Journal of Stem Cells & Regenerative Medicine - Volume 3 Issue1; 2007

Evolving science enhanced with iPS

Webmaster-JSRM — 30 Oct 2007 04:46:03 GMT

Journal:
2007 Vol. 3 (1): p1

Published on:
Nov 2007

Author(s):
Editorial

Dear Friends,

Greetings from all in the team. With the stage set for online submissions and the review-response-revision-resubmission process standardized, we have come with the first regular issue and from now there will be quarterly issues of the journal.

Since the starting of the JSRM in a short span there have been a lot of developments, which we would rather say as "evolutions" keeping in mind, the recent iPS! This evolution we would like you to see from a background of the various developments in the art and science of medicine throughout in the past three centuries. We have come across the era of investigative tools such as bamboo made laryngoscopes to era of vaccines and antibiotics followed by the era of revolutionary non-invasive procedures and recently the nano technology based drugs and now the iPS! Macro to Micro, but still more to go.

All through the influence of the society, religions, philosophies have been playing a very important role in every step the science of biology moves ahead. Starting with the contraception, assisted reproduction then the gene modified plants....and now the embryonic stem cells! With the advent of the iPS, though the issues of oncogenes, teratoma yet to be ruled out, we have found there is a way which can bypass the ES cells! Hats off to those scientists who have burnt their midnight oil to have found this way out!

The lesson we learn is to explore things with an open mind and continue to proceed further without spending much time fingers crossed.

Happy reading!

Yours sincerely,

The Editorial team.

Stem cell therapy for neuropathic pain treatment

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p2-11

Published on:
Oct, 2007

Author(s):
Dario Siniscalco, Francesco Rossi, Sabatino Maione

Abstract:
Pain initiated or caused by a primary lesion or dysfunction in the nervous system is defined as neuropathic pain.

About 75 -150 million people in the United States are suffering for chronic pain disorder. Neuropathic pain has a great impact on the human wellbeing. It is very debilitating and often has an associated degree of depression that contributes to decreasing the quality of life. Moreover, the management of chronic pain is costly to the health care system. Pain is a national healthcare priority in US: the United States Congress has declared the present decade (2001-2010) as the "Decade of Pain Control and Research".

Neuropathic pain is a very complex disease, involving several molecular pathways. Due to its individual character, its treatment is extremely difficult. Current available drugs are usually not acting on the several mechanisms underlying the generation and propagation of pain.

Nowadays, pain research is focusing on newer molecular ways, such as stem cell therapy, gene therapy, and viral vectors for delivery of biologic anti-nociceptive molecules. These methods could provide a new therapeutic approach to neuropathic pain relief.

Key words:
neuropathic pain, stem cell therapy, gene therapy, virus vector.

Simple Laboratory methods to measure cell proliferation using DNA synthesis property

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p12-14

Published on:
Oct, 2007

Author(s):
Madhavan H.N

Introduction

This is a mini-review on the techniques to measure proliferation of cells by estimation of DNA synthesis. This is not an exhaustive review of literature, but a bird's eye view of a few selected articles which may provide the technical details to the readers.

The nucleus of a cell occupies about 10-30% of the cells space, depends on the type of genetic material (DNA -DeoxyriboNucleic Acid). DNA is a long, double-stranded, helical molecule which carries the genetic information. Duplication of the DNA takes place by the phenomena of replication. One copy of double-stranded DNA molecule forms two double-stranded DNA molecules. DNA replication is the fundamental process used in all living organisms as it is the basis for biological inheritance. This process is known also as Mitosis in somatic cells. In Mitosis, the duplication process results in two genetically identical "daughter" cells from a single "parent" cell. The resulting double-stranded DNA molecules are identical; proof reading and error-checking mechanisms exist to ensure near perfect pair. Mitosis is divided into six phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

Methods of detecting and assay of DNA synthesis

In a cell, DNA replication must happen before cell division can occur and the biochemical pathway correlates well with DNA synthesis which is relatively specific for cell division. Measurement of new DNA synthesis is, therefore, essentially synonymous with measurement of cell proliferation. Direct measurement generally involves the incorporation of a labeled nucleoside into genomic DNA. Examples include the tritiated thymidine ([3H]dT) and BrdU (bromodeoxyuridine) methods 1, 2. Radioactive tagging of newly synthesized DNA with 3H-labeled Thymidine (3H-T) is most frequently applied technique. In recent days, usage of BrdU non radioactive labeling has increased. Gratzner in 1982 2 developed a monoclonal antibody to BrdU and for identifying BrdU-labeled S-phase cells with immunoperoxidase, immunofluorescence, or avidin-biotin complexes 3, 4, 5, 6. Monoclonal antibody (MAb) techniques for detection of BrdU have the advantages of simplicity and speed over standard autoradiography. Therefore, for a long period of time the cell biologists have been using the technique of thymidine labeling, whereby a group of chemicals such as BrdU are incorporated into the DNA of dividing cells. The advantages of non-radioactive immune techniques using BrdU or anti-BrdU are undisputed but the scope and limitations of the systems for detecting BrdU cannot always be clearly defined. Besides enabling microscopic detection at the cellular level, the enzyme immune assay technique represents a method for routine screening. Two different detection systems are available: 1) the conventional colorimetric test and 2) a chemiluminescence assay also based on the anti-BrdU technique. One of these test methods will be suitable for use depending on the problem and the available laboratory facilities.

