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Journal of Indian Association of Pediatric Surgeons, Vol. 11, No. 4, October-December, 2006, pp. 211-217 Original Article Therapeutic use of stem cells in congenital anomalies: A pilot study Sharma Shilpa, Gupta DK, Venugopal P, Kumar Lalit, Dattagupta S, Arora MK Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi Code Number: ip06043 Abstract Introduction: Stem cells with potential to transform into healthy cells and repair damaged cells may prove beneficial in various congenital malformations.
Keywords: Biliary atresia, spina bifida, stem cell therapy INTRODUCTION The inciting factor leading to developmental arrest during the process of embryogenesis, and thus leading to various congenital anomalies has been far from understood in many anomalies. The surgical outcome in the best hands is far from optimal when compared to a normal child in some diseases like biliary atresia with hepatocellular failure and spinabifida with neurological deficit. This has called for a need to explore any means to continue the developmental process from the stage where it had been arrested or to reverse the changes caused by the anomaly to improve the functional status of the organs and tissues involved.PURPOSE OF THE STUDY It was thought 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. In such situations, there is nothing much left to offer for the full functional recovery. Stem cells with their growth potential of healthy cells, repair of damaged cells, and ability to transform into healthy cells may prove beneficial in the following diseases: Extra hepatic biliary atresia The outflow biliary channels from the liver are congenitally blocked thus pre-disposing to cirrhotic liver and hepatocellular failure. These children have a poor life span and only few reach adolescence. Spina bifida with neurological deficiency There is a defect in the midline in the back involving the bones, nerves, muscles, and skin. Though the muscle and skin defect can be repaired with surrounding tissues, there is often variable neurological deficit involving bladder, bowel, and one or both lower limbs. The child is thus crippled for life with these impairments and is a burden to the society and his family. The stem cells were used in liver cirrhosis and meningomyelocele with the following aims.
MATERIALS AND METHODS During the period from July 2005 to July 2006, stem cells were used in 27 patients; 12 with liver cirrhosis and 15 with meningomyelocele. A special informed and written consent was sought from the parents for this purpose. The stem cells were injected during definite surgery or post-operatively if the definitive surgery had been done earlier. No separate surgical procedure was required for offering the therapy, except to harvest the stem cells from the patient's own bone marrow. The bone marrow was collected under local anesthesia, from the tibia (autologous) in a volume varying from 3 to 25 mL (average 12-15 mL). Mononuclear cells were separated by a Ficoll-Hypaque gradient, washed, and resuspended. Flow cytometric enumeration of CD34+ cells was performed. The median number of CD34+ cells was 30x10 6 (20-60x10 6) in a concentration from 4-30x10 6 cells/mL with a 1-5 mL ready solution for infusion. Cell viability before transplantation was superior to 95% in all samples. The procedure from the bone marrow collection, stem cells harvestation, and transportation of stem cells to the operating theatre took 2-3 hours. Autologous stem cells were injected into the hepatic artery and portal vein or hepatobiliary radicles for liver cirrhosis or into the spinal cord and caudal space for meningomyelocele. Stem cells in extra hepatic biliary atresia (EHBA) were injected 20% in hepatic artery and 80% in the portal vein. The pre-operative status of the patient was taken to serve as the control for that patient. Post-operative monitoring included temperature charting and looking for any evidence of infection or other complications. Antibiotic coverage was given for 7-10 days for meningomyelocele and 3 months in biliary atresia. The patients with liver cirrhosis were evaluated by their clinical status, liver function tests, liver biopsy, and nuclear imaging. The patients with meningomyelocele were evaluated for their clinical status and muscle charting by two unbiased separate physiotherapists. They were examined for any change in power, sensations, 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 liver cirrhosis was due to EHBA; Obstructive cholangiopathy due to neonatal hepatitis and Choledochal cyst in 8; 2 and 2 patients, respectively. The male and female ratio was 1:1. Kasai and stem cell infusion was done in five cases, Cyst excision with hepaticoduodenostomy and cystoduodenostomy along with stem cell infusion were done each in two cases of choledochal cyst, flushing of the biliary tree with stem cell infusion was done in the two cases of neonatal hepatitis and only stem cells were given in three cases. There was smooth post-operative recovery in all of them with no post-operative complications noted. Five patients expired due to ongoing cirrhosis due to late presentation at 1-6 months after stem cell therapy. The 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 functions (6/7) and improved appetite (6/7) [Table - 1]. Hepatobiliary scan was excretory in 6/7 with improved uptake in 4/7. Histopathology repeated 6 months after stem cells in three patients demonstrated comparative improvement in fibrosis in all three. In one patient, it was again repeated at 12 months follow up and further improvement was noted [Figure - 1]. The ages of meningomyelocele patients were between 0 and 1 month; 1-5 months and 1-4 year in 5; 8 and 2 cases, respectively. Five had history of rupture and three had undergone meningomyelocele repair in the past but had neurological deficiency. Redo surgery for tethered cord was done in one, meningomyelocele repair and stem cells injection was done in 