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 introduced no karyotypical abnormalities, and the cells maintained a normal 46XY karyotype 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 MSCs proposed by the International Society for Cellular Therapy (ISCT, Table 2).
Patient outcomes as a case report
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 body weight (BW) of 720 grams. Soon after birth, the patient developed signs of respiratory distress syndrome with retraction followed by apnea, 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 (PDA), which closed after one course of paracetamol (15 mg/kg/6 h) for seven days, and no evidence of pulmonary artery hypertension (PAH) was observed 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); the patient was then switched to continuous positive airway pressure therapy (CPAP) at 7 cmH2O and 50% FiO2 for the following 1.5 months. At 3.5 months postnatal age, the patient was diagnosed with BPD and continued to receive oxygen support via a nasal cannula at 0.5 – 1 L/min. Nebulized corticosteroids at 100 mcg/kg 4 times/day were administered for a 1-month period. 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 supplementation with vitamins E, A, K) were initiated for 2 months. However, at 4.5 months postnatal age, the patient’s BPD was not improved, with the SpO2 off oxygen support dropping to 90%. The chest CT scan and X-ray at 3.5 months 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 allo-UC-MSC administration, 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 on a maximum pulmonary artery pressure (PAP) of 40 mmHg and illustrated on echocardiogram, 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 was 4 months old. The allo-UC-MSC administration was performed at 144 days postnatal age (47 weeks gestational age). No signs of serious adverse events were observed during the two interventions. Three days after the second infusion, the patient could breathe spontaneously with an SpO2 of 96% without oxygen support. The patient was discharged at 161 days postnatal age (17 days postadministration).
At the first follow-up visit, the patient was alert, had a BW of 4 kg and was spontaneously breathing, with an SpO2 of 96% without oxygen support. Blood gas analysis revealed a reduction in the saturated 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. The pro-BNP level had dropped to 351.9 ng/ml, leading to the termination of PAH treatment at 4 months postadministration. At the 1-month follow-up examination, the patient was conscious and active, and her BW had increased to 4.3 kg, with air fully entering both lungs. She spontaneously breathed and had an SpO2 of 97% on room air without oxygen support. The laboratory tests revealed that her pH (7.37), PaCO2 (46.3 mmHg), HCO3- (27 mmol/L), and PaO2 (42 mmHg) remained stable post administration. Hematological analysis also confirmed the absence of inflammation and sepsis, as indicated by the Hgb level (129 G/L), white blood cell count (WBC, 6.1 G/L), and neutrophil level (6.4%). At the 6-month visit, the patient no longer required oxygen support, with her SpO2 reaching 100%, and she exhibited 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 reduction in lung fibrosis was observed on chest CT without PAH (Figure 2B). At 12 months postdischarge, the patient’s condition was improved. Her BW had increased to 7.5 kg. ABG results were in 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. Lung fibrosis was reduced 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.
The second premature neonate was the twin of patient 1 and 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 by apnea, bradycardia, and cyanosis with an SpO2 between 50% and 60%. The patient was positive pressure-ventilated by a bag mask and then intubated and placed on a ventilator in SIMV 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 3rd days after birth. A large PDA was detected by cardiac echography, requiring one course of indomethacin (0.2 mg/kg/12 hours) within the first postnatal week as previously described (22). After 2 months on SIMV, ventilation support was switched to CPAP with a PEEP of 6 cmH2O and 40% FiO2. After treatment, the PDA size was reduced (1 mm), and the shunt size was small; however, the size increased gradually and reached 3.6 mm at 3 months. The PDA was maintained at a large size and required surgical closure at 3.5 months of age. Although the PDA was closed without complications, PAH was observed (38 mmHg), and the pro-BNP level was 2223 ng/ml, leading to treatment with 1 mg/kg/6 hours sildenafil and 2 mg/kg/8 hours bosentan. After the operation, the patient was on CPAP at 6 cmH2O at an FiO2 of 30% before switching to nasal cannula oxygen at a rate of 1 L/min after 1 month 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 having BPD 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 did not improve, and she remained dependent on oxygen support, leading to allo-UC-MSC administration at 151 days postnatal age. Prior to intervention, 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 administration, the patient was discharged with oxygen support via nasal cannula at 1 L/minute, a respiration rate of 64-67 breaths/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 was 145 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, HCO3- of 32.3 mmol/L, PaO2 of 36 mmHg, and 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 reductions in both herPaCO2 and HCO3-, while her SpO2 was maintained at 94-98% on oxygen via a cannula at 0.5 l/min. Two months after the first hUC-MSC administration, 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 positive changes 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 that all parameters were within the normal limits without oxygen support. The chest CT scan at the 6-month visit revealed a 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 PAP 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 PAP was 37 mmHg, and the pro-BNP level was reduced to 202 ng/mL; therefore, a lower dose of sildenafil (0.5 mg/kg/12 h) was given.
