Efficacy and Safety of Sequential Infusion of Hypoxic 3D-Cultured Umbilical Cord Mesenchymal Stem Cells in Haploidentical Hematopoietic Stem Cell Transplantation for Severe Aplastic Anemia: A Multicenter, Randomized, Phase 1 Trial
Phase: PHASE1
Conditions: Severe Aplastic Anemia (SAA), Severe Aplastic Anemia, Refractory, Severe Aplastic Anemia
Sponsor: Chinese PLA General Hospital

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Efficacy and Safety of Sequential Infusion of Hypoxic 3D-Cultured
Umbilical
Cord
Mesenchymal
Stem
Cells
in
Haploidentical
Hematopoietic Stem Cell Transplantation for Severe Aplastic Anemia:
A Multicenter, Randomized, Phase 1 Trial
Methods
Study Design and Participants
This trial was a multicenter, randomized, phase 1 clinical trial was conducted across in
China. The study evaluated the safety and preliminary efficacy of conventional
haplo-HSCT combined with UC-MSCs cultured under 3D hypoxic conditions in the
treatment of SAA. Patients were recruited after receiving approval from the Institutional
Review Board of the People's Liberation Army General Hospital. Written informed
consent was provided by the participants, their legal guardians, or next of kin.
The inclusion criteria were (1) age 6 to 60 years; (2) diagnosis of SAA or very SAA
according to the International Aplastic Anemia Study Group (3) without any severe
pulmonary, cardiac, liver, or renal diseases or any active infection; and (4) adequate
performance status (Eastern Cooperative Oncology Group score 0-2). Fanconi anemia
was excluded after evaluation of diepoxybutane-stimulated peripheral blood (PB)
chromosome breakages. Patients with congenital aplastic anemia like dyskeratosis or
Diamond-Blackfan anemia were also excluded.
Preparation of UC-MSCs and 3D dynamic culture of UC-MSCs.
UC-MSCs were purchased from Daji Kanghua Biotechnology Co., Ltd. (Beijing, China).
The 3D aggregate culture method of UC-MSCs in this study is similar to the 3D culture
method of BMSCs in previous articles. Briefly, UC-MSCs at passages 3 to 4 were
suspended in ACF, and 5.0 × 105 UC-MSCs in 2 mL ACF were seeded in a 60 mm
ultra-low-attachment (ULA) culture dish (Corning) at 37 °C in a 5 % CO2 humidified
atmosphere. The ULA plates were placed on a rocking base of a Digital OrbitaI Shaker in
a standard humidified incubator (37 °C, 5 % CO2) under controlled rocking speeds (i.e.,
40–100 rockings/min, or rpm) for 18–48 h. H-3D- UC-MSCs were incubated at 37 °C
under 3 % O2, 5 % CO2, and 92 % N2 for 24 h in a gas-tight humidified chamber
(modular incubator chamber; Billups Rothenberg, Del Mar, CA, USA). N-3D- UC-MSCs
were incubated under 21% O2, 5 % CO2.

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Procedures
The enrolled patients with an HLA-haploidentical relative for HSCT received the
fludarabine (Flu) + cyclophosphamide (Cy) + antithymocyte globulin (ATG) conditioning
regimen. For the patients with acute SAA (SAA-I), intravenous administration of 30
mg/(m2 day) of Flu and 500–800 mg/(m2 day) of Cy was performed from days -5 to -2,
and 5μg/(kg day) of ATG was administered from days -4 to -1. For the patients with
chronic SAA (SAA-II), the same treatment of ATG and Cy was applied as above with the
supplement of 0.6 mg/(kg 6 h) of busulfan (BU) from days -8 to -5 prior to
transplantation. Donor selection and hematopoietic stem cell mobilization and collection
were conducted based on the consensus of The Chinese Society of Hematology regarding
indications, conditioning regimens, and donor selection for allogeneic hematopoietic
stem cell transplantation. On day 0, HSCs were infused intravenously. Both groups
received 5 × 10⁵/kg UC-MSCs at 4 h before HSC infusion. The control group was
transfused with conventional 2D-cultured UC-MSCs, whereas the experimental group
received 3D hypoxia-preconditioned UC-MSCs. Standard GVHD prophylaxis consisted
of mycophenolate mofetil, cyclosporine A, and methotrexate (MTX).
End Points
The primary end point was incidence and severity of adverse events (AEs) within the first
150 days after haplo-HSCT. Secondary end points included the 1-year cumulative
incidence of severe cGVHD, the incidence and severity of aGVHD, rates of overall
survival (OS), and GVHD-free and relapse-free survival (GRFS; survival without III to
IV aGVHD, cGVHD requiring systematic treatment, AA relapse, or death) and AA
relapse. Organ scoring and global assessment of cGVHD were conducted based on the
2014 National Institutes of Health consensus criteria, whereas acute GVHD was assessed
based on the Mount Sinai Acute GVHD International Consortium criteria. AEs were
graded according to the Common Terminology Criteria for Adverse Events version 5.0.
For the OS analysis, death was counted as death of any reason after transplantation.
Random Assignment and Statistical Analysis
At each participating center, consented participants meeting the inclusion criteria
underwent 1:1 randomization to either the 3D hypoxia-preconditioned UC-MSC group or
the control group. The randomization sequence was generated and implemented by
dedicated statisticians who remained independent of patient recruitment, clinical
management, and endpoint evaluation, thereby maintaining allocation concealment
throughout the study.

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The Mann-Whitney U test, χ2 test, and Fisher's exact test were used to compare the
baseline patients’ characteristics and AEs between the 3D hypoxia-preconditioned
UC-MSC and control groups. The competing risk model (Fine and Gray model) was used
to estimate the Secondary end point (1 year severe cGVHD cumulative incidence
completed by death and relapse) and hazard ratios (HRs) with 95% CIs, and the incidence
and severity of cGVHD, aGVHD and AA relapse. The rates of GRFS and OS were
analyzed using Kaplan-Meier analysis and presented as percentages with 95% CIs and
tested by log-rank test between the two groups. Statistical analyses were conducted using
R (version 4.4.2, R Foundation for Statistical Computing, Vienna, Austria).