Burns et al, 2005 7 showed evidence for BrDU labeled dead cells after introducing them into the brains of mice. BrdU is used in experiments to mark cell splitting at a particular time. This helps researchers to examine where those cells migrated and what new cell types they differentiated into. It has been shown that cells from one tissue marked with BrdU extracted and were transplanted into a second tissue with the assumption that the host cells had taken up the cells which could later be identified. Experimental evidences suggests that many thymidine-labeled stem cells die after being transplanted (as done for studies on stem cell transplantation research where many labeled stem cells die after being transplanted into the mice brain7). The released chemical were taken up by neighbouring cells, (dividing and non-dividing) which may be mistaken as transplanted cells. 7, 8

The tritiated thymidine ([3H]-T) labeling index has been used for estimation of the proportion of S-phase cells in asynchronous cell populations as described by Steel 9. Most investigators have found that BrdU has been equally good as [3H]-T in providing assays of S-phase fractions in labeling-index assays and by bivariate flow cytometry 10, 11, 12. Incorporation of [3H]-T and BrdU were studied which showed, the same cells developed positive for both precursor analogues. 13, 14 Monoclonal antibody (MAb) techniques for detection of BrdU have the advantages of simplicity and speed over standard autoradiography required for radio-isotopes as used in 3H Thymidine technique. Lin& Allison (1993) stated that simultaneous labeling of cells with BrDU and [3H]-T was rather difficult because both these precursor analogues were incorporated by the salvage pathway which was the competitive binding for Thymidine kinase. 15 This study shows that Feulgen hydrolysis denatured the DNA of fixed cells sufficiently to allow detection of incorporated BrdU with monoclonal antibodies. Cells were then double-labeled with [3H]-T and BrdU, placed on slides, and Feulgen stained. The absorption cytometry of DNA content was measured on randomly selected cells. The removal of the BrdU or r31-4T grains after DNA measurements did not interfere with subsequent detection of the grains from the other label. BrdU and r3HJ-T can be used reliably in combination for identification of S-phase cells. This method eventually allows the microscope-based image analysis for selectively measuring DNA contents.

Macallan and colleagues developed a method for measuring DNA synthesis analyzing within the cells. 16 It consisted of administering [6, 6-2H2] Glc or [U-13C] Glc, in this method genomic DNA was isolated, enzymatically hydrolyzed to free deoxyribonucleosides and it was prepared for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) revealed the fraction of newly synthesized DNA, by application of the precursor-product relationship. The technique was different from the widely used [3H]thymidine or BrdU techniques in the de novo nucleotide synthesis pathway, rather than the nucleoside salvage pathway was used to label DNA and deoxyribose rather than the base moiety was labeled; purine rather than pyrimidine deoxyribonucleosides were analyzed; and stable isotopes rather than radioisotopes were used. The method can be applied for both in-vitro (cell culture) and in-vivo (animal model). This method has several advantages over previously available techniques for measuring the cell turnover which involves no radioactivity or potentially toxic metabolites, and is suitable for use in humans. The availability of a reliable and safe method for measuring cell proliferation in humans opens up a number of fundamental questions to direct experimental testing, including basic problems related to cancer, AIDS, and other pathologic states.

Conclusion

This is a small review which may be helpful for the beginners, to learn technical details.

References

1 . Waldman, F. M., Chew, K., Ljung, B. M., Goodson, W., Hom, J., Duarte, L. A., Smith, H. S. & Mayall, B. 1991 Mod. Pathol. 4, 718-722.

