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Stem Cells as a Therapeutic Modality in Pediatric Malformations
Stem Cells as a Therapeutic Modality in Pediatric Malformations D.K. Gupta, S. Sharma, P. Venugopal, L. Kumar, S. Mohanty, and S. Dattagupta ABSTRACT Aim. The aim of this study was to explore stem cell use in congenital anomalies. Patients and Methods. During July 2005 through July 2006, autologous stem cells were used in 29 patients: 12 with liver cirrhosis and 17 with meningomyelocele. Stem cells were injected into the hepatic artery and the portal vein or into the hepatobiliary radicals for liver cirrhosis, or into the spinal cord and caudal space for meningomyelocele. Preoperative status served as the control condition. Observations and Results. The ages of patients with liver cirrhosis ranged between 1.5 and 9 months (mean, 4.12 months). The etiology was extra hepatic biliary atresia (EHBA) versus neonatal cholestasis and choledochal cyst in 8; 2 and 2 patients, respectively. Five patients died due to ongoing cirrhosis. Follow-up at 3 to 12 months (N ⫽ 7) showed absence of cholangitis (4/7), yellow stools (5/7), decreased liver firmness (3/7), improved liver function (6/7), and better appetite (6/7). Hepatobiliary scan was excretory in 6 of 7 with improved uptake in 4 of 7. Histopathology demonstrated comparative improvement in fibrosis among 3 patients. Meningomyelocele patients were between 0 and 1 month, 1–5 months, and 1– 4 years in 5, 8, and 2 cases respectively. Five had a history of rupture. Three had undergone meningocele repair in the past with neurological deficits. Redo surgery for a tethered cord was performed in 1 patient. Follow-up at 3 to 11 months in 14 cases showed improved power in 7 with dramatic recovery in 3 (22%) and status quo in 7 (50%). Conclusion. Initial stem cell use in liver cirrhosis and meningomyelocele has suggested beneficial results. However, long-term evaluation in randomized controlled trials is essential to draw further conclusions.
T
HE inciting factor leading to developmental arrest during embryogenesis and various congenital anomalies is far from understood for many anomalies. The surgical outcome in the best hands is far from optimal in some diseases such as biliary atresia with hepatocellular failure and spina bifida with neurological deficits. This need called for a exploration of any means to continue the developmental process from the stage where it had been arrested, or to reverse the changes caused by the anomaly, seeking to improve the functional status of the involved organs and tissues. Stem cells with the potential to transform into healthy cells and repair damaged cells may prove beneficial in various congenital malformations. We sought to use stem cells in selected congenital malformations for which the present treatment options are either limited or not available when the irreversible changes have already taken place at an early stage. These include extra hepatic biliary atresia (EHBA) and spina bifida with neurological deficiency. In 0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.01.060 700
EHBA, the outflow biliary channels from the liver are congenitally blocked, thus predisposing to cirrhotic liver and hepatocellular failure. These children have a poor life span and only a few reach adolescence. In spina bifida with neurological deficiency, there is a defect in the midline in the back involving the bones, nerves, muscles, and skin. Although the muscle and skin defect can be repaired with surrounding tissues, there often are variable neurological deficits, involving bladder, bowel, and 1 or both lower limbs. The child crippled for life with these impairments is a burden to society and to the family. We sought to use stem
From the Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India. Address reprint requests to Prof D.K. Gupta, Professor and Head, Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India. E-mail: profdkgupta@gmail. com; [email protected] © 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 39, 700 –702 (2007)
STEM CELLS IN PEDIATRIC MALFORMATIONS
cells in biliary atresia and choledochal cysts to prevent further degeneration of hepatocytes, regenerate new hepatocytes, and reverse the degeneration of hepatocytes. We also sought to use stem cells in spina bifida with neurological deficiency to repair the damaged neurons to improve the existing deficits. MATERIALS AND METHODS During July 2005 to August 2006, stem cells were administered to 29 patients: 12 with liver cirrhosis and 17 with meningomyelocele. We obtained a special informed written consent. Stem cells were injected during definitive surgery or postoperatively if the definitive surgery had been performed earlier. No separate surgical procedure was required to offer the therapy. Autologous bone marrow was collected under local anesthesia. Mononuclear cells were separated using a Ficoll-Hypaque gradient, washed, and resuspended. We performed flow cytometric enumeration of CD 34⫹ cells, the median number of which was 30 ⫻ 106 (20 ⫺ 60 ⫻ 106) in concentrations from 4 ⫺ 30 ⫻ 106 cells/mL with a 1–5 mL ready solution for infusion. Cell viability before transplantation was ⬎95% in all samples. Autologous stem cells were injected into the hepatic artery (20%) and portal vein (80%) or into the hepatobiliary radicals for liver cirrhosis, or into the spinal cord and caudal space for meningomyelocele. The preoperative status of the patient served as the control condition for that patient. Postoperative monitoring included temperature charting and seeking evidence of infection or other complications. Antibiotic coverage was given for 7 to 10 days for meningomyelocele and 3 months in biliary atresia. The patients with liver cirrhosis were evaluated based on clinical status, liver function tests, liver biopsy, and nuclear imaging. The patients with meningomyelocele were evaluated for clinical status and muscle charting by 2 unbiased separate physiotherapists. They were examined for changes in power, sensation, and neurological status of bladder and bowel.
