that using non-myeloablative conditioning 
followed by HSCT to treat MS can reboot the immune system and significantly slow 
the progression of relapsing-remitting MS. Mechanistic studies suggest that HSCT may 
disrupt the activation cycle between astrocytes and microglia, alleviating the chronic 
inflammatory state in CNS, while also partially mitigating mitochondrial dysfunction 
and slowing neurodegeneration [11, 12]. A research team led by Professor Bo Peng 
from Fudan University and Shanghai Sixth People’s Hospital Affiliated to Shanghai 
Jiao Tong University School of Medicine published a breakthrough finding in the 
journal Science. By replacing pathogenic microglia in the CNS through HSCT, they 
successfully halted disease progression in adult-onset leukoencephalopathy with axonal 
spheroids and pigmented glia, with patients’ motor and cognitive function scores 
stabilizing or even showing improvement [13]. Given the close association between 
ALS and neuroinflammation, HSCT holds promise as a crucial avenue for improving 
the immune environment in ALS and breaking through the current therapeutic 
bottleneck. 
3.3 Advantages of Unrelated Umbilical Cord Blood Transplantation 
Unrelated umbilical cord blood transplantation offers advantages such as low HLA 
matching requirements, a low risk of graft-versus-host disease (GVHD), a potent graft-
versus-leukemia (GVL) effect, and immediate availability, leading to its increasingly 
widespread clinical application [14]. Compared to HSCs from adult donors, cord blood-
derived HSCs have lower immunogenicity and allow for HLA mismatches. Typically, 
transplantation can proceed with an HLA 4/6 match (HLA-A, -B, -DR) between the 
cord blood unit and the recipient, significantly broadening the donor pool. The immune 
cells in cord blood are relatively “naïve” and have a weaker ability to recognize and 
attack recipient tissues, which can significantly reduce the risk of severe GVHD. The 

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incidence and severity of acute and chronic GVHD after cord blood transplantation are 
generally lower than those following unrelated matched or mismatched peripheral 
blood hematopoietic stem cell transplantation. This results in lower transplant-related 
mortality and allows patients to achieve a better long-term quality of life. Furthermore, 
unrelated cord blood units are cryopreserved in public or private banks. Once a match 
is identified, they can be provided promptly, greatly reducing the time needed to find 
and procure stem cells. This is crucial for ALS patients whose condition is progressing. 
To evaluate the safety and efficacy of umbilical cord blood transplantation for ALS 
patients, investigators plan to conduct an exploratory clinical trial. The preliminary plan 
is to enroll eight adult subjects. Following successful neutrophil engraftment (defined 
as an absolute neutrophil count ≥ 0.5×10⁹/L for three consecutive days) and 
confirmation of complete donor chimerism, the trial will focus on assessing 
transplantation-related complications and patient tolerance. The study duration includes 
subject recruitment, transplantation, and post-transplant follow-up period for 
comprehensive evaluation. Upon completion of this study, subjects will be invited to 
continue participating in long-term follow-up (via telephone or hospital visits). This 
study is a single-arm, single-center, open-label human clinical trial. It will strictly 
adhere to the Declaration of Helsinki, the International Ethical Guidelines for 
Biomedical Research Involving Human Subjects by the Council for International 
Organizations of Medical Sciences, as well as relevant Chinese regulations including 
the Good Clinical Practice and the Interim Measures for the Administration of Clinical 
Research on Stem Cells. 
To provide clinical investigators with a more comprehensive and in-depth 
understanding of unrelated umbilical cord blood transplantation for ALS, detailed 
introductions to the clinical data of the ALS patient cohort, basic experimental results 
such as drug sensitivity tests, and animal studies are presented herein for clinical trial 
reference. 

