03 mrinal k dewanjeescienceandculture-isna.org/sept-oct-2016/03 mrinal k... · 2018. 1. 30. ·...

278 SCIENCE AND CULTURE, SEPTEMBER-OCTOBER, 2016

STEM CELL THERAPY ITS CHALLENGES, SUCCESSES, FAILURESAND POTENTIAL APPLICATIONS : ON-GOING TRANSLATIONAL

RESEARCH

MRINAL K. DEWANJEE*

Critical cells in human organs of injured genetically diseased or aged populations degeneratedue to various reasons, mainly injury, genetic mutations and chronic inflammation. The stem cell-niche in most human organs has a limited ability for cell regeneration. In an adult human body,about a billion cells die every day. In some organs and blood pools, injured or lost cells andtissue sheets (e.g. skin graft) could be replaced and in blood loss, it could be replenished bytransfusion. For cellular components of blood, we have developed superb tools, specifically taggingthem with gamma-emitting radionuclides, for measuring the cellular half-lives, their survival times,turn-over rates and size of blood pools in healthy volunteers and patients. It is possible now toincrease the platelet circulating time in blood of splenomegaly patients by splenectomy, thusdecreasing the episodes of bleeding complications resulting in fewer visits to blood bank for platelettransfusions. However, these measurements of cell-survival parameters of blood cells are not possiblefor other cell types, particularly in some retinal- and neuro-degenerative diseases, where only onespecific cell type, e.g. neurons, may be lost. This specific cell could be derived from the embryonicor induced pluripotent stem cells (ESC or iPSC) by differentiation and could be delivered to theaffected organs provided the cell-loss is not diffuse over a large area and the cells are not migratory.iPSCs now could be derived easily from skin fibroblasts or blood lymphocytes by transfecting withonly four genes. In spite of significant and pioneering developments of cell access from ESCs andiPSCs in the last decade, their optimized differentiation methods and their large scale production,cell therapy faces a tremendous challenge in near future due to poor cell-integration in host organs.At present, attention has been diverted to drug discovery. Successes, failures and potentialapplications of cell therapy in certain diseases are discussed in this article to draw attention of theenergetic young minds, scientists and clinicians to solve this challenging puzzle and give hope tothe stroke, heart-attack, diabetic and demented patients for a longer and a better quality of life.

ARTICLE

* National Eye Institute, National Institutes of Health, Bethesda,MD 20892, U.S.A. e-mail : [email protected]

Introduction

Derivation of human embryonic stem cells (hESCs,29) and developments of induced pluripotent stemcells (iPSCs, 30, 38) offered us the opportunity

of generating all cell types in large numbers. With a varietyof recombinant growth factors and morphogens, a variety

of differential protocols were developed. Combination ofthe activators and inhibitors of essential developmentalpathways (Wnt, Shh, Activin/Nodal, BMP, TGF signalingand dual SMAD inhibition, 42) with a few small molecules,gave us alternative efficient method of deriving these cellsin a period spanning 30-200 days. History of cell-labeling,their turn-over studies in blood, their potential for cell-fatemapping by nuclear and magnetic resonance imaging (MRI)and recent cell therapy applications are referred in our early

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methods of iPSC inductions(direct and indirect), majorareas of neurodegenerationand human diseases in fiveslides.

Issues of cell scarcityare being resolved as bettermethods of harvestinghealthy fibroblasts andreprogramming them intoiPSCs were developed29,38.The desired cells are nowproduced on a large scaleusing the sequential additionof growth factors or theirinhibitors in tissue culturemedia. For tissue repair orregeneration, a model mustbe developed to estimate thesize or pool of injuredtissue. We must replenishadequate number of cells ina correct configuration, cellsuspension (very fragile),cell-sheet or cell/tissuecluster in a correctproportion. Since the injuredtissue is in a higherinflammatory state, theremay be higher cell lossinitially (Figure 8). For thesuccessful integration oftransplanted cells or tissuesin the host organ, a viablecell or tissue delivery modelmust be developed. Most ofthe experiments are carriedout on an empirical basis.Most of the investigatorsshow a few slices of dozenstained cells out of a fewhundreds of thousands ormillions of injected cells.Unfortunately, there is nosuitable non-invasivemethod of measuring thecell death rate. Most of

Figure 1. US FDA guideline for cell therapy. The desired cells derived from stem cells must be characterizedfor their viability, karyotyping and desired 7 function before regional delivery. Presence of undifferentiatediPSCs even at a low level may induce cancer.