RESULTS
The age range of the patients with liver cirrhosis was between 1.5 and 9 months (mean, 4.12 months). The etiology was EHBA, neonatal cholestasis, and choledochal cyst in 8, 2, and 2 patients, respectively. The male:female gender ratio was 1:1. The procedures included: For the cases of EHBA, Kasai ⫹ stem cell (n ⫽ 5), post-Kasai (stem cells only n ⫽ 2) and trans-hepatic stem cells (n ⫽ 1). For choledochal cyst excision ⫹ hepaticoduodenostomy (n ⫽ 1) or cystoduodenostomy (n ⫽ 1) for choledochal cyst, and for neonatal cholestasis flushing ⫹ stem cell (n ⫽ 2). There was a smooth postoperative recovery in all subjects with no complications. Five patients died at 1– 6 months after stem cell therapy due to ongoing cirrhosis due to late presentation. Follow-up results evaluated at 3–12 months (N ⫽ 7) showed absence of cholangitis (4/7); yellow colored stools (5/7); decreased liver firmness (3/7); improved liver function of (6/7); and better appetite (6/7). Hepatobiliary scan was excretory in 6 of 7 with improved uptake in 4 of 7. Histopathology repeated after stem cells transplant in 3 patients demonstrated comparative improvement in fibrosis in all 3. Ages of meningomyelocele patients were 0 –1 month, 1–5 months, and 1– 6 years in 6, 8, and 3 cases, respectively. Five
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patients had a history of rupture. Four patients had undergone meningomyelocele repair in the past but had persistent neurological deficiency. Redo surgery for a tethered cord was performed in 1. Meningomyelocele repair and stem cell injection was performed in 13, and only stem cell injection in 3. Only 2 patients had meningoceles. There was a smooth postoperative recovery in all subjects with no noted complications. Follow-up of 3–11 months in 14 cases showed improved power in 7 with a dramatic recovery in 3 (22%) and status quo in 7 (50%). Three patients are still under observation. Among the 7 patients with status quo, motor power was already good in 5. DISCUSSION
The goal of stem cell therapy is to repair damaged tissue that has lost the property to heal itself. This might be accomplished by transplanting stem cells into the damaged area and directing them to grow new, healthy tissue. Autologous bone marrow stem cells are a type of adult stem cells, a multipotent unit still capable of differentiating into only a few specialized cells. Studies investigating liver regeneration under conditions that preclude hepatocyte proliferation have reported the proliferation of a subpopulation of small, oval-shaped cells, initially in the portal triad.1 These cells, termed liver progenitor oval cells, have been shown to participate in liver regeneration in a variety of rodent models of chronic liver damage. They express markers common to hepatocytes and cholangiocytes, suggesting that they are a common precursor of both liver cell lineages.1 In a rat experiment of injection of bone marrow– derived cells native liver stem cells cause liver regeneration when proliferation of native hepatocytes is blocked by retrorsine. Mobilization from the bone marrow is a prerequisite, the mechanism of which is complex. In this study, histopathology could only be done at 6 months after the stem cell infusion, in the 3 patients who were alive with adequate follow-up. A comparative improvement in fibrosis was seen in all 3. Although the hypothesis for this documented improvement is lacking studies in humans, there is some evidence from experimental studies. After injecting bone marrow mesenchymal stem cells isolated from bone marrow of male donor mice into the liver remnants of hepatectomized female mice, Y chromosome and albumin positive cells were observed with increased liver weight in the transplanted mice.2 Thus bone marrow mesenchymal stem cells may be induced to differentiate into hepatocytes. Stem cells were used in spina bifida with neurological deficiency with an aim to repair the damaged neurons and improve the existing neurological deficits. Recovery of central nervous system disorders is hindered by the limited ability to regenerate lost cells, replace damaged myelin, and re-establish functional neural connections. Both hematopoietic stem cells and marrow stromal cells have been shown to have the potential to restore the injured spinal
702