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4 Clinical Data of the ALS Cohort 
4.1 Analysis of Basic Clinical Characteristics Based on Questionnaires from 805 
ALS Patients 
In the preliminary phase of this study, basic clinical data from 805 ALS patients 
were collected through an electronic questionnaire system. The data encompassed key 
indicators such as age, body mass index (BMI), history of smoking and alcohol 
consumption, past history of chronic diseases, time of ALS onset and diagnosis, type 
of initial symptoms, and ALSFRS-R scores. Analysis of the results revealed that the 
patients in this cohort were predominantly middle-aged (concentrated between 45-55 
years old). A higher BMI was relatively common, suggesting that overweight or obese 
body types are somewhat prevalent among ALS patients. The majority of patients 
(62.33%) presented with limb-onset disease, accompanied by multi-site muscle atrophy. 
Approximately half (47.83%) of the patients exhibited bulbar symptoms. Some patients 
had a history of chronic conditions such as hypertension, diabetes, and coronary heart 
disease. Among these, blood pressure control was generally acceptable overall, while 
glycemic control levels were generally moderate. A smaller number of patients had a 
history of rheumatic diseases. Half of the patients experienced sleep disturbances. 
Almost all patients presented with psychiatric or cognitive symptoms, primarily 
manifested as low mood and anxiety. 
This cohort provides a preliminary sketch of the clinical baseline characteristics 
of ALS patients in China, offering important epidemiological reference for the 
subsequent screening of potential patients suitable for HSCT. 
4.2 Laboratory Results and In-depth Clinical Assessment of 8 ALS Patients 
To further assess the systemic involvement of ALS patients and the feasibility of 
transplantation, this study adopted a multidisciplinary collaboration model to recruit 
ALS patients with an ALSFRS-R score ≥ 35 points. These patients underwent a 
systematic, in-depth evaluation primarily encompassing: disease confirmation, immune 
function testing, bone marrow-related examinations, CSF analysis, and imaging studies. 

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Specific test items included: complete blood count, liver and kidney function tests, bone 
marrow morphology, bone marrow flow cytometry, chromosomal analysis, CSF 
cytokine profiling, electromyography, cardiac ultrasound, pulmonary function tests, 
magnetic resonance imaging (MRI), and computed tomography (CT). The eight 
initially enrolled patients were all male, aged between 35 and 48 years, with a time 
since diagnosis ranging from 5 to 31 months. 75 percent (6 cases) presented with limb-
onset symptoms. Two patients had a history of diabetes, and two had a history of 
hepatitis B. All cases were sporadic. Laboratory tests revealed that the proportion and 
absolute count of reticulocytes and immature granulocytes in the patients’ peripheral 
blood were elevated, with nearly all patients exceeding the normal reference range 
(Figure 1). Consistent with this finding, bone marrow flow cytometry results indicated 
an increased proportion of myeloid lineage cells in 7 patients. Notably, only one patient 
showed an elevated myeloid cell proportion in the bone marrow smear morphology 
results. This suggests that flow cytometry may be more sensitive in detecting early or 
subtle myeloid bias. 
 
Figure 1. Percentage and absolute of reticulocytes, immature granulocytes in 
ALS patients 

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    Previous studies have indicated that immune dysregulation plays a significant role 
in the pathogenesis and progression of ALS. Therefore, we focused on examining the 
immune function of enrolled ALS patients. Significant abnormalities were observed in 
the immune cell subsets of all eight patients. Notably, the proportions of naive and 
effector CD4+ T cells were decreased, while the proportions of activated, effector, and 
central memory CD8+ T cells were also lower than normal ranges (Figure 2). 
Furthermore, although peripheral blood levels of inflammatory cytokines showed no 
significant abnormalities, analysis of CSF cytokine levels in four patients revealed that 
IL-8 concentrations were more than twice the normal upper limit (Figure 3). This 
finding indicates the presence of significant neuroinflammation in these ALS patients. 
 

