hUC-MSC characterization
Our data showed that the UC-MSC line exhibited plastic adherent properties and a spindle- and fibroblast-like morphology (Figure 1A), with a population doubling time of 24 ± 0.6 hours (n=3, mean ± SEM). Propagation of UC-MSCs up to passage 6 did not introduce any karyotypical abnormality, and the cells maintained normal 46XY as indicated by the G-banding technique (Figure 1B). These cells were also able to form 519 ± 80 CFU/1000 cells (mean ± SEM, n=3) (Figure 1C). Further analysis of the differentiation potential confirmed that the UC-MSC line could undergo adipogenic, chondrogenic, and osteogenic differentiation processes, illustrated by positive staining with Oil O Red, Alcian Blue, and Alizarin Red, respectively (Figure 1D). Analysis of the expression patterns of positive markers, including CD73, CD90, and CD105, showed that more than 99% of the cells expressed all these markers and less than 2% expressed negative markers, including CD11b, CD19, CD34, CD45, and HLR-DR (Figure 1E). These results fulfilled the minimum criteria for mesenchymal stem cells proposed by the ISCT (Table 2).
Patient Outcomes as Case Report
Patient 1
An extremely premature girl (24 weeks and 5 days, first born of twins) was born by C-section due to premature rupture of the placental membrane with a bodyweight (BW) of 720 grams. Soon after birth, the patient developed signs of respiratory distress syndrome with retraction followed by apnea and cyanosis (SpO2 ranged from 60% to 70%) and bradycardia with a heart rate below 100 bpm. She was immediately intubated and placed on mechanical ventilation, with a peak inspiratory pressure (PIP) of 18 cmH2O and a positive end-expiratory pressure (PEEP) of 5 cmH2O. Chest X-ray showed a stage 2 hyaline membrane requiring one dose of surfactant (Curosurf) at 200 mg/kg BW. Heart ultrasound detected patent ductus arteriosus, which closed after one course of paracetamol (15 mg/kg/6 h) for seven days, with no evidence of pulmonary artery hypertension (PAH) on echocardiogram after treatment. In addition, the patient suffered from septicemia caused by Staphylococcus epidermidis,resulting in necrosis at the distal phalanx of the left little and ring fingers and requiring antibiotic treatment. In the first 2 months, the patient was supported with synchronized intermittent mandatory ventilation (SIMV) and switched to continuous positive airway pressure therapy (CPAP) at7 cmH2O and 50% FiO2 for the following 1.5 months. At 3.5months postnatal age, the patient was diagnosed with BPD and continued to receiveoxygen support via anasal cannula at 0.5 – 1 L/min.Nebulized corticosteroids at 100 mcg/kg 4 times/day for a 1-month period wereadministered. A combination of diuretics (furosemide at 1 mg/kg/12 h), spironolactone (2 mg/kg/12 h) and bronchodilators (inhaled b2-agonists) together with nutrient enhancement (high-calorie nutrition and vitamins E, A, K supplementation) were initiated for 2 months. However, at 4.5 months postnatal age, the patient’s BPD was not improved, with the SpO2offoxygen support dropping to 90%. The chest CT scan and X-ray at 3.5months postnatal age confirmed the formation of diffuse fibrosis, atelectasis in the upper lobes of both lungs and significant air trapping in both lower lobes (Figure 2A and 3A).
Before transplantation, chronic hypercapnia was confirmed by ABG analysis with the following measured values: pH of 7.31, PaCO2 of 68 mmHg, HCO3- of 41.3 mmol/L, and PaO2 of 73 mmHg. PAH was determined based ona maximum pulmonary artery pressure (PAP) of 40 mmHg and illustrated onechocardiogram, and the pro-BNP level was high (1942 ng/mL). Oral sidenafil (1.5 mg/kg/6 h) and bosentan (1 mg/kg/8 h) were administered when the patient was4 months old. The allo-UC-MSC transplantation was performed at 144 days postnatal age (47 weeks gestational age). No signs of serious adverse events were observed during the two transplantations. Three days after the second transplantation, the patient could breathe spontaneously withan SpO2 of 96% without oxygen support. The patient was discharged at 161 days postnatal age (17 days posttransplantation).