cord and promote functional recovery in mice.3– 6 This positive effect was most pronounced using mesenchymal stem cells. Progressive complete functional motor recovery with evident nervous tissue regeneration has been achieved following administration of bone marrow stromal cells in traumatic central spinal cord cavities of adult rats with chronic paraplegia due to a previously injured spinal cord.7 In our experience with favorable responses in 50% of cases, it is hoped that stem cells may prove beneficial to improve the neurological deficits associated with cases of spina bifida. The hypothesis for the mechanism of action has been difficult to elucidate. A possible explanation has been that the stem cells provide a trigger factor for the growth of neurons. Thus bone marrow– derived stem cells may ultimately be optimal for spinal cord repair, if the mechanism of possible transdifferentiation can be elucidated.8 The fact that the stem cells reach the desired site of action has been shown by animal experiment although it is difficult to do in humans. To conclude, stem cell therapy may prove beneficial in cases of liver cirrhosis due to congenital anomalies by reversing or delaying the onset of end-stage liver disease and decreasing the need for liver transplantation and immunosuppression. The intial use of stem cells in meningomyelocele has shown promising results. However, longterm evaluation with randomized controlled trials is essential to draw conclusions. Major concerns remain: what prompts stem cells to assume specific functions and which factors would dictate them to stop multiplication once the
GUPTA, SHARMA, VENUGOPAL ET AL
aim is achieved. An uncontrolled proliferation may carry the risk of teratoma formation at any stage after stem cell therapy. The clinical scope of stem cell therapy could be endless. Only further research and wider clinical applications will address many practical and theoretical queries related to stem cells. REFERENCES 1. Matthews VB, Yeoh GC: Liver stem cells. IUBMB Life 57:549, 2005 2. Wu X, Zhao L, Xu Q, et al: Differentiation of bone marrow mesenchymal stem cells into hepatocytes in hepatectomized mouse. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 22:1234, 2005 3. Koda M, Okada S, Nakayama T, et al: Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice. Neuroreport 16:1763, 2005 4. Koshizuka S, Okada S, Okawa A, et al: Transplanted hematopoietic stem cells from bone marrow differentiate into neural lineage cells and promote functional recovery after spinal cord injury in mice. J Neuropathol Exp Neurol 63:64, 2004 5. Sykova E, Jendelova P, Urdzikova L, et al: Bone marrow stem cells and polymer hydrogels-two strategies for spinal cord injury repair. Cell Mol Neurobiol 26:1111, 2006 6. Zhao ZM, Li HJ, Liu HY, et al: Intraspinal transplantation of CD34⫹ human umbilical cord blood cells after spinal cord hemisection injury improves functional recovery in adult rats. Cell Transplant 13:113, 2004 7. Zurita M, Vaquero J: Bone marrow stromal cells can achieve cure of chronic paraplegic rats: functional and morphological outcome one year after transplantation. Neurosci Lett 402:51, 2006 8. Enzmann GU, Benton RL, Talbott JF, et al: Functional considerations of stem cell transplantation therapy for spinal cord repair. J Neurotrauma 23:479, 2006
T
HE inciting factor leading to developmental arrest during embryogenesis and various congenital anomalies is far from understood for many anomalies. The surgical outcome in the best hands is far from optimal in some diseases such as biliary atresia with hepatocellular failure and spina bifida with neurological deficits. This need called for a exploration of any means to continue the developmental process from the stage where it had been arrested, or to reverse the changes caused by the anomaly, seeking to improve the functional status of the involved organs and tissues. Stem cells with the potential to transform into healthy cells and repair damaged cells may prove beneficial in various congenital malformations. We sought to use stem cells in selected congenital malformations for which the present treatment options are either limited or not available when the irreversible changes have already taken place at an early stage. These include extra hepatic biliary atresia (EHBA) and spina bifida with neurological deficiency. In 0041-1345/07/$–see front matter doi:10.1016/j.transproceed.2007.01.060 700
EHBA, the outflow biliary channels from the liver are congenitally blocked, thus predisposing to cirrhotic liver and hepatocellular failure. These children have a poor life span and only a few reach adolescence. In spina bifida with neurological deficiency, there is a defect in the midline in the back involving the bones, nerves, muscles, and skin. Although the muscle and skin defect can be repaired with surrounding tissues, there often are variable neurological deficits, involving bladder, bowel, and 1 or both lower limbs. The child crippled for life with these impairments is a burden to society and to the family. We sought to use stem
From the Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India. Address reprint requests to Prof D.K. Gupta, Professor and Head, Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India. E-mail: profdkgupta@gmail. com; [email protected] © 2007 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 39, 700 –702 (2007)