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Figure 2. Percentage of immune cells in peripheral blood in ALS patients 
 

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Figure 3. CSF results in ALS patients 
In addition, all eight enrolled patients underwent a comprehensive assessment of 
organ function, including pulmonary function tests, cardiac ultrasound, and vascular 
ultrasound examinations. Imaging evaluations revealed that all patients exhibited 
varying degrees of pulmonary imaging abnormalities, suggesting possible interstitial 
lung involvement. Pulmonary function testing indicated that four patients had moderate 
restrictive ventilatory dysfunction, with a significant decrease in their measured Forced 
Vital Capacity (FVC). Four patients were diagnosed with fatty liver via abdominal 
ultrasound, and three were found to have atherosclerosis with plaque formation based 
on vascular ultrasound. Cranial magnetic resonance (MR) imaging showed that four 
patients had intracranial demyelinating lesions. Furthermore, one patient was diagnosed 
with deep vein thrombosis in the lower limbs and subsequently initiated on 
anticoagulant therapy with Rivaroxaban. These collective findings reflect the frequent 
multisystem involvement and comorbidities in ALS patients, providing crucial clinical 
evidence for screening patients suitable for HSCT in this study. Considering the specific 
requirements of HSCT for overall organ reserve capacity, particularly pulmonary 
function, this series of assessments preliminarily identified a patient subgroup capable 

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of tolerating transplantation-related stress. For individuals whose key indicators, such 
as pulmonary function, do not meet transplantation criteria, further comprehensive 
evaluation by a multidisciplinary team is required to assess the associated treatment 
risks and benefits. 
5 Experimental Results 
5.1 Single-Cell Sequencing 
Compared to healthy donors, the bone marrow of ALS patients contained a lower 
proportion of hematopoietic stem and progenitor cells (HSPCs), particularly HSCs and 
multipotent progenitors (MPPs). Furthermore, the overall HSC/MPP population in ALS 
patients exhibited characteristics closer to long-term HSCs (LT-HSCs), with 
significantly weaker lineage commitment, particularly toward myeloid and lymphoid 
lineages. An increasing trend was observed in the proportion of B-cell precursors within 
the bone marrow HSPCs of ALS patients. Additionally, analysis of mature cell 
populations revealed divergent trends in myeloid and lymphoid subsets between 
peripheral blood and bone marrow in ALS patients. In bone marrow, the proportion of 
monocytes was relatively low, while that of CD4+ T cells was relatively high. This 
suggests a potential inflammatory state within the bone marrow, possibly promoting the 
homing of peripheral CD4+ T cells to this site. Conversely, in peripheral blood, the 
proportions of monocytes and CD8+ T cells were elevated (Figure 4). 

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Figure 4. Comparison of cell subtypes between ALS and healthy control based on 
RNA-seq 
5.2 Flow Cytometry Results 
In peripheral blood, ALS patients exhibit a decreased proportion of T cells and an 
increased proportion of myeloid cells in peripheral blood based on flow cytometry 
analysis, although these differences did not reach statistical significance. In contrast, 
within bone marrow, ALS patients showed a significantly higher proportion of T cells 
and a significantly lower proportion of myeloid cells compared to healthy controls 
(Figure 5). The underlying mechanisms driving these distinct cellular compositional 
differences between bone marrow and peripheral blood in ALS patients require further 

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investigation. 
 
Figure 5. Comparison of cell subtypes between ALS and healthy control 
based on flow cytometry 
5.3 Colony-Forming Cell (CFC) Assay 
In CFC assay, investigators compared the ability of CD34+ cells from ALS and 
control groups to form various clonal colonies in semi-solid culture medium. Primary 
CFC assay showed a declining trend in the number of CFU-GM colonies formed by the 
same quantity of cells from the experimental group. However, the secondary CFC assay 
indicated an increase in myeloid lineage colonies in the patients. These results suggest 
that HSPCs from ALS patients may experience a differentiation block (Figure 6). 
Furthermore, investigators observed that within the ALS experimental group, as the 
patients’ condition worsened, the colony-forming capacity of their HSPCs 
progressively declined. The number of clonal colonies was significantly lower in ALS 
patients who had already developed paralysis. 

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Figure 6. CFC assay (primary, second) 
5.4 Drug Sensitivity Testing 
Prior to undergoing HSCT, ALS patients require pharmacological or 
radiotherapeutic intervention to eliminate aberrant microglia and other cells. Based on 
findings from previous research, investigators designed drug sensitivity assays to 
evaluate the efficacy of different agents in killing microglial cells. The results 
demonstrated that the combination of Melphalan and Fludarabine exhibited superior 
efficacy in eliminating microglial cells, followed by Thiotepa (Figure 7). This provides 
a theoretical basis for the myeloablative conditioning regimen prior to transplantation. 