2 Grattner HG. Monoclonal antibody to >-bromo- and -iododeoxyuridine: a new reagent for detection of DNA replication. Science 1982;218:47

3. Gundut N. The use of FIX-conjugated monoclonal antibodies for determination - of S-phase cells with fluorescence microscopy. Cytometry 1985;6:597

4. Sasaki K, Ogino T, Uash M. Immunological determination of labeling index on human tumor tissue sections using monoclonal anti-BrdUrd antibody. Stain Techno1 1986;61:155

5. Langer EM, Rottgers HR, Schliermann MB, Meier EM, Miltenburger HG, Schumann J, Gohde W. Cyding S-phase cells in animal and spontaneous tumours. Acta Radio1 Oncol 1985;24:545

6. Wilson GD, McNally NJ, Dunphy E, Karcher H, Pfragner R. The labeling
index of human and mouse tumours assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry. Cytometry 1985;6:641

7. Burns TC, Ortiz-Gonzalez XR, Gutierrez-Perez M, Keene CD, Sharda R
Demorest ZL, Jiang Y, Nelson-Holte M, Soriano M, Nakagawa Y, Luquin MR, Garcia-Verdugo JM, Prosper F, Low WC, Verfaillie CM. Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution. Stem Cells. 2006; 24:1121-7.

8. Kuan CY, Schloemer AJ, Lu A, Burns KA, Weng WL, Williams MT, Strauss KI, Vorhees CV, Flavell RA, Davis RJ, Sharp FR, Rakic P. Hypoxia-ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain. J Neurosci. 2004; 24:10763-72

9. Steel GG. Growth kinetics of tumours. Oxford: Clarendon Press, 1977

10. Gundut N. The use of FIX-conjugated monoclonal antibodies for determination of S-phase cells with fluorescence microscopy. Cytometry
1985;6:597

11. Meyer JS, Coplin MD. Thymidine labeh index, flow CytomeUic S-phase
measurement, and DNA index in human tumors. Am J Clin Pathol 1988;89:586

12. Meyer JS, Nauert J, Koehm S, Hughes J. Cell kinetics of human tumors
by in vitro bromodeoxyuridine labeling. J Histochem Cytochem 1989;37:1449

13. Hume WJ, Thompson J. Double labeling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine:
the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously. Cell Tissue King 1989;22:393

14. Thornton JG, Wells M, Hume WJ. Measurement ofthe S-phase duration in normal and abnormal human endometrium by in vitro double labeling with bromodeoxyuridine and tritiated thymidine. J Pathol 1989;157:109

15. Lin P, Allison DC. Measurement of DNA Content and of Tritiated Thymidine and Bromodeoxyuridine Incorporation by the Same Cells J Histochem. Cytochem 41:1435-1439, 199

16. Macallan DC, Fullerton CA, Neese RA, Haddock K, Park SS, Hellerstein MK. Measurement of cell proliferation by labeling of DNA with stable isotope- labeled glucose: Studies in vitro, in animals, and in humans Proc. Natl. Acad. Sci.- 95, 708?713, 1998.

Bone marrow stem cell injection for the treatment of critical limb ischemia

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p15-18

Published on:
Oct, 2007

Author(s):
Shunya Shindo

Background

Vascular reconstruction remains a treatment of choice for critical limb ischemia. Bypass surgery has been accepted as the most effective therapy to achieve increase of blood flow and cure the ischemic symptoms. On the other hand, continuous increase of the patients suffering diabetes mellitus enhances the increase of the number of the patients with peripheral arterial disease (PAD). This tendency is also observed in Japan and expected to continue from now on. The reports of the Ministry of Health, Labor and Welfare of Japan indicated that more than 6% of population was treated for diabetes mellitus and more than 12% of population was supposed to be diabetic in 2002.1) This report also gave a warning for rapid and continuous increase of the diabetic patients at present and in the future.? As is well known, diabetes mellitus is one of the strong risk factors to cause an arteriosclerosis obliterans (ASO). Therefore, the patients with PAD will increase continuously and more severe ischemic limb will need to be treated. Moreover, diabetic macroangiopathy is characterized as showing diffuse stenosis and occlusion down to the foot arteries.2) Monckeberg's medial calcification is also frequently observed in the ASO with diabetes mellitus.3) Therefore, along with the increase of the patients with PAD, the vascular reconstruction becomes technically demanding due to co-morbid diabetes with diffuse lesion and severe calcification. The patients, who show critical limb ischemia but are excluded from the operative candidate, are eventually performed major amputation. To prevent this disastrous sequel, therapeutic angiogenesis has been investigated.

In 1996, Isner reported a new therapy to treat ischemic limb by using angiogenetic gene; vascular endothelial growth factor (VEGF).4) Since then, angiogenetic therapy started to be investigated by using gene transfer with VEGF, fibroblast growth factor (FGF)5) or hepatic growth factor (HGF).6) Animal study showed increase of the vascular network, disappearance of local coldness and recover of limb function.7,8) Human study also started by using these genes.? Major concern to use gene transfer is possible infection or carcinogenic effect via adenovirus which is used as a vector. To avoid this problem, plasmid is also used although the efficacy of transfer is less. The human application revealed that skin temperature increased, pain was cured and vascular network seemed to increase in angiography. However, the long-term effect remains undetermined.