Figure 2. The schematics of handling of stem cells, their differentiation, their use in drug screening andregional delivery for potential therapy.

publications1-42. For a quick reference, I also high-lightedthe main themes of cell therapies, their expectations,

organs must be sliced and tagged cells must be countedunder a microscope for estimating survival time or its half-


Figure 3. Methods of differentiation of iPSC-derived cells into oculocytes for therapyof retinal regeneration.

life. The fMRI of whole brain is based on changesin blood-oxygen-level-dependent (BOLD) signals.BOLD signals modulated by opto-geneticstimulation of endogenous neurons could identifythe site of functional graft integration in thenervous system43. Currently there are more than600 iPSC cell lines and 60 clinical trials are indifferent phases of progress for cell therapies.

I.Conventional Cell Therapy in BloodTransfusion (Red cells, Platelets andWhite cells) for Blood Loss, Bleedingand Repeated Infection

Conventional therapy of blood celltransfusion is an established procedure. Almosthomogenous cells could be separated by size bysedimentation procedure. Turn-over studies ofblood cells are optimally carried out in blood byrepeated blood sampling after intravenousinjection. All the blood cells had been tagged witha variety of water-soluble radionuclides inappropriate chemical forms [(32P-tagged Di-isopropyl fluorophosphate, 75Seleno-methionine,31Chromium chromate, 99mTechnetium-pertechnetate (Na99mTcO4), 111Indium(Tropolone)3] and 89Zirconium (Oxine)3 within thelast five decades. I developed a freeze-dried kit(Stannous-citrate) for rapid preparation of Tc-RBCs and used for measurement of ejectionfraction of human hearts. In those days, we didnot even use a laminar flow-hood for bloodhandling at Tufts-NE Medical Center in 1974. Wewere lucky that there were no adverse reactions:from bacterial contaminations in these earlystudies with multiple alcohol swabs and rapidsterile handlings. The radioactive 111In-taggeddebris of dying cells in blood are continuouslyremoved by the reticuloendothelial cells in liver,spleen and bone marrow. However, the iron-oxidenanoparticle tagged neural stem cells disappearedrapidly from the mouse brain as observed byPrussian-Blue staining. Lung air prevents MRI oflung tissue cell-retention. The fluorescent proteintags play a critical role in following multi-cellfates in all aspects of biology: development,growth, maintenance and death in a variety ofinvertebrate and vertebrate models. However, theyare very antigenic in human and could not be usedin human. After intravenous injection, most of the

Figure 4. Anatomy of human knee joint with a cushion of cartilage cells. Afterinjury, attempts were made for regeneration after regional injection of suspendedcells and/or a sheet of cartilage cells. However the sheet-attachment is a challenge.

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large tagged cells or their aggregates stickto lungs for 3 to 24 hours. They are thensequestered mainly in liver and spleen anda percent in bone marrow (Figure 22).

We know the blood pools orcompartments, the half-life of red bloodcells, platelets and many types of whitecells (PMN neutrophils, monocytes, T andB lymphocytes) systemically during thelast decades of twentieth century. Half-life of PMN neutrophils had a darkunethical history, where the AfricanAmerican prisoners at Utah were used asguinea pigs and tagging was carried outwith 32Phosphorus tagged DIFP and half-life and survival rate of human PMNneutrophils were estimated. Among thegamma-emitting radionuclides, 111Indiumwas the best for imaging of deep-seatedorgans due to high photon abundance andappropriate tissue-penetrating gamma rays(171- and 245-keV, half-life: 68 hours).However, these 111In-tagged cells are notapplicable for turn-over studies of othercells delivered in solid organs, e.g. heart,pancreas, brain or retina. After leavingNIST and NIH at 2005, I joined FDAStem Cell and Gene Therapy group of Dr.Raj Puri for helping them to develop anduse imaging tools for following the fateof transplanted cells in mouse model.When we used dual-tagging with In-111and iron nanoparticles (Fe-NPs) andinjected 111In-tagged neural stem cells(500,000 to 1 million NSCs) in mousebrain under stereotactic guidance, weobserved a brain clearance rate ofradioactivity of ~60 hours. There washardly any clearance of radioactivity fromthe injection site (Figure 17). Recentstudy suggests that dying cells protrudedeath-domain protein (DD1) ligand,which is recognized by the abundantphagocytic cells in blood69 but not insolid organs. Clearance studies oftransplanted cells could not be carried outfor solid organs as we did so successfullyin blood. However, the 99% of Fe-nanoparticle tagged neurons disappeared

Figure 5. ECMPs play a critical role in every organ for integration of developing or transplantedcells into a 3D geometry.

Figure 6. Tissue-engineering Approach of ECMP Modulation GF binding to ECM proteins.