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Figure 7. Drug sensitivity test 
6 Animal Experimental Data 
Research conducted by the team at the University of Pittsburgh, Epperly et al. 
(2019), demonstrated that HSCT combined with myeloablative conditioning 
significantly extended paralysis-free survival in the SOD1G93A mouse model, a widely 
recognized model of ALS. The research team used SOD1G93A mice that had 
developed grade I paralysis at 90 days of age. The mice received a total body irradiation 
(TBI) dose of 9 Gy (Cesium source, 340 cGy/min), followed by intravenous injection 
of 1×10⁶ C57BL/6 GFP+ donor bone marrow cells. The results showed that the mice’s 

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survival was extended from 129 days to 233 days (p=0.0045), and the paralysis-free 
period was prolonged to over 250 days. The donor-derived bone marrow cells 
differentiated into M2 microglia, exerting a neuroprotective effect by localizing to the 
degenerating motor neuron regions in the spinal cord anterior horn. These M2 microglia 
likely inhibited neuroinflammation through the secretion of neurotrophic factors or 
anti-inflammatory cytokines [15]. Traditional non-gene therapies (such as antioxidants, 
anti-inflammatory drugs, and neurotrophic factors) only extended the survival of SOD1 
mice by 6-10 days, showing a minimal effect. The transplantation strategy employed 
by Epperly et al. extended the survival of ALS mice by over 100 days, demonstrating 
significant therapeutic potential. 
7 HSCT Protocol and Associated Risks 
7.1 Inclusion Criteria 
(1) Patients with a confirmed diagnosis of ALS, with an ALSFRS-R score ≥ 35 
and a maximum vital capacity ≥ 65% 
(2) Aged > 35 years and < 50 years, with no gender restrictions 
(3) Karnofsky Performance Status (KPS) score ≥ 70, and Eastern Cooperative 
Oncology Group (ECOG) Performance Status ≤ 2 
(4) Unrelated umbilical cord blood and the recipient demonstrate high-resolution 
HLA (-A, -B, Cw, DR, DQ) matching at ≥ 4/6, 5/8, or 6/10 loci, and the post-thaw 
CD34+ cell count in the cord blood unit is ≥ 1.2×10⁵/kg (recipient body weight) 
(5) Willing and able to comply with the study procedures and conditions, 
demonstrating good compliance 
(6) Willing to receive at least two years of treatment and follow-up, with detailed 
medical records maintained 
(7) Patients and/or their legal guardian voluntarily participate in this clinical trial, 

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sign the informed consent form, and are capable of completing all follow-up 
assessments as required by the protocol 
7.2 Exclusion Criteria 
(1) Patients with positive results in the following etiological tests: Human 
Immunodeficiency Virus (HIV-1/2), Human Cytomegalovirus DNA (HCMV-DNA), 
Epstein-Barr Virus DNA (EBV-DNA), Hepatitis B (positive for Hepatitis B surface 
antigen (HBsAg) or Hepatitis B virus DNA (HBV-DNA)), Hepatitis C antibody (HCV-
Ab), or Treponema pallidum antibody (TP-Ab) 
(2) Clinically significant active bacterial, viral, fungal, or parasitic infections as 
judged by the investigator during screening 
(3) Patients who have previously received gene therapy or allogeneic 
hematopoietic stem cell transplantation 
(4) First-degree relatives with known or suspected familial cancer syndromes 
(including but not limited to hereditary breast and ovarian cancer syndrome, hereditary 
nonpolyposis colorectal cancer syndrome, familial adenomatous polyposis, etc.) 
(5) Diagnosis of major psychiatric disorders or predisposition to such conditions 
that would significantly impair the ability to participate in the clinical study 
(6) History of major organ impairment, including: Liver disorders: Liver function 
tests showing AST or ALT > 3 × ULN; total serum bilirubin > 2.5 × ULN; for cases 
consistent with Gilbert syndrome, total bilirubin > 3 × ULN and direct bilirubin > 2.5 
× ULN; history of hepatic bridging fibrosis, cirrhosis, or active hepatitis; Cardiac 
disorders: Left ventricular ejection fraction (LVEF) <45% at screening; New York 
Heart Association (NYHA) Class III or IV congestive heart failure; severe arrhythmias 
requiring treatment; poorly controlled hypertension (systolic blood pressure > 160 
mmHg and/or diastolic blood pressure > 100 mmHg despite antihypertensive therapy), 
or prior history of hypertensive emergencies, hypertensive encephalopathy, or unstable 
angina; history of myocardial infarction, coronary artery bypass graft surgery, 