Bone marrow monocyte injection

In place of gene transfer, bone marrow stem cell has been studied whether causing angiogenesis or vasculogenesis. Angiogenesis, which means development from capillary endothelial cells, has been considered as a main cause to increase blood vessels in the adults. On the other hand, angioblast or endothelial progenitor cells (EPC), which differentiates to the endothelial cells, enhances vasculogenesis in the embryogenic stage. These cells surround hematopoietic cells and form a blood island, then develop to blood vessels in the embryo.9) Bone marrow monocytes were reported to include these cells even in the adults. Therefore, vasculogenesis, the development of vascular network independent to capillaries, could occur in the adults.10) They also secrete angiogenetic cytokines such as VEGF or FGF.11) From these observations, injection of the monocytes collected from the bone marrow was expected to cause vasculogenesis and angiogenesis in the ischemic limb. The animal experiment using EPC demonstrated occurrence of angiogenesis and development of collateral flow in the ischemic hind limb of the rats.12) The advantages using bone marrow monocytes are that infection or immune reaction can not occur different from the technique using gene transfer. The disadvantage is necessity to collect bone marrow blood with a volume of 500 to 600ml under general anesthesia. The EPC and angioblast also exists in the peripheral blood, so they could be collected by using apheresis.13) However, the numbers of these cells are much lower than in the bone marrow and the efficacy of collection is less. To investigate the effects to human ischemic limb by bone marrow monocyte injection, Japan Trial for Therapeutic Angiogenesis Using Cell Transplantation (J-TACT) started since 2000.14) Our institute also started therapeutic angiogenesis to use bone marrow monocytes since 2004, independently.

Methods

The patients who are scheduled to be treated by angiogenetic therapy using bone marrow monocyte are fully consent before treatment. Those who show critical ischemia in the extremities and are diagnosed as inoperable are candidates for this therapy. The angiography is performed to clarify the vascular network in the ischemic limb. The patients are also examined for cancer or diabetic retinopathy to exclude adverse side effects. Those who have these diseases or other severe morbidity are excluded because this treatment may worsen the disease. After the ethical committee of each institute agrees, angiogenetic therapy starts. The bone marrow blood is collected from the iliac bone of the patient under general anesthesia. Usually the necessary volume of the collected blood is from 500ml to 600ml. Then this blood is transferred to the laboratory, and monocytes are collected and purified from the bone marrow blood. Usually 1.0x109 of monocytes is obtained. These monocytes are divided to small syringes, and then injected to the muscles of ischemic limb.

Results

The results of J-TACT demonstrated the increase of ankle brachial pressure index (ABPI), increase of walking distance with tread-mill and relief from pain with significant difference in comparison with the control group.14) ABPI increased 0.097 in the injection group and 0.024 in the control group. Walking distance increased to 2.6 times and pain relief was observed in 90% of the study group. However, in the midterm at 2 years, these parameters decreased again to the pretreatment value. Therefore, the effects may discontinue at least in 2 years and the long-term results remained undetermined. In our institute, 4 patients were treated with bone marrow-derived monocyte injection as a therapeutic angiogenesis; 3 patients are effective and 1 patient is not effective in the short term. The all effective cases were operated for inflow reconstruction and received concomitantly monocyte injection to the area of non-operative below-knee lesion. As the increase of ABPI after therapeutic angiogenesis was slight, the combination of the angiogenetic therapy and concomitant inflow reconstruction seemed to be effective from these clinical experiences

At present and in the future

As the results showed, therapeutic angiogenesis using bone marrow monocytes seemed to be effective in the short-term. The pain relief and prolongation of walking distance are impressive, while increase of ABPI is slight. Angiography after monocyte injection seemed to show the increase of the vascularity in the ischemic leg, but it is unclear whether it is significant or not because of the difficulties of quantification of angiographic demonstration. With regard to pain, relief does not necessarily mean the increase of blood flow. Cytokines secreted from monocytes may lower the threshold of the pain through direct effect to the nerve. The continuation of the effects by bone marrow monocyte injection remains unclear especially in the long-term. Theoretically, the vasculogenesis around the injection field does occur. However, the conjunction from the newly formed vessels to the inflow artery is necessary for the certain increase of the blood flow. It has not been proven yet whether this phenomenon occurs regularly or not by angiogenetic therapy. Otherwise, the formation of the new vessels may be temporary phenomena, similar to inflammation.

The increase of the blood flow after bypass surgery has been recognized as the most effective treatment for the ischemic leg. The vascluogenetic therapy by the bone marrow monocyte injection could not achieve as much effects as vascular reconstructive surgery. Therefore, the vasculogenetic therapy alone is considered not to increase the blood flow enough to cure the severe ischemia completely. The combination of inflow reconstruction by the bypass surgery and therapeutic angiogenesis to the inoperative distal area would be a realistic application at present. The future investigation should focus on how to connect the new vessels formed by vasculogenesis to the inflow artery; the development from vasculogenesis to arteriogenesis. Otherwise, the long-term effect of the present vasculogenetic treatment could not prove the clinical value, whether the gene transfer or bone marrow stem cell injection is applied.