Figure 7. Multitude of high-resolution imagers revolutionized the non-invasive diagnosis ofhuman pathology. They also are critical for the evaluation of fate of transplanted cells inanimal models and patients.


Figure 8. Inflammatory response play a critical role in early clearance of celldebris, their repair by regional cell proliferation that results in a completewound healing at the site of injury. A variety of cytokines, growth factors,ECMPs and matrix-degrading enzymes repair the injury without a major scarat an early age. This does not happen at older age or in the degeneratingtissues71.

Figure 9. Platelet-tagging with 111In-radionuclide is ideal for following theturnover of the homogenous cell population in all animals and human patients,not true for other cells4.

within a day and the rest declined after a few days. Icounted the Prussian-Blue stained NSCs after three andseven days after cryo-section of mouse brain. Thesurvival curve for retinal and intracranial transplants isa bi-exponential one with approximetely 99% and 1%cells dying with half-life of 1 day and 3-7 daysrespectively. Histochemical analyses indicate that a fewphotoreceptors try to hang onto the outer segment-retinalpigment epithelial interface. In the absence of reliablecell fate measurement, many false claims of cell-viabilityand improvements of organ functions were made in bothanimal models and patients45,49. Dentate gyrus andsubventricular zones are the source of neural stem cellsin brain. Their turnover decrease with age significantly.Bhardwaj et al. used the 14Carbon spike fromatmospheric nuclear weapon tests by USA and Russiain the 1940-1960s and BrdU tracer to estimate thetumover of animal and human neurons in human andanimal brains and in human and found it low 1.8% peryear in the hippocompus, about 800 new neurons perday). For cardiomyocytes, the turnover is ~1%/ year.

Mean platelet survival times (days) in healthyhuman volunteers were determined after intravenousinjection of autologous platelets tagged with twodifferent radionuclides of 51Cr and 111In-complexes ofoxine and tropolone. We used the linear, exponentialand multiple hit mathematical models for the calculationof human platelet survival times6 (Table 1).

II. Cell Therapy for Skeletal Joints,Cartilage, Bone Repair and Pain-relief

Mesenchymal stem cells (MSCs) are self-renewing,and multipotent stem cells giving rise to all the skeletaltissues63 and constitutes ~0.1% of total adult marrowcells. They are present in the mononuclear fraction ofBM, separated by Ficoll-Hypaque gradient separationand also present in all tissues. They could be producedon a large scale using current good-manufacturingpractice (cGMP). After transplantation, they formminiature bone and marrow organoids. The MSC ofstromal cell type is defined differently than the classicalMSC and are isolated from cultures of bone marrowstromal cell types characterized by their adhesivecharacteristics to polymeric materials of culture dishes.There are more than four hundred MSC-clinical trials(www.ClinicalTrials.Gov). The desirable properties ofMSCs, e.g. anti-apoptosis (HGF, IGF-1, bFGF, TGF²),

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TABLE 1. Platelet survival times in healthy human volunteers with taggedautologous platelets

All sampling <2½ hoursMathematical model Group times included excluded

[51Cr] Disodium chromate (N = 28)Linear Total 10.0 ± 0.9 10.1 ± 0.9Exponential Total 6.9 ± 1.0 6.8 ± 0.9Multiple hit Men 8.8 ± 1.6 9.1 ± 1.1

Women 9.7 ± 1.6 9.8 ± 1.6111In-oine (N = 28)

Linear Total 9.0 ± 0.6 8.8 ± 0.6Exponential Total 5.8 ± 0.6 5.1 ± 0.6Multiple hit Men 8.2 ± 0.8 7.7 ± 0.9

Women 8.6 ± 0.8 8.1 ± 1.211In-tropolone (N = 8)

Linear Men 10.6 ± 0.8 10.2 ± 0.7Exponential Men 6.8 ± 0.9 5.4 ± 0.7Multiple hit Men 10.4 ± 1.0 8.9 ± 1.4

* Data shown as mean ± 1 SD.

Figure 10. Gd-(MPO)3 complex was designed for positive signal MRI with Gd-tagged cells.Iron-oxide nanoparticles internalized by the phagocytic cells give a negative signal, difficult tovisualize the fate of transplanted cells using MRI.

Figure 11. Anesthetized mouse head is immobilized with three arms of Kopf head-holder forintracranial cell delivery under stereotactic guidance.

angiogenesis (VEGF, PlGF, IL-6), anti-inflammation, growth factorproduction, neuroprotection, anti-fibrosis (HGF, bFGF), and chemo-attraction CCL2-6, CXCL2,8,10) areexploited in many disease therapyprotocols by allogeneic transplantation.After working with platelets, Ipredicted that considering the easyaccess from Blood banks, homogeneityand platelet-granules derived fromactivated (ADP, Thrombin) humanplatelets, may provide the nutrients thetransplanted cells need. We attemptedtheir use in enhancing the survival oftransplanted photoreceptors in mouseretina. Next to platelet-granules, theMSCs might be used as supplement forcell growth and maintenance.