At the first follow-up visit, the patient was alert, hada BW of 4 kg and was spontaneously breathing, with an SpO2 of 96% without oxygen support. Blood gas analysis revealed a significant reduction in thesaturated CO2 in the blood as follows: pH of 7.5, PaCO2 of 33.6 mmHg, HCO3- of 26.9 mmol/L, BE of 4 mmol/l and PaO2 of 46 mmHg. These results suggested that the hypercapnic condition was ameliorated after stem cell transplantation. The pro-BNP level had dropped to 351.9 ng/ml, leading to the termination of PAH treatment at4 months posttransplantation. At the 1-month follow-up examination, the patient was cognizant and active, and her BW had increased to 4.3 kg with air fully entering both lungs. She was spontaneously breathing and had an SpO2 of 97% onroom air without oxygen support. The laboratory tests revealed that herpH (7.37), PaCO2 (46.3 mmHg), HCO3- (27 mmol/L), and PaO2 (42 mmHg) remained stable posttransplantation. Hematological analysis also confirmedthe absence of inflammation andsepsis, as indicated by the Hgb level (129 G/L), white blood cell count (WBC, 6.1 G/L), and neutrophillevel(6.4%). At the 6-month visit, the patient no longer required oxygen support, with her SpO2 reaching100%, good air entry into the lungs, no sign of dyspnea and ABG results in the normal ranges (pH: 7.37, PaCO2: 38 mmHg, HCO3-: 21.9 mmol/L, PaO2: 41 mmHg, and SpO2: 100%); the pro-BNP level was 283.1 ng/ml. A significant reduction in lung fibrosis was observed on chest CT without PAH (Figure 2B). At 12months postdischarge, the patient’s condition was improved. Her BW had increased to 7.5 kg. ABG results werein the normal ranges (pH: 7.34, PaCO2: 35.5 mmHg, PaO2: 87 mmHg, BE: -6; HCO3-: 19.3 mmol/l), and her pro-BNP level was 154.2 without PAH treatment. A significant reduction in lung fibrosis was observed on chest X-ray at the 12-month visit (Figures 3B). The detailed progression of the patient’s condition is described in Supplement Table 1.
Patient 2
The second premature neonate was patient 1’s twin, who was enrolled in this study with a BW of 650 grams. Similar to her twin sister, the patient suffered from respiratory distress syndrome, including gasping, followed byapnea, bradycardia, and cyanosis with an SpO2 between 50% and 60%. The patient was positive pressure ventilated by bag-mask and then intubated and placed on aventilator inSIMV mode (with ventilator parameters similar to those in the first case). Chest X-ray revealed a stage 3 hyaline membrane, and a surfactant was given at a dose of 200 mg/kg BW on the 1st and 3rddays after birth. A large patent ductus arteriosus (PDA) was detected by cardiac echography, requiring one course of indomethacin (0.2 mg/kg/12 hours) within the firstpostnatalweek as previously described(20). After 2 months onSIMV, ventilation support was switched to CPAP with a PEEP of6 cmH2O and 40% FiO2. After treatment, the PDA size was reduced(1 mm), andthe shunt size was small; however, the size increased gradually and reached 3.6 mm at 3 months. The PDAwas maintained at a large size and required surgical closure at 3.5 months of age. Although the PDA was closed without complications, PAH wasobserved (38 mmHg), and the pro-BNP level was 2223 ng/ml, leading to treatment with 1 mg/kg/6 h sildenafil and 2 mg/kg/8 h bosentan. After the operation, the patient was on CPAP at 6 cmH2O at a FiO2 of 30% before switching to nasal cannula oxygen after 1 month at a rate of 1 L/min to maintain a stable SpO2 between 93% and 