References

Reports of surveillance of diabetes mellitus in Japan in 2002. The reports of the Ministry of Health, Labor and Welfare of Japan, 2004.

Joseph WS, LeFrock JL. The pathogenesis of diabetic foot infections-immunopathy, angiopathy, and neuropathy. J Foot Surg 26;S7-11, 1987.

Ballota E, Renon L, Toffano M, et al. Patency and limb salvage rates after distal revascularization to unclampable calcified out flow arteries.? J Vasc Surg 39;539-546, 2004.

Isner JM, Pieczek A, Schainfeld R, et al. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patients with ischaemic limb. Lancet 348;370374, 1996.

Masaki I, Yonemitsu Y, Yamashita A, et al. Angiogenetic gene therapy for experimental critical lomb ischemia; acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2. Circ Res 90;966-973, 2002.

Morishita R, Aoki M, Hashiya N, et al. Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease. Hypertension 44;203-209, 2004.

Takeshita S, Zheng LP, Brogi E, et al. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invst 93;662-670, 1994.

Banai S, Jaklitsch MT, Shou M, et al. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation 89;2183-2189, 1994.

Risau W.? Mechanism of angiogenesis. Nature 386;671-674, 1997.

Shintani S, Murohara T, Ikeda H, et al. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation 103;897-903, 2001.

Rehman J, Li J, Orschell CM, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation 107;1164-1169, 2003.

Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 105;1527-1536, 2000.

Minamino T, Toko H, Tateno K, et al. Peropheral-blood or bone-marrow mononuclear cells for therapeutic angiogenesis Lancet 360;427-435, 2002.

Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischemia by autologous transplantation of bone-marrow cells; a pilot study and a randomized controlled trial. Lancet 360;427-435, 2002.

Bioengineering of cultured epidermis from adult epidermal stem cells using Mebio gel suitable as autologous graft material

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p19-20

Published on:
Oct, 2007

Author(s):
Lakshmana K Yerneni

Closure of burn wound is the primary requirement in order to reduce morbidity and mortality that are otherwise very high due to non-availability of permanent wound covering materials. Sheets of cultured epidermis grown from autologous epidermal keratinocyte stem cells are accepted world over as one of the best wound covering materials. In a largely populated country like ours where burn casualties occur more frequently due to inadequate safety practices, there is a need for indigenous research inputs to develop such methodologies. The technique to culturing epidermal sheets in vitro involves the basic Reheinwald-Green method with our own beneficial inputs. The technique employs attenuated 3T3 cells as feeders for propagating keratinocyte stem cells that are isolated from the epidermis of an initial skin biopsy of about 5 cm2 from the patient. The cultures are then maintained in Dulbecco's modified Eagle's medium strengthened with Ham's F12 formula, bovine fetal serum and various specific growth-promoting agents and factors in culture flasks under standard culture conditions. The primary cultures thus established would be serially passaged to achieve the required expansion. Our major inputs are into the establishment of (1) an efficient differential trypsinization protocol to isolate large number epidermal keratinocytes from the skin biopsy, (2) a highly specific, unique and foolproof attenuation protocol for 3T3 cells and (3) a specialized and significant decontamination protocol. The fully formed epidermal sheet as verified by immuno-histochemical and light & electron microscopic studies, is lifted on to paraffin gauze by incubating in a neutral protease. The graft is then ready to be transported to the operating theatre for autologous application. We have a capability of growing cultured epidermal sheets sufficient enough to cover 40 per cent burn wound in 28 days. The preliminary small area clinical applications undertaken so far revealed quicker healing proving the importance and usefulness of the method.

With this new approach a large number of moderate to severely burned patients could be saved in several burn centers across our country with reduced hospitalization period. However, the cell based therapeutic option in burn-wound healing by the application of in vitro - cultivated sheets of epidermis from autologous epidermal keratinocyte stem cells uses no matrix. This technque is sufficient for burn wounds of 2nd & 3rd mixed degree. The burn wounds predominantly of 3rd?and 4th?mixed degree can not be healed by the thin cultured epidermis, thus requiring a cellular or cellular scaffold that more or less mimic for graft take in deeper burns.

With this aim, we are presently attempting to create such a scaffold using Mebiol gel, which could support the cultured epidermis for better transfer to the wound bed. Additionally, the usefulness of Mebiol gel in growing epidermal sheets without the necessity of FBS and/or animal origin feeder cells but using human feeder cells will also be tested.