Oral surgeons use platelet-enriched plasma (PRP) to regeneratebone and soft tissue in the jaw. Formaking autologous PRP, blood isdrawn in a syringe and spun to aspiratethe supernatant of PRP and theninjected in the injured joint or used incombination with surgery (Figure 9).PRP is replete with multiple growthfactors and other small molecules thatpromote healing. It reduces pain ininflamed tendons, tennis elbow, andarthritic joint by yet unknownmechanisms. In the alternativeapproach, Veterinarians use the bonemarrow aspirate concentrate (BMAC),extracted from hip under localanesthesia for cartilage repair in racehorses or orthopedists in patients forcartilage repair; BMAC forms a clotto fill gaps in cartilage. Stem cells andgrowth factors nurture the graftedtissue. Collagen sponge soaked inpatient’s blood is also used to promoterepair of injured meniscus and anteriorcruciate ligament (ACL). But nothingis as good as your cartilage in yourknees. Injury prevention is crucial; forunfortunate victims of complex joints,there may be better choice of cocktails


Figure 12. Scintiphoto of tagged autologous neutrophils at 2 hours after intravenous injection.Most of the neutrophils, marginate to the network of capillaries in the large surface of lunglobes; after a few hours, they ultimately migrate to liver, spleen and a small fraction to bonemarrow. Similar dynamics of migration pattern was observed with 89Zr-oxine tagged bonemarrow derived cells (3).

Figure 13. My cartoon is suggesting the non-adherent and repelling nature of retina, or otherintended target organs to transplanted photoreceptor cells in young mouse retina. The cells indeveloping retina arrive in two phases (right).

of cells and a 3D scaffold for repair in near future. Disksof cartilage cells were made from bone marrow extracts,BMPs are used for differentiating ESCs or iPSCs intocartilage cells. However, attachment is a challenge. Smallcartilage plugs or single cell suspension is used for jointinjection for repair after joint injury.

Regional injection of MSCs (6-18 million) gavesubstantial and sustained back-and arthritic joint pain relieffor a period of two years (Mesoblast Inc., Australia, 2015).

Athersys Company is using the fat-derivedMSCs delivered intravenously in theischemic stroke patients44, 45. No responsewas observed in sMRI of brain or spinalcord.

III. Stem Cell therapy in BoneMarrow Transplantation

Bone marrow stem cell (SC) therapyby transplantation evolved during the last60 years. They are used in patients wherethe bone marrow is not adequate toprovide all the blood cells in adequateabundance or in blood cancer patients(Leukemia: Too many white cells in bloodmake blood color white). Source of SCmay be autologous, directly from thepatient, syngeneic, SC from identical twinor allogenic from another person. Toavoid the graft-versus host disease(GVHD), SCs must be matched. Bloodforming cells may be derived from bonemarrow, umbilical cord or blood stream.Patients with different types of leukemia(acute lymphoblastic leukemia: ALL),lymphoma or multiple myeloma aretransplanted with BMSCs. For leukemiaor lymphoma patients, patient’s defectivebone marrow is treated with radiation orchemotherapy or a combination of both.The BMSCs are delivered intravenouslyand slowly (1-5 hours). Our imagingstudies in animal models and patientssuggest that there is temporarymargination in the lung. After some time,most of these migrate to the niche inrecipient’s marrow, liver, and spleen andstart producing red cells, white cells andplatelets. By blood sampling and

differential white cell counts, rapid appearance of RBCsand platelets could be monitored. Immune cells appear aftera few months, earlier for auto than allogenic SC transplants(1-2 years). Adverse effects, e.g. bleeding, infection couldtreated with transfusion of platelets and neutrophils.Immune rejection could be treated with steroids,cyclosporine or Rapamycin. BMSCs recipients must visita regional transplant center for consultation, HLA matching,transplantation and follow-up procedures.

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Figure 14. Courtesy of Dr. T. Michael Redmond, NEI, NIH. RPE-65 protein recyclesthe cis- and trans- retinals (derived from Vitamin A) between retinal pigment epithelial(RPE) cells and photoreceptors.

Double bonds of Vitamin A make it a photon trap. The theme of slidewas essential for RPE-65 based gene therapy in animal models andHuman patients.

