Introduction
Niemann Pick (NP) diseases is a group of autosomal disorders caused by a series of mutations of the sphingomyelin phosphodiesterase-1 gene (SMPD1) which encodes the acid sphingomyelinase, (ASM) involved in the degradation of sphingomyelin (1). The acid sphingomyelinase defect leads to the accumulation of sphingomyelin in the cells of the liver, spleen, bone marrow, lungs, and in some patients, brain (2–5).
Niemann Pick type A is a severe neurodegenerative disease with little or no enzyme activity; it develops in infancy with abdominal enlargment due to hepatosplenomegaly, feeding difficulties, cherry red macula. In addition it envolves with progressive loss of acquired motor skills. The increased accumulation of sphynomyelin in ganglion cells of the central nervous system leads to neurologic disturbances and mental retardation generally resulting in death by 3 years of age (6, 7).
Niemann Pick type B is a slow paced disease with the same gene defect but it has more residual enzyme activity. The disease develops in pre-teen years with the enlargment of the liver and spleen; while in adulthood pulmonary difficulties and ataxia are the major complications although the CNC is not involved (8).
So far there are no specific pharmacological treatments that assure a cure for these diseases.
However since 1986 we have cured a series of patients with Niemann Pick type B by means of a subcutaneous injection of amniotic membrane cells (9). Amniotic membranes share many features with mesenchymal cells.
Recently we put forward a child with Niemann Pick type A for a bone marrow transplant, who had failed to improve lysosomal storage in his organs and to slow down the worsening of the CNS symptoms.
Later we infused I.V. and intrathecally the mesenchymal cells isolated from the marrow of the same donor. The result was then very interesting.
Patient’s history
A 10 month old child was admitted to the Bone Marrow Transplantation Department of Trieste in 2008. He suffered from a rapid worsening of psychomotor skills; he was not able to sit down, or even to keep his head in an upright position unaided. The liver and spleen were enlarged.
In the bone marrow smear and in the liver biopsy there were large foamy cells typical of Niemann Pick disease. A defect of sphyngomyelinase was demonstrated in the leukocytes.
The parents consented to a BMT even though they were aware of the slim chance of success.
Bone marrow transplantation
Since there was no HLA compatible sibling, and the search of a matched unrelated donor (MUD) would have taken too much time, we chose his haploidentical father as a donor, given the patient’s rapidly deteriorating condition. The conditioning regimen was: fludarabine, busulphan, thiotepa, cyclophosphamide and ATG.
The donor’s marrow was treated with vincristine and methylprednisolone as has been performed in Trieste since 1986 for mismatched BMTs (10).
The engraftment of the marrow was rather quick, with minor problems of GVHD (grade 1). The engraftment of the donor’s cells was confirmed by means of HLA control and DNA polymorphism.
Unfortunately after three months the size of the liver and spleen was even larger. In the liver biopsy and in the marrow smear there were still foam cells. The number of platelets after a first growth not sustained by transfusions fell and never exceeded 20,000/microl.
At that moment we, along with the parents, decided to proceed with a further attempt with mesenchymal cells.
Cellular therapy
A large core (“carrot”) of bone marrow stroma cells was harvested from the iliac crest of the father.
The spongy bone sample was fragmentated with surgical forceps in PBS. The cell suspension was then centrifugated and the pellet resuspended in culture medium (D-MEM, 10% fetal bovine serum (FBS), glutamine and antibiotic/antimycotic. The mixture was distributed between four 75 cm2 Falcon flasks and incubated in a CO2 incubator for 24 hours. Then the non adherent cells and bone fragments were poured out and the adherent cells re-suspended in culture medium which was cultivated for three weeks. The medium was changed every three days. At the end the cells were detached from the plastic wall with trypsine, washed and cryopreserved in aliquots in a 10% DMSO solution.
This system of harvest and the culture allowed elimination of all the haemopoietic stem cells, and at the end of the culture the phenotype of the cells was CD90+, CD73+, CD 105−, CD44+, CD31−, CD34−, CD45−. We underline that CD105 is usually weak/negative in the first three weeks of culture, while it becomes positive in the fourth week.
On the day of the treatment one aliquot was washed twice with PBS and used for IV injection; two million cells were diluted in 50 ml of PBS and infused IV within 10 minutes.
Another aliquot was washed, resuspended in PBS and 10% patient’s serum, and incubated for 40 mins with 6 ml of a solution of retinoic acid in ethanol (10 mg in 10 ml).
This incubation started the initial differentiation of the stem cells into the neurological line, as demonstrated by means of the expression of neurological markers (assay at 2 hours) as III tubulin and neurofilaments M (NF-M) in real time experiments with reverse transcriptase.
After incubation, the cells were centrifugated and re-suspended in 2 ml of PBS solution, and immediately injected into the lumbar spine of the patient.
The same treatment was repeated after 2 months.
Outcome
The patient had no untoward effects due to these injections.
After 25 days the number of platelets rose to > 100,000/microl. The size of the liver and spleen was reduced to one half.
After one month the child was able to keep his head in an upright position unaided. His relationship with his parents improved and the achievements brought him up to a psychomotor level compatible with a child of 7–8 months old.
We did not consider it ethically acceptable to perform a further liver biopsy.
We were not able to continue the treatment for 2 years. The psychomotor skills maintained a plateau.
After 2 years the patient showed a clear worsening mainly from the neurological perspective. He became unable to swallow and was then fed by naso-gastric tube. An anesthesiologist refused to put him under anesthesia to insert a PEG, due to the high anesthesiological risk.
In 2012, in the Hospital of Brescia, he restarted another cycle with intratecal and I.V. mesenchymal cells. After one month from the first injection he was able to swallow normally, and he is currently fed with a spoon. The liver and spleen remain within normal size.
His psychomotor skills (movements, relationship with the parents) improved, but he still remains a severely handicapped child.
The injections continue every two months; to date he has had 4 injections of the new series.
No untoward affect was seen after this treatment.
Discussion
We understand that this patient was submitted to the treatment with mesenchymal cells too late. Maybe if we had chosen this treatment much earlier the outcome would have been different. The effect of mesenchymal cells lasted less than 2 years, after which the symptoms reappeared. The treatment was given again, with significant - but not complete- results. Since we used the father as a donor, this could be a particular situation from the immunological aspect, maybe more favourable than in cases when there could be an HLA mismatch. However, since these cells have no HLA-DR on their surface and are immunosuppressive of their own, the choice of the donor may have been irrelevant.
The crucial point of our method seems to be the short incubation with retinoic acid, which starts a differentiation along the neurological line, without achieving the complete maturation expected to occur in vivo. As long as the cells maintain the features of stem cells, they are able to move across the blood brain barrier and to “feel” the chemical messages of cellular damage. Mature neurons are unable to move more than one mm from the site of injection (11).
Our method seems much more efficient than treatments that use no differentiated mesenchymal cells in neuro-degenerative diseases (12).
Cellular therapy with mesenchymal cells could be the right answer for the treatment of Niemann Pick diseases, but even other storage diseases not yet curable by gene therapy or enzyme replacement could deserve such an innocuous treatment.