Proceedings of Annual Symposium on Regenerative Medicine

Novel approach in the management of an oral premalignant condition - A case report

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p21

Published on:
Oct 2007

Author(s):
Sankaranarayanan.S, Ramachandran C, Padmanabhan J, Manjunath S, Baskar S, Senthil Kumar R, Abraham S

Oral submucous fibrosis is a progressive oral disease first described by Pindborg and Sirsat 3 decades ago. It is a premalignant condition. The signs and symptoms depend on the involvement of the different sites in the oral cavity. The patient feels burning sensation for normal diet and trismus which may be so severe. If not properly treated the risk of malignant change in advanced cases of OSMF is relatively high. Wide ranges of treatment such as medical management, surgical therapy and physiotherapy have been attempted in the past, but none of them has proved to be a cure for this chronic fibrotic disease.

Histopathologically as the disease progresses, (i) change in the morphology of collagen, (ii) increased accumulation of amorphous collagen, and (iii) decreased collagen degradation results in decrease in number of blood vessels are observed in the affected area compared to the normal area. With an aim of bringing more blood supply to the affected area which is expected to bring ?more nutrients and help in collagen degradation, earlier application of vasodilators and studies with curcumin have been done, but still with - no significant outcome.

As an alternative approach to improve the blood circulation, we have tried Autologous bone marrow stem cells which have been earlier applied in several diseases such as ischemic peripheral vascular diseases, ischemic heart diseases etc with proven improvement in angiogenesis.

A 38 year old patient with oral submucosal fibrosis, proven by histopathology, and endothelial markers was injected 175 million BMMNCs into the area affected. The paramaters such as blanching, fibrous band have significantly improved, 4 weeks after the injection. We could observe positive changes clinically to prove the improvement. The mouth opening has improved to 35 mm from the previous 30.0 mm. Further histopathology and SEM studies with larger samples are done for establishing stem cell therapy's safety and efficacy.

Proceedings of Annual Symposium on Regenerative Medicine

Tissue Engineering Based Therapy for Articular Cartilage Defects - A New Approach

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p22-23

Published on:
Oct, 2007

Author(s):
Arumugam S, Manjunath S, Abraham S

Background: Articular cartilage, the load-bearing tissue in diarthrodial joints, when damaged due to trauma could lead to osteoarthritis. At present Autologous Cartilage Implantation is an established method in which patients own chondrocytes are isolated and then implanted after in vitro expansion over the affected area with bovine or porcine collagen matrix. This procedure results in more of Collagen Type I during in vitro expansion, which eventually becomes fibrocartilage. Also it requires growth factors. We have in this study tried growing human Chondrocytes without growth factors using synthetic scaffolds to grow more Collagen Type II

Materials and Methods: Human cartilage specimens were harvested through arthroscopy from the non-weight bearing area of the knee joint from 13 patients who underwent surgical procedures of the knee joint after getting their informed consent. The tissues were transported in saline taking 1 hour to laboratory and subjected to digestion with Collagenase type II for 16~18 Hrs. The chondrocyte cells obtained after dissociation were divided into two groups for culture. Gr. I were embedded in a Thermogelation polymer (TGP) and Gr. II in basal culture media (DMEM + Ascorbic Acid) without using any growth factors. The Group II cells were viable only for 4 weeks and then started degenerating. The TGP-Chondrocytes scaffolds were grown for 16 weeks and the specimens were harvested at 4, 8, 12 and 16-week intervals and their morphology and molecular characteristics were studied by H&E staining, S-100 protein analysis and RT-PCR.

Results: Human chondrocytes could be cultured in both TGP (group I) and Basal culture media (group II). The Gr. I cells were viable upto the 16th week while the Group II chondrocytes started degenerating after the 4 week. Both the groups were proven positive for S-100 protein, a Chondrocyte specific marker protein; Gr. II specimens after 4 weeks, and Gr. I specimens after 4, 8, 12 and 16 weeks. RT-PCR study of the cells of group I were positive for TGF beta 3 (Proliferation, differentiation, and other functions), GR beta, GR alpha (Development, metabolism and immune Response) (glucocorticoid receptor alpha), AGGF (Apoptosis), VDR (Vitamin D3 Receptor), Col II (Type II Collagen).

Conclusion: We have established a methodology by which Human chondrocytes could be cultured in vitro without any growth factors for a period of 16 weeks in a polymer-hydrogel scaffold. Upon further confirmation of their characteristics, the TGP grown chondrocytes can be used for autologous implantation to repair damaged cartilage area as the Collagen Type II which grows better without growth factors in the scaffold, eventually will become Hyaline cartilage is expected to give a longer disease free duration than the present method of ACI.

