Viability and neural differentiation of mesenchymal stem cells derived from the umbilical cord following perinatal asphyxia

Abstract

Objective:

Hypoxia-ischemia is the leading cause of neurological handicaps in newborns worldwide. Mesenchymal stem cells (MSCs) collected from fresh cord blood of asphyxiated newborns have the potential to regenerate damaged neural tissues. The aim of this study was to examine the capacity for MSCs to differentiate into neural tissue that could subsequently be used for autologous transplantation.

Study Design:

We collected cord blood samples from full-term newborns with perinatal hypoxemia (n=27), healthy newborns (n=14) and non-hypoxic premature neonates (n=14). Mononuclear cells were separated, counted, and then analyzed by flow cytometry to assess various stem cell populations. MSCs were isolated by plastic adherence and characterized by morphology. Cells underwent immunophenotyping and trilineage differentiation potential. They were then cultured in conditions favoring neural differentiation. Neural lineage commitment was detected using immunohistochemical staining for glial fibrillary acidic protein, tubulin III and oligodendrocyte marker O4 antibodies.

Result:

Mononuclear cell count and viability did not differ among the three groups of infants. Neural differentiation was best demonstrated in the cells derived from hypoxia-ischemia term neonates, of which 69% had complete and 31% had partial neural differentiation. Cells derived from preterm neonates had the least amount of neural differentiation, whereas partial differentiation was observed in only 12%.

Conclusion:

These findings support the potential utilization of umbilical cord stem cells as a source for autologous transplant in asphyxiated neonates.

Introduction

Hypoxia-ischemia in newborns leads to acute morbidity, significant mortality and long-term disability. In neonates who develop moderate to severe acute encephalopathy following delivery room resuscitation, approximately half die and a third of the survivors develop long-term neurological deficits.1, 2 From a global perspective, birth asphyxia accounts for more than 800 000 deaths every year, with an equally high number of surviving neonates who suffer from long-term neurological handicaps.3

Preterm delivery is another major source of neonatal brain injury.4 Developmental delay and cerebral palsy are frequently seen in preterm infants with intracranial hemorrhage and periventricular leukomalacia, although they also often present in the absence of grossly noticeable brain lesions.5

Mesenchymal stem cells (MSC) have the capacity for self-renewal and differentiation into bone, cartilage, neuron and fat cell types.6 These features are attractive to investigators working to develop cell-based therapies to treat human diseases. Upon exposure to specific growth factors,7 MSCs are able to differentiate to neuron-like cells, and thus have potential applications in the treatment of neurological diseases.8 In addition to the direct trans-differentiation into neural cells, MSCs can perform regenerative functions in neural tissues through a number of mechanisms including secretion of trophic factors, inhibition of apoptosis, stimulation of endogenous stem cells and immunomodulation resulting in decreased inflammation.9 Umbilical cord blood (UCB) contains a high percentage of various stem cell subsets: hemopoeitic, mesenchymal and endothelial stem cells. Therefore, UCB represents a repository of cellular material with outstanding regenerative potential.10, 11

Because hypoxia is often present in the physiological environment for a variety of stem cell types, researchers have incorporated hypoxic conditions in tissue culturing.12 Reports have shown that laboratory-induced hypoxia can regulate the proliferation and differentiation of stem cells and, especially when mild, has salutary effects on stem and progenitor cells.13 On the other hand, severe hypoxia can reduce these processes.14 The influence of in vivo hypoxia, as in birth asphyxia, on the proliferation and differentiation of human UCB stem cells has not been studied. Additionally, characterization of stem cells obtained from UCB of preterm infants has not been previously reported. We aimed to examine the biological properties, including the number and viability, of various UCB stem cell populations collected from term neonates who had experienced significant fetal depression in comparison with full-term controls and preterm neonates. Furthermore, we compared the proliferative and neural differentiation properties of MSCs isolated from UCB of these populations. We hypothesized that, when treated in a suitable environment, MSCs in the UCB of preterm and depressed full-term neonates are capable of proliferation and neural differentiation.

Methods

Patients

This prospective study was conducted at Cairo University Medical Center in Cairo, Egypt, during the period from January 2008 to January 2010.

The study population included three groups of newborns.

Group I: Full term with perinatal depression. Subjects in this group were born at 38 weeks gestation and experienced both fetal and neonatal compromise. The following events were considered manifestations of in utero fetal compromise: a) increased placental resistance with loss or reversal of diastolic blood flow by Doppler, b) obstructed vaginal delivery as diagnosed by obstetrician, c) fetal bradycardia, and/or d) late decelerations with loss of beat to beat variability. Neonatal compromise was defined by absence of spontaneous breathing and need for cardiopulmonary resuscitation after birth.