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peripheral arterial bypass graft implantation, or stent placement within 12 months prior 
to enrollment; clinically significant valvular disease; calculated eGFR < 60 
mL/min/1.73m²; Pulmonary function: FEV1/FVC < 60% and/or diffusing capacity less 
than 60% of predicted; clinically significant evidence of pulmonary hypertension 
requiring medical intervention 
(7) Uncorrectable coagulation dysfunction or history of severe bleeding disorders 
(8) Any other condition deemed by the physician to render the subject unsuitable 
for hematopoietic stem cell transplantation 
(9) Known hypersensitivity to the investigational drug or its components 
(10) Participation in or ongoing participation in other interventional clinical 
studies within 3 months prior to screening 
(11) Vaccination with live vaccines within 6 weeks prior to screening 
(12) Pregnant or breastfeeding women 
(13) History of solid organ transplantation 
(14) Poor compliance of the subject with the study protocol 
(15) Any other condition considered by the investigator as unsuitable for 
participation in this clinical trial 
(16) Unwillingness of the subject to provide pre-existing valid diagnostic evidence 
before treatment or to undergo bone marrow, lumbar puncture, blood tests, and other 
examinations after treatment 
7.3 Myeloablative Conditioning Regimen and GVHD Prophylaxis Protocol 
(I) Reduced-Intensity Myeloablative Conditioning Regimen 
Based on preliminary research and the results of drug sensitivity assay, this study 
employs a reduced-intensity myeloablative conditioning regimen (conditioning 
intensity score TCI = 3.5) consisting of Fludarabine (Flu), Melphalan (Mel), Thiotepa 

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(TT), and Total Marrow Irradiation (TMI). The goal is to maximally eliminate 
abnormally activated immune cells and potentially pathogenic microglial cells, thereby 
creating the necessary immune and bone marrow niche for umbilical cord blood stem 
cell engraftment. 
The specific drug administration schedule is as follows: 
TT: Total dose: 10 mg/kg. Administered at 5 mg/kg/day, qd via intravenous 
infusion (iv) on Days -6 and -5. 
Flu: Total dose: 150 mg/m². Administered at 30 mg/m²/day, qd, iv from Day -6 to 
Day -2. 
TMI: Total dose: 400 cGy. Administered as a single dose of 400 cGy on Day -2. 
Mel: Total dose: 100 mg/m². Administered qd, iv on Day -1. 
(Note: Doses will be adjusted based on the patient’s renal and hepatic function 
during implementation) 
Considering that the conditioning process may induce significant oxidative stress, 
the antioxidant food supplement sulforaphane will be co-administered prophylactically 
to mitigate potential oxidative damage. Preliminary clinical studies have indicated that 
sulforaphane not only possesses antioxidant properties but may also promote umbilical 
cord blood stem cell engraftment. Sulforaphane will be administered orally at a dose of 
1 tablet, three times daily, starting from the beginning of the conditioning regimen (Day 
-6) until Day +7 post-transplantation. If the umbilical cord blood unit used has been 
cryopreserved for > 10 years, the sulforaphane dose will be increased to 2 tablets, three 
times daily, from Day -1 to Day +7. 
(II) GVHD Prophylaxis Protocol 
GVHD prophylaxis will be implemented using a combination of Cyclosporine A 
(CsA) and short-course Mycophenolate Mofetil (MMF). CsA: Initial dose of 2.5 
mg/kg/day. Administration will begin on Day -1 (one day before transplantation) as a 