Proceedings of Annual Symposium on Regenerative Medicine

Autologous Stem Cell Injection for Spinal Cord Injury - A Clinical Study from India.

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p24-25

Published on:
Oct, 2007

Author(s):
Ravikumar R, Narayanan S, Baskar S, Senthil Nagarajan R, Abraham S

Abstract:
We studied 100 patients with Spinal Cord injury (SCI) after Autologous Stem cell Injection in the Spinal fluid with a Follow up of 6 months post Stem cell injection. There were 69 males and 31 females; age ranging from 8 years to 55 years. Time after Spinal Injury ranged from 11 years - 3 months (Average: 4.5 years). The Level of Injury ranged from Upper Thoracic (T1-T7) - 34 pts, Lower thoracic (T7-T12) -45 pts, Lumbar -12, Cervical-9 pts. All patients had an MRI Scan, urodynamic study and SSEP (somatosensory Evoked Potential) tests before and 3 months after Stem cell Injection.

80% of patients had Grade 0 power in the Lower limbs and rest had grade 1-2 power before stem cell injections. 70% of cases had complete lack of Bladder control and 95% had reduced detrusor function.

We Extracted CD34 and CD 133 marked Stem cells from 100 ml of Bone marrow Aspirate using Ficoll Gradient method with Cell counting done using flowcytometry.15 ml of the Stem cell concentrate was injected into the Lumbar spinal fluid in aseptic conditions. The CD 34/CD45 counts ranged from 120-400 million cells in the total volume.

6 months after Injection, 8 patients had more than 2 grades of Motor power improvement, 3 are able to walk with support. 1 patient with T12/L1 injury was able to walk without support. 12 had sensory tactile and Pain perception improvement and 8 had objective improvement in bladder control and Bladder Muscle contractility. A total of 18 patients had reported or observed improvement in Neurological status. 85% of patients who had motor Improvement had Lesions below T8. MRI, SSEP and Urodynamic Study data are gathered at regular intervals.

Conclusion:
This study shows that Quantitative and qualitative Improvement in the Neurological status of paralyzed patients after Spinal cord injury is possible after autologous bone marrow Stem cell Injections in select patients. There was no report of Allodynia indicating the safety of the procedure. Further studies to (i) quantify the neurological and vascular damage and to standardize the dosage, (ii) Identify Mechanism of action of each group of Stem cells (HSCs, MSCs, Naive cells, Purified subsets etc) on Nerve Tissue both In-vivo and In-vitro will be necessary to Confirm Above results.

Proceedings of Annual Symposium on Regenerative Medicine

Autologous Bone Marrow Stem Cell Infusion (AMBI) therapy for Chronic Liver Diseases

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p26-27

Published on:
Oct, 2007

Author(s):
Rajkumar JS, Baskar S, Senthil Nagarajan R, Murugan P, Terai S, Sakaida I, Abraham S

Background: Liver Cirrhosis is the end stage of chronic liver disease which may happen due to alcoholism, viral infections due to Hepatitis B, Hepatitis C viruses and is difficult to treat. Liver transplantation is the only available definitive treatment which is marred by lack of donors, post operative complications such as rejection and high cost. Autologous bone marrow stem cells have shown a lot of promise in earlier reported animal studies and clinical trials. We have in this study administered in 22 patients with chronic liver disease, autologous bone marrow stem cell whose results are presented herewith.

Materials and Methods: In 22 patients with chronic liver diseases of Child B-C category, Autologous Stem Cell has been transfused, upon getting approval from the ethics committee and informed consent. Under short GA, 200-300ml of bone marrow was tapped. The bone marrow stem cells were isolated using density gradient fractionation method and processed, suspended as per protocols earlier published (Terai et al., doi:10.1634/stemcells.2005-0542). The processing was done in cGMP facility under stringent aseptic precautions. Endotoxin test clearance was obtained (<0.25EU) and CD34+ analysis was performed using FACS. A cell count of 240 to 1068 X 106 was administered intravenously through the median cubital vein. Liver function tests, ultrasound and CT were performed before the administration, thereafter at 4 and 8 weeks of infusion. The protocols used were the same as used by Terai et al., Yamaguchi University, Japan.? Acites, albumin, bilirubin, radio lucency of liver and overall quality of life were studied in all these patients. Liver biopsies were not done due to lack of patient compliance. Standard work up of chronic liver disease by viral marker, copper, Alfa Feto Protein etc., were performed.

Results: The administration of the transfusion did not have any adverse reactions in the patients. 67% of patients showed an increase of serum albumin that was significant, 73% showed significant reduction of ascites, 32% showed drop in bilirubin. The overall quality of life of index (QULI) was significantly improved in 82% of the patients.