Group II: Full term control. Subjects in this group were healthy neonates who did not have any of the above mentioned signs for fetal stress and did not receive cardiopulmonary resuscitation in the delivery room.

Group III: Preterm neonates. Subjects in this group were born between 30 and 36 6/7 weeks gestational age without evidence of fetal or neonatal compromise.

Infants with any of the following criteria were excluded from the study: a) major congenital anomalies, b) previous siblings diagnosed with inborn errors of metabolism, c) family history of hemoglobinopathies or coagulopathies, or d) suspected funisitis, chorioamnionitis, or neonatal sepsis.

This study was approved by the Institutional Review Board, and was conducted in compliance with the Declaration of Helsinki and regulations of the University of Cairo. Informed consent for each subject was obtained. Subjects in groups II and III were identified and recruited before the time of delivery, whereas subjects for group I were consented prenatally when fetal compromise was suspected, and enrolled only if neonatal depression occurred after birth.

Blood sampling

Samples of UCB were collected aseptically immediately after delivery and before separation of the placenta. A site at the distal end of the UCB was wiped with gauze to remove any blood, and then wiped three times with iodine swabs toward the placenta at 15 s intervals. A total volume of 20 ml of cord blood was collected using preservative-free heparinized syringes. A sample was also obtained for analysis of a cord blood gas. Samples were collected by the obstetrician in the presence of one of the co-investigators (DA) who was responsible for ensuring compliance with the specified protocol.

Separation of mononuclear cells

UCB was diluted to a 1:2 ratio with phosphate buffer saline, layered over Ficoll Hypaque (density 1.077, Biochrom KG, Berlin, Germany) for density gradient separation and centrifuged at 1800 r.p.m. for 20 min. Collected mononuclear cells were washed twice with phosphate buffer saline, resuspended and counted. Viability testing was done using trypan blue exclusion test.

Flow cytometric analysis

To enumerate the various stem cell subsets in UCB, we used Coulter Epics Elite flow cytometer (Beckman Coulter, Miami, FL, USA), with stem cell markers including CD34 and CD38 (R&D, Minneapolis, MN, USA and Dako, Glostrup, Denmark). Single- and dual-expression CD34 and CD38 were assessed for hematopoietic commitment and reduction of plasticity. CD34 monoclonal antibody was phycoerythrin-labeled and CD38 was fluorescein isothiocyanate labeled. Cells positive for both markers (dual expression) were located in the upper right quadrant of the output histogram.

MSC isolation

Mononuclear cells were incubated for 2 weeks and subcultured in a complete medium containing nerve growth factor in fibronectin-coated flasks.15 After 2 to 4 weeks, viability was tested and immunohistochemical staining was done to examine neural differentiation.

Assessment of proliferative capacity of UCB MSCs

DNA index was done for cultured cells to assess their proliferative capacity using flow cytometry. DNA indexing was related to half of the DNA content in human cells (DNA=1n); and cells were grouped according to the DNA content into three types as follows: a) normal quiescent cells that contain DNA=2n, b) cycling cells that contain DNA >2n, but <4n, and c) apoptotic cells that contain DNA <2n.

Immunohistochemistry

Microscopic examination was performed after preparation with primary antibodies from neural stem cell functional identification kit (StemCell Technologies, Vancouver, Canada; catalog number 5716). The kit included neural class III B-tubulin antibody, glial fibrillary acidic protein antibody and oligodendrocyte marker O4 antibody.

Statistical analysis

Data analysis was performed using the SPSS (version 10.0; Chicago, IL, USA). Descriptive statistics were expressed by means±s.d. Student's unpaired t-test, one-way analysis of variance and Mann-Whitney Wilcoxon U-tests were used for group comparisons where appropriate. Nominal data was expressed by frequency (%) and were analyzed using χ2-test.

Results

A total of 55 subjects were included in the three study groups, with 27, 14 and 14 subjects in groups I, II and III, respectively. Deliveries in group I, asphyxia group, were complicated by severe fetal distress (n=15), meconium-stained amniotic fluid (n=9) and eclampsia (n=3). Groups I and II were similar in gestational age, and there was a nonsignificant trend for male predominance in group I (67 vs 43%, P=0.14). Results of the blood gases measured in UCB are presented in Table 1. When compared with the other two groups, group I had significantly lower pH and PO2 (P<0.001 and P=0.04, respectively) and higher PCO2 (P=0.01). In group I, cord blood pH was <7.15 in 17 subjects; 4 of them had pH <7, whereas none in the other two groups had such significant acidemia.