97%. X-rays and chest CT scans at 4 months of age indicated diffuse fibrosis in the lung structures with atelectasis in the upper lobes of both lungs and significant air trapping in both lower lobes (Figure 2A and 3A). The patient was confirmed as havingBPD and treated with nebulized corticosteroids (100 mcg/kg 4 times/day), diuretics (furosemide 1 mg/kg/12 hours), spironolactone (2 mg/kg/12 hours), bronchodilators (inhaled β2-agonists) in combination with ipratropium bromide, and other supportive measures (high-calorie nutrition, vitamins E and A, etc.) for 6 weeks. However, the patient’s condition didnot improve, and she remained dependent on oxygen support, leading to the indication forallo-UC-MSC transplantation at 151 days postnatal age. Prior to transplantation, ABG analysis revealed a pH of 7.6, PaCO2 of 37.9 mmHg, PaO2 of 35 mmHg, increased HCO3- of 29.1 mmol/L and BE of 8 mmol/L. Three days after transplantation, the patient was discharged with oxygen support via nasal cannula at 1 L/minute, a respiration rate of 64-67 times/minute, and an SpO2 of 83% (FiO2: 21%).
At the first follow-up visit, the patient’s body weight had increased to 4.3 kg, her heart rate was145 bpm, and she still required oxygen support at 0.5 l/min to maintain an SpO2 over 92% (83% without oxygen support). The ABG results showed a PaCO2 of 67 mmHg and an HCO3- of 32.3 mmol/L, a PaO2 of 36 mmHg, and a BE of 6 mmol/l. The total hemoglobin level, WBC level, and neutrophil percentage were 129 G/L, 6.1 G/L, and 6.4%, respectively. The patient’s condition had improved by her 1-month follow-up visit, with both reductions in both herPaCO2 and HCO3-, while her SpO2 was maintained at 94-98% onoxygen via a cannula a 0.5 l/min. Two months after the first hUC-MSC transplantation, home oxygen monitoring results confirmed that the patient could breathe normally, and her SpO2 had reached 95%.
From the 6-month follow-up onwards, the patient’s health had stabilized under normal conditions, with her BW reaching 8 kg at the 12-month visit. All ABG tests were within normal parameters at the 6-month visit, further confirming the recovery of the patient from BPD. Her SpO2 was maintained at 95% at the 6-month visit and reached 100% at the 12-month follow-up. Blood gas analysis at 12 months showed all parameters were within the normal limits without oxygen support. The chest CT scan at the 6-month visit revealed a significant reduction in lung fibrosis (Figure 2B). A normal chest X-ray was observed at the 12-month follow-up (Figure 3B). It is important to note that the maximal PA recorded at the 6-month visit was 46 mmHg,with a pro-BNP level of 511 ng/ml, leading to the administration of sildenafil (1 mg/kg/12 h). At the 12-month visit, the maximum PA was 37 mmHg, and her pro-BNP levelwas reduced to 202 ng/mL; therefore, a lower dose of sildenafil (0.5 mg/kg/12 h) was given.
Patient 3
A 34-week-old male infant was prematurely born due to premature rupture of the placental membrane and had a BW of 2.4 kg at birth. The patient was diagnosed with hyaline membrane disease and required ventilator support. After 3 consecutive treatments with a surfactant, he was successfully weaned off of mechanical ventilation at 3months postnatal age. However, he still depended on oxygen support at a rate of 1 L/min via a sponge cannula. The diagnosis of BPD with vocal cord cirrhosis and laryngomalacia combined with periventricular leukomalacia was confirmed using nasopharyngoscopy, CT and MRI.