STEM CELLS IN PEDIATRIC MALFORMATIONS
cells in biliary atresia and choledochal cysts to prevent further degeneration of hepatocytes, regenerate new hepatocytes, and reverse the degeneration of hepatocytes. We also sought to use stem cells in spina bifida with neurological deficiency to repair the damaged neurons to improve the existing deficits. MATERIALS AND METHODS During July 2005 to August 2006, stem cells were administered to 29 patients: 12 with liver cirrhosis and 17 with meningomyelocele. We obtained a special informed written consent. Stem cells were injected during definitive surgery or postoperatively if the definitive surgery had been performed earlier. No separate surgical procedure was required to offer the therapy. Autologous bone marrow was collected under local anesthesia. Mononuclear cells were separated using a Ficoll-Hypaque gradient, washed, and resuspended. We performed flow cytometric enumeration of CD 34⫹ cells, the median number of which was 30 ⫻ 106 (20 ⫺ 60 ⫻ 106) in concentrations from 4 ⫺ 30 ⫻ 106 cells/mL with a 1–5 mL ready solution for infusion. Cell viability before transplantation was ⬎95% in all samples. Autologous stem cells were injected into the hepatic artery (20%) and portal vein (80%) or into the hepatobiliary radicals for liver cirrhosis, or into the spinal cord and caudal space for meningomyelocele. The preoperative status of the patient served as the control condition for that patient. Postoperative monitoring included temperature charting and seeking evidence of infection or other complications. Antibiotic coverage was given for 7 to 10 days for meningomyelocele and 3 months in biliary atresia. The patients with liver cirrhosis were evaluated based on clinical status, liver function tests, liver biopsy, and nuclear imaging. The patients with meningomyelocele were evaluated for clinical status and muscle charting by 2 unbiased separate physiotherapists. They were examined for changes in power, sensation, and neurological status of bladder and bowel.
RESULTS
The age range of the patients with liver cirrhosis was between 1.5 and 9 months (mean, 4.12 months). The etiology was EHBA, neonatal cholestasis, and choledochal cyst in 8, 2, and 2 patients, respectively. The male:female gender ratio was 1:1. The procedures included: For the cases of EHBA, Kasai ⫹ stem cell (n ⫽ 5), post-Kasai (stem cells only n ⫽ 2) and trans-hepatic stem cells (n ⫽ 1). For choledochal cyst excision ⫹ hepaticoduodenostomy (n ⫽ 1) or cystoduodenostomy (n ⫽ 1) for choledochal cyst, and for neonatal cholestasis flushing ⫹ stem cell (n ⫽ 2). There was a smooth postoperative recovery in all subjects with no complications. Five patients died at 1– 6 months after stem cell therapy due to ongoing cirrhosis due to late presentation. Follow-up results evaluated at 3–12 months (N ⫽ 7) showed absence of cholangitis (4/7); yellow colored stools (5/7); decreased liver firmness (3/7); improved liver function of (6/7); and better appetite (6/7). Hepatobiliary scan was excretory in 6 of 7 with improved uptake in 4 of 7. Histopathology repeated after stem cells transplant in 3 patients demonstrated comparative improvement in fibrosis in all 3. Ages of meningomyelocele patients were 0 –1 month, 1–5 months, and 1– 6 years in 6, 8, and 3 cases, respectively. Five
701
patients had a history of rupture. Four patients had undergone meningomyelocele repair in the past but had persistent neurological deficiency. Redo surgery for a tethered cord was performed in 1. Meningomyelocele repair and stem cell injection was performed in 13, and only stem cell injection in 3. Only 2 patients had meningoceles. There was a smooth postoperative recovery in all subjects with no noted complications. Follow-up of 3–11 months in 14 cases showed improved power in 7 with a dramatic recovery in 3 (22%) and status quo in 7 (50%). Three patients are still under observation. Among the 7 patients with status quo, motor power was already good in 5. DISCUSSION
The goal of stem cell therapy is to repair damaged tissue that has lost the property to heal itself. This might be accomplished by transplanting stem cells into the damaged area and directing them to grow new, healthy tissue. Autologous bone marrow stem cells are a type of adult stem cells, a multipotent unit still capable of differentiating into only a few specialized cells. Studies investigating liver regeneration under conditions that preclude hepatocyte proliferation have reported the proliferation of a subpopulation of small, oval-shaped cells, initially in the portal triad.1 These cells, termed liver progenitor oval cells, have been shown to participate in liver regeneration in a variety of rodent models of chronic liver damage. They express markers common to hepatocytes and