Table 1 Values of the initial cord blood gases in the three study groups (n=55)

Delivery room resuscitation for group I included positive pressure ventilation (n=15), endotracheal intubation (n=6), chest compressions (n=2) and administration of inotropic medication (n=2). Neurological examination of the newborns in group I showed hypoxic-ischemic encephalopathy in 17 subjects (63%), which was mild (n=10), moderately-severe (n=5) or severe (n=2), using the Sarnat's grading system for encephalopathy.16 Abnormal neurological findings included: convulsions (n=8, 30%), hypertonia (n=8, 30%), hypotonia (n=3, 11%), abnormal reflexes (n=17, 63%) and impaired consciousness (n=6, 22%). There were no neurological abnormalities in group II. Physical and neurobehavioral examinations of group III were appropriate for their gestational age.

Mononuclear cells and viability results among studied group

There was a borderline significant difference between the three groups (P=0.051), with the greatest cell count in group II and the least count in group I. Viability tests were done after culture that demonstrated no significant difference between groups (P=0.54). Viability in each of the three studied groups ranged between 98 and 100% (Table 2).

Table 2 Mononuclear cells in the three study groups: count, viability, immune-phenotyping and immune-histochemical results

Flow cytometric analysis of Mononuclear cells

Single- and dual-CD34 (hemopoeitic stem cell marker) and CD38 (commitment marker) expression were done to assess stemness and ongoing differentiation. CD38 expression was the highest in group II (P<0.001), but did not differ between groups I and III. CD34 and dual expression did not differ among the three groups (Table 2).

MSCs identification

Examination of the culture flasks under an inverted microscope revealed typical fibroblast-like morphology. MSCs showed ability to differentiate into chondrocyte, osteoblast and adipocyte lineages, and expressed typical immunophenotype (i.e., positive for CD44, CD90, CD105; negative for CD45, CD34).

To examine the proliferative capacity, DNA index was done following the culture of cells. The vast majority of cells (93 to 98%) were quiescent. Few cells were cycling in each of the three groups, without significant difference among groups (P=0.51).

Morphological evaluation of neural differentiation

Examination of MSC subjected to nerve growth factor medium revealed the appearance of dendrites and star-shaped cells. Immunohistochemical studies were done on 35 subjects (n=16, 11 and 8 in groups I, II and III, respectively). Thirteen (37%) subjects demonstrated neural differentiation into all three neural cell types (astrocytes, neurons and oligodendrocytes) in more than 80% of MSCs; of them, 11 subjects belonged to group I. Additionally, 14 subjects (40%) showed differentiation in 50 to 80% of MSCs; of them, 5 subjects were in group 1, and 8 were in group II. Undifferentiated cells were seen in eight subjects; the majority of them (seven out of eight) were preterm infants in group III. Therefore, group I had the most and group III had the least neuronal differentiation (P=0.01; Figures 1).

Figure 1
figure1

Neural differentiation of mesenchymal stem cells (MSCs) in the umbilical cord of full-term hypoxemic neonates. (a) Immunocytochemical staining with mouse anti-human Nestin, showing non-differentiating MSCs examined under fluorescence microscopy ( × 100 magnification). (b) Immunocytochemical staining with mouse anti- human Nestin, showing some differentiation of MSCs (neurospheres) examined under fluorescence microscopy ( × 100 magnification). (c) Immunocytochemical staining with mouse anti-human neuron-specific β-III tubulin, showing complete differentiation of MSCs into neurons with dendrites examined under fluorescence microscopy ( × 100 magnification).

Discussion

This study demonstrates that MSCs in the cord blood collected from hypoxic infants showed comparable viability and cell cycle state as normoxic infants whether term or preterm. Almost 90% of the cells in all groups were quiescent as indicated by the DNA index. The potential for in vitro neural differentiation was best observed in hypoxic infants. Complete MSCs differentiation to astrocytes, neurons and oligodendrocytes occurred in this group (69%), whereas it was hardly identified in healthy term (18%) or preterm neonates (0%).

Viability of MSCs, collected from UCB in this study, was not compromised by perinatal asphyxia. In vivo stem cells derived from early-stage blastocysts have a low oxygen environment. This hypoxia maintains pluripotency and minimizes spontaneous differentiation.17, 18 Thus, the link between hypoxia in the microenvironment and the capability of MSCs to maintain viability, as well as ability to proliferate and differentiate, led researchers to use hypoxic environments in vitro.19

To the best of our knowledge, this work is the first to test the ability of cord blood MSCs derived from asphyxiated neonates to differentiate into neural lineage. Previous work examined the ability of MSCs to proliferate and differentiate when exposed to in vitro hypoxic conditions.20, 21 Infusion of MSCs at 3 and 10 days post-hypoxic-ischemic injury was recently shown to enhance cell proliferation, survival and differentiation.10 It was originally hypothesized that regeneration occurs directly by incorporation of MSC-derived neurons and oligodendrocytes to restore damage.22, 23 However, there is now evidence to support that treatment with MSCs after hypoxic-ischemic encephalopathy results in enhanced endogenous cell proliferation and differentiation. In addition, MSC treatment may also be associated with the inhibition of injurious processes.24, 25, 26