Upon admission toVinmec International Hospital, the patient was supported with oxygen at a rate of 1 L/min via nasal cannula to maintain the target SpO2 above 92%. The SpO2 dropped dramatically to 60% without oxygen support or crying. The patient suffered from severe chronic hypercapnia with pH, BE, PaCO2 and HCO3- levels maintained at 7.35, 12 mmol/L, 63.6 mmHg and 67.2 mmol/L, respectively, whereas his SpO2 and PaO2 were relatively low (60% at a FiO2 of 21%, and 44 mmHg, respectively). No cardiovascular malfunction or PAH was detected on echocardiogram, with a pro-BNP level of 176.5 ng/ml. The patient was diagnosed with CMV infection, with a viral load of 1.44 x105 copies/ml in the endotracheal fluid. When CMV treatment with 3 weeks with valganciclovir was complete, the chest CT scan and radiograph revealed lung fibrosis with significant air trapping in both lungs and lung inflammation (Figure 2A and 3A), and the patient could not be weaned off ofoxygen. He was dependent on oxygen at a rate of 1 L/min via nasal cannula to maintain an SpO2 between 94-96%.
Before transplantation, the patient still suffered from chronic hypercapnia with the following parameters: pH 7.51, PaCO2 59 mmHg, HCO3- 47.2 mmol/L, and PaO2 57 mmHg. He required oxygen support via nasal cannula at 1 L/min to maintain an SpO2between 92%-97%; without oxygen support, his SpO2was as lowas 70% (FiO2: 21%). The PCR results and hematological analysis (WBC: 23.9 G/L, neutrophils: 20.9%, and Hgb: 95 G/L) confirmed that the patient no longer carried CMV; he did not suffer from inflammation, nor did he have sepsis. Allo-UC-MSC transplantation was performed at 173 days postnatal age with no signs of severe adverse events. The patient was discharged 13 days after the first transplantation with oxygen support via nasal cannula at 0.5 L/min with an SpO2ranging between 93% and 98%.
At the first visit, the patient’s general condition was fair, and he was cognizant, with his BW slightly increased to 5.3 kg. He was still receiving oxygen at 0.5 l/min via cannula to maintainan SpO2at92-98%. The patient’s hypercapnic condition was reduced, with the following ABG test results: pH of 7.46, PaO2 of 45 mmHg, PaCO2of 52.6 mmHg and HCO3- of 38 mmol/L. His SpO2 without oxygen support had increased to 85% on room air. The total blood count results remained in the normal ranges. The blood CRP level was 0.2 mg/L, confirming that the patient hadnot developed aninflammatory response. At the 1-month follow-up, the patient was still dependent on oxygen support at a rate of 0.5 L/min to maintainan SpO2 level between 95% and 98. Two months posttransplantation, the patient was independent of active oxygen support, with an SpO2 of 96-98%.
The clinical team observed improvements at the 6-month visit. The patient was cognizant and was able to crawl, laugh, and actively respond to his parent’s voice. Due to the complication of periventricular leukomalacia, an additional Denver II test was conducted at the 6-month examination, and the results confirmed that the patient’s gross motor function was similar to that expected at 3 months, his language ability was equivalent to that expected at 5-6 months, his fine motor adaptive skills were equivalent to those expected at 3 months, and his personal-social skills were equivalent to those expected at 5 months. Moreover, improved respiratory function was also documented, with better airflow in both lungs, no crackles or rales, and no signs of retraction ornasal flaring atthe 6-month visit. All ABG results remained stable at the 12-month visit, with no sign of respiratory distress syndrome, an improved saturated oxygen level (SpO2: 100%) and a normal CO2 level in the blood (pH of 7.4, PaO2 of 72 mmHg, PaCO2 of 34.8 mmHg, HCO3- of 21.5 mmol/L; BE of -3 mmol/l) (Supplemental Table 4). Investigation of the patient’s lungswithCT at the 6-month visit indicated areduction infibrosis and the recovery offunction. Chest X-rays at the 12-month visit further confirmed the significant improvement (Figure 3B).