Conclusion: Autologous bone marrow stem cells administered in chronic liver disease patients has yielded significantly good results and has been safe. Although a pilot clinical trial, the study shows promises for newer exciting cell based therapies for chronic liver disease.

Proceedings of Annual Symposium on Regenerative Medicine

Use of Bone Marrow derived Stem Cells in patients with Cardiovascular Disorders

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p28-29

Published on:
Oct, 2007

Author(s):
Madhu Sankar N, Karthik Vaidyanathan, Rajesh V, Prasad GN, Kirtivasan V, Naveen AT, Abraham S, Cherian KM

Patients with end stage heart failure have very few treatment options. The long waiting times for transplant and the complications associated with immunosuppression has led to the search for alternatives. Subsequent to the isolation and characterization of stem cells, tremendous advances have been made and the safety and feasibility of autologous bone marrow derived stem cells has been proven in preclinical studies. Clinical studies have also shown mobilized cells repair the infracted heart, improving function and survival. We have started a clinical study to evaluate the efficacy of bone marrow derived stem cells. Bone-marrow was aspirated from the right iliac crest and the stem cells were isolated by density gradient method and suspended according to the mode of delivery.

From Jan 2007 till date 10 patients (8 adults, 2 children, age) with end stage cardiovascular disorder of varied etiology (Ischemic left ventricular dysfunction - 6 patients, Primary pulmonary hypertension - 2 patients, Dilated cardiomyopathy -1 patient, Biventricular non-compaction -1 patient) underwent stem cell therapy. All patients were evaluated and cardiac function was measured by using echocardiography and thallium scintigraphy. There were no procedure related complications. These patients are being regularly followed-up and one patient who has completed 6-month follow-up has shown improvement in perfusion as well as increase in ejection fraction of 10%. Stem cell therapy in patients with end-stage cardiovascular disorder might be a promising tool by means of angiogenesis and other paracrine mechanisms.

Proceedings of Annual Symposium on Regenerative Medicine

Our Experience in treating Ischemic Ulcer of a Lower Limb in 4 diabetic patients with Autologous Bone Marrow Stem Cells

30 Oct 2007 04:40:33 GMT

Journal:
2007 Vol. 3 (1): p30-31

Published on:
Oct, 2007

Author(s):
Subrammaniyan SR, Rajkumar M, Amalorpavanathan J, Shankar R, Manjunath S, Senthil Kumar R, Abraham S

Background: Chronic limb ischemia is an outcome of peripheral arterial occlusive disease. When conventional medical and surgical treatments are not feasible, amputation is the only option left. Recent studies report that the injection of bone marrow mononuclear cells and Peripheral blood mononuclear cells rich in CD34+ cells have resulted in symptomatic recovery, improved functional activity of the ischemic limb as well as healing of the ulcers. Here we report our experience with 4 patients of such case where autologous bone marrow mononuclear cells were injected and the patient followed up for 6 months. Materials and Methods: Four patients with critical limb ischemia with ulcers were referred for amputation of their limb. A 68-year-old female with critical limb ischemia with an ulcer in the left leg measuring 30X12 cm over the posterior portion of the leg and extending to the medial aspect of the foot measuring 14X10 cm, a 65-year-old male with necrotic wound in his lower foot, a 69-year-old male with a deep wound in his lower foot and a 61-year-old male with ulcer in his toe amputated with all the toe fingers. The first two patients were given injections for more than one sitting at appropriate intervals specified by the clinician. Under short general anesthesia, 110 ml of Bone marrow was aspirated each time, transported in Acid Citrate Dextrose and was processed for mononuclear cells (MNC) by Ficoll density gradient centrifugation, following the cGMP protocols. The MNC concentrate was injected at various sites in the Gastrocnemius muscle and the surrounding area after necessary debridement. Skin grafting was performed in the first two patients and followed up for a period of at regular intervals of 6 to 9 months. The patients have been followed up at regular intervals for six months after the treatment with investigations such as Ankle-Brachial Index, Doppler and Angiogram.

Results: All the patients showed improvements with healthy granulation gradually started appearing in the areas which were previously unhealthy and ischemic. Slow granulation was found in-patient 3 and but the patient 4 died because of other factor such as renal failure, peritoneal dialysis and cardiac failure. Patients 1 and 2 had healthy granulation, uniform revascularization and after a period of 9 months, healing was completely possible.

Conclusion: Stem cell therapy is definitely useful where, revascularization is not feasible at the same time, renal failure, cardiac failure, etc do present some difficulties. All the parameters need to be taken care. Growth factors or plastic surgery need not be used for stem cell therapy thus considering only the appropriate time of injections. As Autologous Bone Marrow stem cell therapy helps in neoangiogenesis and wound healing process in case of chronic ischemic wounds it can be applied in cases as reported herewith.

Proceedings of Annual Symposium on Regenerative Medicine