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 3 months 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 scarring and laryngomalacia combined with periventricular leukomalacia was confirmed using nasopharyngoscopy, CT and MRI.
Upon admission to Vinmec 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 an FiO2 of 21% and 44 mmHg, respectively). No cardiovascular malfunction or PAH was detected on echocardiogram, and the pro-BNP level was 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. After completion of CMV treatment with valganciclovir for 3 weeks, 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 of oxygen. He was dependent on oxygen at a rate of 1 L/min via nasal cannula to maintain an SpO2 between 94-96%.
Before administration, 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 SpO2 between 92%-97%; without oxygen support, his SpO2 was as low as 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 administration was performed at 173 days postnatal age with no signs of severe adverse events. The patient was discharged 13 days after the first intervention with oxygen support via nasal cannula at 0.5 L/min with an SpO2 ranging between 93% and 98%.
At the first visit, the patient’s general condition was fair, and he was conscious, with his BW slightly increased to 5.3 kg. He was still receiving oxygen at 0.5 l/min via cannula to maintain an SpO2 at 92-98%. The patient’s hypercapnic condition was reduced, with the following ABG test results: pH of 7.46, PaO2 of 45 mmHg, PaCO2 of 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 C-reactive protein (CRP) level was 0.2 mg/L, confirming that the patient had not developed an inflammatory response. At the 1-month follow-up, the patient was still dependent on oxygen support at a rate of 0.5 L/min to maintain an SpO2 level between 95% and 98. At two months postadministration, the patient was independent of active oxygen support, with an SpO2 of 96-98%.
The clinical team observed the progression in patients’ condition at the 6-month visit. The patient was conscious 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 or nasal flaring at the 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 lungs with CT at the 6-month visit indicated a reduction in fibrosis and the gradual recovery of lung function. Chest X-rays at the 12-month visit further confirmed the progression (Figure 3B).
A premature female infant was born at another hospital at 28 weeks gestation due to premature rupture of the placental membranes with a birth weight of 1400 grams. 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 Dexamethasone: A Randomized Trial (DART) protocol was advised for one week to further improve the patient’s condition. The patient was successfully weaned from oxygen support and 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 Vinmec Hospital at 4 months old with malnutrition (BW of 3 kg). Although oxygen support was maintained at 1 L/min via nasal cannula, her SpO2 was relatively low (80%). Auscultation showed poor air entry into the lungs with crackles and rales. 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 on a ventilator in SIMV 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, PaCO2 of 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. An echocardiogram 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 hours sildenafil. The viral tests confirmed a CMV infection (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 substantial air trapping in both lungs (Figure 2A and 3A).
Two UC-MSC administrations were carried out without adverse events when the patient was 160 days old. Four days after the first administration, the patient could breathe spontaneously at 55 – 62 breaths/minute. On the day of discharge (a week after the second administration), 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 respiration rate of 53 breaths/minutes. An increase in the SpO2 level to 95% was also recorded. The pro-BNP level was reduced to 136.7 ng/ml. Hematological analysis confirmed that no sepsis or inflammatory reaction had occurred after MSC administration, 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. One month postdischarge, the patient was conscious and active, with a BW of 4 kg. The SpO2 was increased 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 SpO2 had stabilized at 97%. The 12-month follow-up corroborated the conclusion that the patient had recovered from BPD, with a 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 intervention, it is worth mentioning that the patient had recovered well with regard to both her SpO2 and PaO2 levels, which were 100% and 72 mmHg, respectively. Evaluation of the lung structure on CT scans demonstrated that the fibrotic area was reduced (Figure 2B), while alveolation and maturation of the lung had become obvious. 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).