Patient 4
A premature female infant was born at another hospital at 28 weeks gestation with a birth weight of 1400 grams due to premature rupture of the placental membranes. She rapidly developed respiratory distress syndrome and required mechanical ventilation. A single dose of surfactant was given (100 – 200 mg/kg) on the first day. After that, the patient was placed on CPAP for a month, followed by oxygen support at 0.5 – 1 L/min until she reached 36 weeks old. Dexamethasone treatment using the DART protocol was advised for one week to further improve the patient’s condition. The patient was successfully weaned from oxygen supportand discharged at 37 weeks with an SpO2 ranging between 93% and 95%. However, 2 days postdischarge, the patient developed dyspnea with acute respiratory distress and returned to the hospital, where she stayed for the next 2 months.
The patient was referred to VinmecHospital at 4 months old with malnutrition (BW of 3 kg). Although the oxygen support was maintained at 1 L/min via nasal cannula, herSpO2 was relatively low (80%). Auscultation showed poor air entry into the lungs with crackles andrales. Her heart rate was high (200 – 220 bpm), with evident cyanosis and an SpO2 of 80% on 24%oxygen. The patient was intubated immediately and placed ona ventilator inSIMV mode (PIP at 23 cm H2O, PEEP at 5.5 cm H2O, and FiO2 at 50%). Five days after the treatment, ventilation support was switched to sponge cannula with oxygen flowing at 1 L/min. The ABG examinations revealed the following: pH of 7.49, PaCO2of 38.6 mmHg, HCO3- of 29.5 mmol/l, and PaO2 of 60 mmHg with FiO2: 40%. Furthermore, a complete blood count showed a low platelet count (53 G/L), while the WBC, neutrophil, and Hgb results were 5.8 G/L, 1.3 G/L and 112 G/L, respectively. Anechocardiogram was performed when the patient was stable and showed a pressure gradient through the tricuspid valve at 28 mmHg. The pro-BNP level was 8065 pg/ml. Hence, the patient was treated with 0.5 mg/kg/8 h sildenafil. The viral tests confirmed a CMVinfection (460 copies/ml), which was treated with valganciclovir for 21 days. The results of a chest X-ray and CT scan indicated severe lung fibrosis and substantialairtrapping in both lungs (Figure 2A and 3A).
Two UC-MSC transplantations were carried out without adverse events when the patient was160 days old. Four days after the first transplantation, the patient could breathe spontaneously at 55 – 62 breaths/minute. On the day of discharge (a week after the second transplantation), the patient breathed spontaneously with an SpO2 of 95% without oxygen support.
At the 7-day examination, the patient still suffered from dyspnea, with a respirationrate of 53 breaths/minutes. An increase in the SpO2 level to 95% was also recorded. The pro-BNP level was reduced significantly to 136.7 ng/ml. The hematological analysis confirmed that no sepsis or inflammatory reaction had occurred after stem cell transplantation, with a WBC count of 9.8 G/L, neutrophil percentage of 12.1%, Hgb level of 112 G/L and platelet count of 61 G/L. Onemonth postdischarge, the patient was cognizant and active, with a BW of 4kg. There was an increase in the SpO2 to 98% without oxygen support, suggesting that the patient’s respiratory function had recovered.
At the 6-month visit, respiratory distress was assessed as mild. The SpO2had stabilized at 97%. The 12-month follow-up corroborated the conclusion that the patient had recovered from BPD, witha normal SpO2 of 97%, pH of 7.34, PaCO2 of 39.8 mmHg, HCO3- of 20.8 mmol/l, BE of -4 mmol/l, and PaO2 of 73 mmHg. At 12 months after transplantation, it is worth mentioning that the patient hadrecovered well with regard to both her SpO2 and PaO2 levels, which were 100% and 72 mmHg, respectively. Evaluation of the lung structure onCT scans demonstrated that the fibrotic area was reduced (Figure 2B), while alveolation and maturation of the lung had becomeobvious. A further assessment of the lung structure using chest X-rays at the 12-month follow-up showed no signs of atelectasis or hyperexpansion in either lung (Figure 3B).