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continuous 24-hour intravenous infusion. The target therapeutic blood concentration 
will be maintained between 200-300 ng/mL. After neutrophil engraftment and recovery 
of gastrointestinal function, CsA will be switched to oral administration at twice the 
intravenous dose. The target trough concentration will be maintained at 150-200 ng/mL 
until 2 months post-transplantation. The dose will be adjusted based on the presence of 
GVHD, infection status, and patient tolerance. CsA will be gradually tapered starting 
at 2 months post-transplantation and completely discontinued by 5-6 months. 
MMF: Dose of 25-30 mg/kg/day. Administration will commence on Day +1. The dose 
will be gradually reduced following neutrophil engraftment and completely 
discontinued by 3 months post-transplantation. 
7.4 Unrelated Umbilical Cord Blood Transplantation Protocol 
(I) Criteria for Determining a Single Umbilical Cord Blood Unit 
(1) HLA matching between donor and recipient should ideally be ≥ 4/6, 5/8, or 
7/10 (high-resolution genotyping). 
(2) Post-thawing (from a small segment), the cord blood unit must have a total 
nucleated cell (TNC) count ≥ 1.5 × 10⁷/kg and a CD34⁺ cell count ≥ 1.2 × 10⁵/kg. The 
CFU-GM count should correlate with the CD34⁺ cell count or demonstrate good colony 
formation. For patients in urgent need of transplantation, cord blood units with a CD34⁺ 
cell count ≥ 0.83 × 10⁵/kg may be considered. 
(3) Donor-specific antibodies (DSA) must be negative (against 12 HLA loci). 
(4) Donor-recipient gender: No specific requirement for single-unit umbilical cord 
blood transplantation. Donor-recipient ABO blood type: No specific requirement for 
single-unit umbilical cord blood transplantation. 
(II) Mobilization and Collection of Recipient’s Autologous Peripheral Blood 
Hematopoietic Stem Cells during the Screening Phase for Cryopreservation and Future 
Use 

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(1) Use a Granulocyte Colony-Stimulating Factor (G-CSF) mobilization protocol 
to stimulate the release of hematopoietic stem cells from the bone marrow into the 
peripheral blood: G-CSF 5 µg/kg, administered subcutaneously every 12 hours (q12h). 
(2) Timing of collection: Monitor complete blood count daily after mobilization. 
The CD34⁺ cell peak typically occurs 4-6 days after G-CSF mobilization. Collection 
begins when the white blood cell count exceeds 5 × 10⁹/L and the platelet count exceeds 
50 × 10⁹/L. 
(3) Stem cell collection: Use a cell separator to collect peripheral blood stem cells. 
The minimum target CD34⁺ cell dose is ≥ 2 × 10⁶/kg (recipient body weight), with an 
ideal target of ≥ 5 × 10⁶/kg. Strictly monitor electrolytes during the collection process. 
(4) Stem cell cryopreservation: Use a cell freezing solution containing the 
cryoprotectant dimethyl sulfoxide (DMSO) (maximum final DMSO concentration 
should not exceed 10%). After controlled-rate freezing, the autologous hematopoietic 
stem cells are cryopreserved in liquid nitrogen at -196°C. 
(III) Umbilical Cord Blood Infusion 
On transplant day 0, the thawed umbilical cord blood unit is infused. 
Dexamethasone 5 mg is administered intravenously 30 minutes prior to infusion to 
prevent infusion-related reactions. Under continuous and close monitoring of vital signs, 
the thawed cord blood hematopoietic stem cell suspension is infused at a constant rate 
via a central venous catheter. Following the infusion, approximately 100 mL of normal 
saline is slowly dripped to flush the infusion line, ensuring all cells are delivered into 
the patient’s circulation. 
7.5 Post-Transplant Monitoring and Management of Potential Complications 
Post-transplantation management, as the core safety component of this study’s 
implementation, focuses on risks arising from conditioning toxicity, HSCT-related 
complications, and the progression risk of the underlying ALS disease. Specific aspects 
include: 