cholangiocytes, suggesting that they are a common precursor of both liver cell lineages.1 In a rat experiment of injection of bone marrow– derived cells native liver stem cells cause liver regeneration when proliferation of native hepatocytes is blocked by retrorsine. Mobilization from the bone marrow is a prerequisite, the mechanism of which is complex. In this study, histopathology could only be done at 6 months after the stem cell infusion, in the 3 patients who were alive with adequate follow-up. A comparative improvement in fibrosis was seen in all 3. Although the hypothesis for this documented improvement is lacking studies in humans, there is some evidence from experimental studies. After injecting bone marrow mesenchymal stem cells isolated from bone marrow of male donor mice into the liver remnants of hepatectomized female mice, Y chromosome and albumin positive cells were observed with increased liver weight in the transplanted mice.2 Thus bone marrow mesenchymal stem cells may be induced to differentiate into hepatocytes. Stem cells were used in spina bifida with neurological deficiency with an aim to repair the damaged neurons and improve the existing neurological deficits. Recovery of central nervous system disorders is hindered by the limited ability to regenerate lost cells, replace damaged myelin, and re-establish functional neural connections. Both hematopoietic stem cells and marrow stromal cells have been shown to have the potential to restore the injured spinal
702
cord and promote functional recovery in mice.3– 6 This positive effect was most pronounced using mesenchymal stem cells. Progressive complete functional motor recovery with evident nervous tissue regeneration has been achieved following administration of bone marrow stromal cells in traumatic central spinal cord cavities of adult rats with chronic paraplegia due to a previously injured spinal cord.7 In our experience with favorable responses in 50% of cases, it is hoped that stem cells may prove beneficial to improve the neurological deficits associated with cases of spina bifida. The hypothesis for the mechanism of action has been difficult to elucidate. A possible explanation has been that the stem cells provide a trigger factor for the growth of neurons. Thus bone marrow– derived stem cells may ultimately be optimal for spinal cord repair, if the mechanism of possible transdifferentiation can be elucidated.8 The fact that the stem cells reach the desired site of action has been shown by animal experiment although it is difficult to do in humans. To conclude, stem cell therapy may prove beneficial in cases of liver cirrhosis due to congenital anomalies by reversing or delaying the onset of end-stage liver disease and decreasing the need for liver transplantation and immunosuppression. The intial use of stem cells in meningomyelocele has shown promising results. However, longterm evaluation with randomized controlled trials is essential to draw conclusions. Major concerns remain: what prompts stem cells to assume specific functions and which factors would dictate them to stop multiplication once the
GUPTA, SHARMA, VENUGOPAL ET AL
aim is achieved. An uncontrolled proliferation may carry the risk of teratoma formation at any stage after stem cell therapy. The clinical scope of stem cell therapy could be endless. Only further research and wider clinical applications will address many practical and theoretical queries related to stem cells. REFERENCES 1. Matthews VB, Yeoh GC: Liver stem cells. IUBMB Life 57:549, 2005 2. Wu X, Zhao L, Xu Q, et al: Differentiation of bone marrow mesenchymal stem cells into hepatocytes in hepatectomized mouse. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 22:1234, 2005 3. Koda M, Okada S, Nakayama T, et al: Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice. Neuroreport 16:1763, 2005 4. Koshizuka S, Okada S, Okawa A, et al: Transplanted hematopoietic stem cells from bone marrow differentiate into neural lineage cells and promote functional recovery after spinal cord injury in mice. J Neuropathol Exp Neurol 63:64, 2004 5. Sykova E, Jendelova P, Urdzikova L, et al: Bone marrow stem cells and polymer hydrogels-two strategies for spinal cord injury repair. Cell Mol Neurobiol 26:1111, 2006 6. Zhao ZM, Li HJ, Liu HY, et al: Intraspinal transplantation of CD34⫹ human umbilical cord blood cells after spinal cord hemisection injury improves functional recovery in adult rats. Cell Transplant 13:113, 2004 7. Zurita M, Vaquero J: Bone marrow stromal cells can achieve cure of chronic paraplegic rats: functional and morphological outcome one year after transplantation. Neurosci Lett 402:51, 2006 8. Enzmann GU, Benton RL, Talbott JF, et al: Functional considerations of stem cell transplantation therapy for spinal cord repair. J Neurotrauma 23:479, 2006