12, only stem cells injection was given in two. Two patients had only meningocele. There was smooth post-operative recovery in all of them with no complications noted. Follow up (3-11 months) in 14 cases showed improved power in four (28%), dramatic recovery in three (22%) and status quo in seven (50%) [Table - 2]. Dramatic recovery was taken as improvement in grade of motor power by more than two or improvement in bladder or bowel continence. One patient is still under observation. Out of seven patients with status quo, the motor power was already good in five. DISCUSSION Stem cell biology is a new field advancing at an incredible pace with immense potential. A stem cell is capable of forming various tissues under definite signals received from the body. The goal of stem cell therapy is to repair a 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. Many experiments have been done in animals using the embryonal stem cells with fruitful results. Harvested stem cells from umbilical cord blood or embryos require proper cross matching and may lead to complications like graft versus host reaction. Also harvesting stem cells from embryos involves ethical issues, as life is considered by some to begin at conception so it may be considered criminal to sacrifice an embryo for experimental and research purposes. With autologous infusion, there are no ethical issues involved with the stem cell therapy. However, being a new therapy with no definite known long term results, the detailed procedure of the stem cell therapy, its source, the route and the possible future impact was explained to each and every patient/parents and a special informed consent was obtained. Autologous bone marrow stem cells are a type of adult stem cells, a multipotent cell, still capable of differentiating into only a few specialized cells. Evidence suggests that, given the right environment, some adult stem cells are capable of being 'genetically reprogrammed' to generate specialized cells that are characteristic of different tissues. This phenomenon is termed adult stem cell plasticity or trans-differentiation. The stem cells were injected at the desired action site for achieving maximal concentration at the target site during the proposed definitive surgery. Thus they were injected in the 20% in hepatic artery and 80% in the portal vein in EHBA taking into consideration the normal blood flow to the liver. In cases in which Kasai had been done previously, they were injected into the hepatobiliary radicles. The site for injection in meningomyelocele cases was into the spinal cord and caudal space. Biliary atresia is an infantile serious condition associated with obliteration of extra hepatic biliary ducts, cirrhosis, portal hypertension, and hepatocellular failure. Meningomyelocele involves defects of the skin, muscles, bone and spinal cord in the midline on the back of varying degree, resulting in neurological deficits involving the urinary bladder, bowel and lower limbs. These conditions are considered to be an arrest in the developmental process. Surgical corrections are being done but with poor outcome in terms of liver cirrhosis in EHBA and irreversible neurological deficit in meningomyelocele. Thus we selected these two areas for the initial use of stem cells. Bone marrow hematopoietic stem cells have been shown to facilitate regeneration in multiple non-hematopoietic tissues by either generating epithelial cells by spontaneous cell fusion or trans-differentiation or altering the inflammatory response.[1] The stem cells were used in cases of liver cirrhosis due to biliary atresia with the aim to prevent further degeneration of hepatocytes, regenerate new hepatocytes and to reverse the degeneration of hepatocytes. Studies investigating liver regeneration under conditions that preclude hepatocyte proliferation report the proliferation of a subpopulation of small, oval-shaped cells, first observed in the portal triad.[2] These cells, termed liver progenitor oval cells are 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 they are a common precursor of both liver cell lineages.[2] Supporting evidence for liver stem cells has also come from cell tracing studies that show transdifferentiation of bone marrow cells into hepatocytes in both human and animal models.[2] Liver regeneration has been shown to occur via native liver stem cells in a rat experiment after injection of bone marrow derived cells by blocking the proliferation of native hepatocytes by retrorsine.[3] Though macrophage fusion, hematopoietic stem cell fusion and transdifferentiation can all contribute to hepatic epithelial lineages, a greater understanding of the factors that regulate the long-term efficacy is needed.[4] Mobilization from the bone marrow is a prerequisite, the mechanism of which is complex. Murine and human studies have shown that the chemokine SDF-1, a potent hematopoietic stem cell chemoattractant produced by the liver and its receptor CXCR4 participate in the mobilization of bone marrow hematopoietic stem cells and migration to injured liver.[1] Additional factors implicated in hepatic migration of hematopoietic stem cells include IL-8, hepatocyte growth factor and MMP-9.[1] Partial hepatectomy in humans has been shown to induce mobilization of a distinct population of myelomonocytic progenitor cells, which have hepatic differentiation potential in vitro and might play a role in liver regeneration.