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(1) Conditioning-Related Toxicities: Primarily manifest as pancytopenia due to 
myelosuppression, leading to risks of secondary infections and bleeding. Other 
potential toxicities include mucositis, organ function impairment, and electrolyte 
imbalances. Active supportive care is essential, encompassing blood product 
transfusions, anti-infective therapy, nutritional support, and organ function support. 
(2) Infusion-Related Adverse Reactions: The cryoprotectant DMSO contained in 
the cord blood product may cause a range of reactions. Common symptoms include 
nausea, vomiting, abdominal pain, bradycardia, blood pressure fluctuations, urticaria, 
and other allergic manifestations. Severe cases may present with dyspnea or 
hemoglobinuria. Management principles involve timely symptomatic intervention, 
such as enhanced hydration and diuresis, antihistamines for pruritus, and 
antiemetics/sedatives. Infusion should be paused and emergency medical support 
initiated for severe reactions. 
(3) GVHD: Although the incidence of acute and chronic GVHD is relatively lower 
after cord blood transplantation, risk remains. Typical acute GVHD may present with 
fever, 
skin 
rash, 
jaundice, 
and 
diarrhea. A 
calcineurin 
inhibitor-based 
immunosuppressive regimen (e.g., tacrolimus) will be employed for prophylaxis, with 
close monitoring for relevant clinical signs and laboratory indicators. 
(4) Rescue Protocol for Graft Failure: Primary graft failure is defined as the failure 
to achieve neutrophil engraftment by day 28 post-transplantation. Complete blood 
counts will be monitored daily post-transplant. Donor-recipient chimerism status will 
be dynamically assessed using STR-PCR (peripheral blood samples on days 7, 14, 21; 
bone marrow sample on day 28). A donor chimerism rate < 50% on day 21 suggests a 
risk of delayed engraftment. A diagnosis of primary graft failure is made if neutrophils 
are not engrafted by day 28 and the bone marrow donor chimerism rate is < 50%. 
Autologous peripheral blood HSCs mobilized and collected with G-CSF during the 
screening phase and cryopreserved for backup will be reinfused to restore 
hematopoietic function upon confirmation of primary graft failure. 

25 
 
(5) Delayed Immune Reconstitution: T-cell recovery: Reconstitution of cord 
blood-derived naïve T cells is slow. Close monitoring for CMV/EBV reactivation is 
required. Letermovir will be routinely administered from day +7 for CMV prophylaxis. 
B-cell function: Hypogammaglobulinemia will be managed with intravenous 
immunoglobulin replacement therapy. 
(6) Risk of Infection Transmission: Although all cord blood units undergo rigorous 
screening per national standards for pathogens including HIV, HBV, HCV, and syphilis, 
and are assessed for bacterial contamination and xenotransplantation-related infection 
risks, a minimal possibility of transmission of unknown or latent pathogens cannot be 
entirely excluded. Systemic infection monitoring and prophylaxis will be implemented 
post-transplantation. 
(7) 
Progression 
of 
Underlying 
Disease 
During 
Transplantation 
and 
Countermeasures: Despite the strict inclusion criterion of FVC ≥ 65% predicted in this 
study, given that ALS is a progressively deteriorating disease, there remains a risk of 
significant pulmonary function decline during the transplantation process. Prior to 
transplantation, patients will undergo non-invasive ventilation (NIV) tolerance training, 
and individualized ventilation plans will be established. Continuous monitoring of ECG, 
respiration, and blood pressure will be maintained throughout the process, with arterial 
blood gas analysis performed as needed. Should acute or subacute respiratory 
deterioration occur, a pre-defined respiratory support protocol will be immediately 
initiated, escalating from supplemental oxygen and NIV to, if necessary, invasive 
mechanical ventilation, to ensure patient safety. This entire management process will 
be jointly executed by a multidisciplinary team comprising Neurology, Critical Care 
Medicine, and Anesthesiology departments to minimize risks posed by ALS 
progression during transplantation. 
8 Evaluation 
To comprehensively assess the safety of unrelated umbilical cord blood 