The increased proliferative capacity of MSCs under low O2 tension is thought to be due to increased expression of hypoxia inducible factor-1α that promotes self renewal and inhibits apoptosis of cells, whereas increased O2 tension promotes differentiation or apoptosis.27 We found that more than 90% of the cells in all groups were quiescent as indicated by the DNA index. A recent study reported a significantly reduced rate of proliferation in severely hypoxic medium with the accumulation of G1 phase cells,28 which is similar to our findings. Others20, 21 have demonstrated that UCB-derived human MSCs can adapt to a physiologically hypoxic environment by altering their energy consumption and metabolism.

We observed complete MSCs differentiation to astrocytes, neurons and oligodendrocytes, mostly in the term asphyxia neonates, but rarely in healthy term neonates or preterm neonates. It is possible that the release of different inflammatory cytokines in response to hypoxia could promote the differentiation of MSCs to the appropriate cell line needed for tissue repair. Such response and differentiation is not limited to the nervous system. In patients with osteoarthritis, for example, hypoxia has been demonstrated to stimulate the commitment of stem cells in the infrapatellar fat pad to chondrogenesis.29 Although hypoxia stimulates MSCs to differentiate, in general, the determination of cell lineage is dependent on the environmental milieu through mechanisms that are still incompletely understood. It is equally unclear why the absence of in vitro neural differentiation was observed in cells collected from preterm infants. It is likely that the in vivo cerebral environment differs by gestational age. This is evidenced by the diverse response between term and preterm infants to hypoxic-ischemic insults, in which neuronal injury predominates in term infants, whereas oligodendroglial and white-matter injury manifest in premature infants.30

Cell therapy has been described as a promising treatment for certain neurodegenerative diseases, as well as various forms of acute injury to the spinal cord and brain.31 MSCs or multipotent mesenchymal stem cells (MSCs) may serve as a substrate for regenerative stem cell therapy due to their ability to self renew, differentiate into multiple tissues32 and immunomodulate.33 Under certain conditions, stem cells from fresh cord blood are able to differentiate into neurons, microglial cells and astrocytes. Animal models have shown that treatment with fresh cord blood resulted in improvements in the progression of neurological disorders including stroke, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease and spinal cord injuries. Further, improved bone healing was noted.34 Intracerebral transplantation of MSCs derived from human UCB was shown to enhance the functional recovery of neonatal rats with hypoxic-ischemic brain injury.35 Although this study provides preliminary data supporting the promise of human UCB MSCs for cell-based repair after perinatal brain injury, it is recognized that it is premature to draw conclusions about the practicality and the efficacy of using these cells for autologous transplantation in the vulnerable neonatal population.

We found that more than 90% of the cells in all groups were quiescent as indicated by the DNA index. A recent study reported a significantly reduced rate of proliferation in severe hypoxic medium with accumulation of G1 phase cells28 that is similar to our findings, whereas others20, 21 demonstrated the umbilical cord-derived human MSCs to adapt their energy consumption and metabolism when cultured in a physiologically appropriate hypoxic environment.

Fresh cord blood is a promising source of non-hematopoietic stem cells. Among others, it contains endothelial cells and mesenchymal stromal cells.36 UCB is more readily available than bone marrow. MSCs can be isolated from UCB within 2 to 3 working days, favorable for time-sensitive situations. Because of the immunological immaturity of the cells, cord blood is better tolerated than bone marrow, although being equally safe and effective.14, 37 In certain circumstances, cord blood can be transplanted successfully even if the donor and the recipient are not a perfect match; this substantially widens the range of patients who are able to receive a transplant.14

Conclusions

This study is the first to demonstrate that perinatal hypoxia influences the potential for MSCs to differentiate into neural cells when later exposed to the appropriate in vitro conditions. Our findings provide evidence for the multipotential nature of UCB-derived MSCs, and support the rationale for future studies in animal models of hypoxic-ischemic encephalopathy and eventual early human trials of autologous MSC-based therapy in neonates with hypoxic-ischemic encephalopathy.

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Aly, H., Mohsen, L., Badrawi, N. et al. Viability and neural differentiation of mesenchymal stem cells derived from the umbilical cord following perinatal asphyxia. J Perinatol 32, 671–676 (2012) doi:10.1038/jp.2011.174

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Keywords

  • mesenchymal cells
  • umbilical cord
  • asphyxia
  • hypoxic-ischemic encephalopathy
  • full term

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