[5] In this study, the 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 functions (6/7) and improved appetite (6/7). Five patients expired at 1-6 months after stem cell therapy due to ongoing cirrhosis. This was due to the fact that most of the cases in this study came late when cirrhosis had already set in. One of these was a case of forme fruste choledochal cyst presenting at the age of 7 months with cirrhotic liver. These patients were not ideal candidates for the definitive surgery. Hepatobiliary scan was excretory in 6/7 with improved uptake in 4/7.The histopathology was repeated at 6 months after the stem cell infusion. It could be done in only three patients that were alive with a follow up of at least 6 months after stem cells. A comparative improvement in fibrosis was seen in all three. In one patient, the histopathology was again repeated at 12 months follow up and further improvement was noted [Figure - 1]. Though the hypothesis for this documented improvement is lacking from studies in humans, there is enough evidence from experimental studies. It has been shown that hematopoietic stem cells become liver cells when cocultured with injured liver separated by a barrier.[6] Wu et al. injected bone marrow mesenchymal stem cells isolated from bone marrow of male donor mouse into the liver remnants of hepatectomized female mouse and found Y chromosome and albumin positive cells with increased liver weight in transplanted mouse.[7] Thus bone marrow mesenchymal stem cells could be induced to differentiate into hepatocytes.[7] Hepatocytes cocultured with rat bone marrow stoma cells have been found to secrete significantly more albumin than those in monoculture in vitro .[8] Bioencapsulated bone marrow stem cells transplanted intraperitoneally into 90% hepatectomized rats have been found to increases both the rates of hepatic regeneration and survival of the animals.[9] However, these animal studies can not be directly applied to cases of EHBA as the process of insult to the liver started at the antenatal stage and much irreversible damage has already been done till the baby presents for definitive surgery. This pilot study serves as only a ray of hope in this direction, whether it is twilight or dawn will only be unraveled with further studies. Stem cells were used in spina bifida with neurological deficiency with an aim to repair the damaged neurons and improve the existing neurological deficit. Bone marrow derived stem cells have great potential as therapeutic agents, since they are easy to isolate and can be harvested from patients without serious ethical and technical problems Recovery in central nervous system disorders is hindered by the limited ability of the vertebrate central nervous system 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 cord and to promote functional recovery in mice.[10],[11] Fluorescent in situ hybridization for the Y chromosome and double immunohistochemistry showed that transplanted cells survived 5 weeks after transplantation and expressed specific markers for astrocytes, oligodendrocytes, and neural precursors, but not for neurons.[10] It has been shown that progressive complete functional motor recovery with evident nervous tissue regeneration can be achieved over a course of time following the administration of bone marrow stromal cells in traumatic central spinal cord cavities of adult rats with chronic paraplegia due to previously injured spinal cord.[12] Injection of autologous expanded mesenchymal stem cells into the spinal cord of amyotrophic lateral sclerosis patients has been shown to be safe, with no significant acute or late toxicity and well-tolerated with encouraging clinical results.[13] In this study, follow up (3-11 months) in 14 cases showed improved power in four (28%), dramatic recovery in three (22%) and status quo in seven (50%) [Table - 2]. One patient is still under observation. Out of the seven patients with status quo, the motor power was already good in five. These results suggest 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 the trigger factor for the growth of the neurons. Thus bone marrow derived stem cells may ultimately be optimal for spinal cord repair, if the mechanism of possible transdifferentiation can be elucidated.[14] The fact that the stem cells do reach the desired site of action has been proved by animal experiments though it is difficult to do so in humans. Transplantation of human neural stem/progenitor cells into the injured spinal cord in primates has been shown to promote functional recovery and histologically differentiate into neurons, astrocytes, and oligodendrocytes.[15] Mesenchymal stem cells delivered by lumbar puncture have been shown to reach the contused spinal cord tissues and exerted a significant beneficial effect by reducing cyst and injury size in rats.[16] This study has been recently applied safely to humans by injecting autologous bone marrow precursor cells into the spinal cord via lumbar puncture in patients with spinal cord injury without any adverse effects. The cerebrospinal fluid examination repeated after 7 days demonstrated absence of mononuclear cells thought to be due to homing of cells toward injured spinal cord.[17] CONCLUSION Stem cell therapy may prove beneficial in cases of liver cirrhosis due to extra hepatic biliary obstruction by reversing or delaying the onset of end stage liver disease and thus decreasing the need of liver transplantation. Autologous bone marrow stem cells have no ethical issues involved and do not require immunosuppressants as required for liver transplantation. The initial use of stem cells in meningomyelocele has shown promising results. However, long-term evaluation with randomized-controlled trials is essential to draw further conclusions. Major concerns still remain that what prompts the stem cell to take up a specific function and what are the factors that would dictate to stop its multiplication once the aim is achieved. An uncontrolled proliferation may carry the risk of teratoma formation at any stage after stem cell therapy. The clinical scope of the use of stem cell therapy could be endless. Only further research and its wider clinical application will solve many practical and theoretical queries related to the use of stem cells. References
Copyright 2006 - Journal of Indian Association of Pediatric Surgeons The following images related to this document are available:Photo images[ip06043t1.jpg] [ip06043f1.jpg] [ip06043t2.jpg] |
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