26 
 
transplantation for the treatment of ALS, this study will employ multidimensional 
assessment tools to evaluate participants. Safety assessments will be conducted 
throughout the entire study period, involving continuous monitoring and documentation 
of all adverse events and serious adverse events. Particular attention will be paid to the 
progression and evaluation of ALS, as well as transplantation-related complications 
such as GVHD, infections, graft failure, and organ toxicities associated with the 
conditioning regimen. 
8.1 Safety Assessment System 
(1) LVGI Safety Score 
Considering the limited assessment conditions within the transplant isolation unit, 
this study has modified and simplified the ALSFRS-R scale and incorporated the 
Inbody score, which provides a comprehensive assessment of muscle mass and body 
fat mass, to form the “LVGI” safety score (details in the table below). Among the 
components, VCmax refers to maximum lung capacity; Grip quantifies upper limb 
strength using a dynamometer, with the Grip Strength-to-Body Weight Index calculated 
as Grip Strength (kg) / Body Weight (kg) × 100. The LSGI assessment is performed 
weekly before transplantation and during the transplantation process for the patient. 
The results of each assessment are compared. A change within ± 20% in the LSGI score 
within the first month post-transplantation compared to the pre-transplantation score is 
considered safe. 
Part 
Item 
Score 
Medulla 
Function 
Language 
4  Normal 
3  Detectable speech impairment 
2  Comprehensible repetitive speech 
1  Speech is non-communicative 
0  Absence of effective speech 
Respiratory VCmax 
4  VCmax > 80% 

27 
 
Function 
3  70% < VCmax ≤ 80% 
2  60% < VCmax ≤ 70% 
1  50% < VCmax ≤ 60% 
0  VCmax ≤ 50% 
Upper 
Limb 
Function 
Grip Strength-to-Body 
Weight Index 
4  Grip > 40 
3  30 < Grip ≤ 40 
2  20 < Grip ≤ 30 
1  10 < Grip ≤ 20 
0  Grip ≤ 10 
Overall 
Muscle 
Status 
Inbody Score 
4  Inbody > 80 points 
3  70 points < Inbody ≤ 80 points 
2  60 points < Inbody ≤ 70 points 
1  50 points < Inbody ≤ 60 points 
0  Inbody ≤ 50 points 
(2) Assessment of Drug-Induced Liver and Kidney Toxicity 
The Roussel Uclaf Causality Assessment Method (RUCAM) will be used to assess 
the occurrence of drug-induced liver injury; Kidney Disease Improving Global 
Outcomes criteria for Acute Kidney Injury will be used to assess the occurrence of drug-
induced kidney injury. 
(3) Assessment of acute GVHD 
The grading criteria for the severity of acute GVHD are established based on its 
impact on non-relapse mortality post-transplantation. This involves separate scoring for 
acute GVHD affecting the skin, gastrointestinal tract, and liver, which are then 
combined to determine an overall severity grade. The assessment primarily follows the 
modified Glucksberg criteria and the grading system established by the Mount Sinai 
Acute GVHD International Consortium. 

28 
 
8.2 Multidimensional Efficacy Assessment 
Overall Survival (OS): Defined as the time from umbilical cord blood infusion to 
death due to any disease-related cause. This endpoint is used to assess the fundamental 
impact of umbilical cord blood transplantation on patient survival. 
Assessment of Disease Functional Progression: Standardized evaluation using the 
ALSFRS-R scale will be conducted at baseline, as well as at 1, 2, 3, 6, 12 and 24 months 
post-transplantation. Changes in scores over time will be analyzed. 
Biomarker of Neuroaxonal Injury: Peripheral blood and CSF samples will be 
collected to assess levels of NFL. The dynamic changes in neuroaxonal injury following 
umbilical cord blood transplantation intervention will be objectively evaluated by 
analyzing changes in NFL relative to baseline. 
Imaging Evaluation: A combined approach using MRI and PET-CT will be 
employed to quantify the degree and rate of upper motor neuron degeneration. Specific 
tracers targeting activated microglia will be used to quantitatively analyze changes in 
neuroinflammatory levels in key areas such as the motor cortex and brainstem. 
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