Figure 15. The layers of cells in retina and the entry of lightfrom inner layer to the outer layer (Arrow, top figure). A mousemodel of cell therapy system for harvesting photoreceptorsfrom post-natal mice (P5), processing for making the singlecell suspension with enzyme (Accutase) and subretinal deliveryis shown in the lower figure. About 250,000 PRs are injectedin one eye. The detached retina could be pushed back gentlywith the needle for reattachment.

IV. Islet Cells Harvested fromCadaver Pancreas in Diabetes: ESCsand iPSCs

At present, there are ~382 million diabeticsin this planet and this number is doubling in 20years. India is the diabetic capital of the worldwith a population of prediabetics and diabetics~110 and ~35 million and China,~115 and ~ 40million respectively. Every hour 370 diabeticsdevelop retinopathy (DR); 20% of them alsoprogress to proliferative DR due to growth ofECs. Choriocapillaris and central retinal arterysupply blood to outer retina and inner retina.Central retinal artery exits from the optic nerveand branches into the macula (sensitive retinaresponsible for central vision). Hyperglycemiacauses endothelial and pericyte damage andbasement membrane thickening, inducingformation of leaky blood vessels. Beta islet cells(BIC) of pancreas regulate glucose level in bloodby releasing insulin. Their loss from Type Idiabetes (TID, autoimmunity) results from rapidBIC loss by T cell-killing in early age andpatients need routine insulin injections. In TypeII diabetes (T2D), patients lose (50-65)% of BICgradually. In the early stage, patients could betreated with oral drugs, e.g. Metformin and /orGlipizide adding insulin when BIC level furtherdecreases. Hyperglycemia could result incomplications of cardiac, kidney diseases,retinopathy, peripheral nerve pain and gangrene-induced amputations from micro- and macro-vascular pathologies, e.g. endothelial celldysfunction (ECD), microaneurysm formation,their rupture causing hemorrhaging in retina andrupture of vulnerable atherosclerotic plaque inlarger arteries. Photocoagulation with laser andanti-VEGF therapy slows down proliferativediabetic retinopathy (PDR). The spongy retinamay swell from a thickness of 250 μm to 1500μm in diabetic macular edema (DME). DMEcould be evaluated by fundus imaging andoptical coherent tomography (OCT). ECD


Figure 16. Courtesy of NEJM. The human corneal sheet harvested and assembled fromlimbal stem cells of injured eye provides an ideal approach for repairing corneal injury,e.g. acid burn, mechanical or blast injury. The five-year success rate is about 70-80%.

Figure 17. Scintiphotos of retention of In-111 tagged cells at 7 days post-intracerebralinjection. Radioactivity was retained mainly at the injection site with slight dispersion.

Figure 18.The positive MRI signal (black arrow) from Gd-tagged NTERA2 cell afterintracranial delivery.

decreases production of nitric oxide, a greatvasodilator, increase vascular inflammationand neovascularization (Proliferative DR).Several genes, FoxO, Akt, Irs2, play criticalroles affecting ECD. In T2D, patients developinsulin resistance, in which muscle, fat andliver cells do not use much insulin andrequires more insulin production. Althoughthe BIC compensates by excess insulinproduction, in later stage, BICs fail andglucose level rises. Being a diabetic myself,I try to reduce weight gain by diet, exerciseand metformin. My participation at theObesity Clinical Trial for five years trainedme in controlling carbohydrate intake andmaking a balanced meal (NIDDK.NIH.GOV).The fathers of T1D children are working hardto make the BIC therapy work!

Considering the potential benefits ofinsulin secretion from cell therapy, BICtherapy was attempted in multiple animalmodels of T2D induced by Streptozotocin.Porcine cells encapsulated in polymercapsules were implanted for long-termdelivery. The leaky capsule membranereleases insulin and the porcine tissue isprotected by attack from immune cells. Aftera while, the capsule is covered with fibroustissues and glucose level rises due to lack ofinsulin secretion and islet cell death. In the“Alberta Protocol”, cadaveric pancreas wasused for harvesting human BICs. After HLA-matching, they are delivered to the liver viaportal vein. Although the glucose level iscontrolled by secreted insulin, after a fewyears, patients need insulin for controllinghyperglycemia.

BICs were derived from several typesof embryonic stem cells (ESCs), inducedpluripotent stem cells (iPSCs), adultmesenchymal stem cells (MSCs) andreprogramming of acinar cells, gut andenteroendocrine cells. Several endodermgenes are activated, e.g. SOX17, FOXA2,PDX1, HNF6, NGN3, NEUROD1. Finally β-cell lineage is characterized by insulin,GLUT2, GCK, NKX6-1 and MAFA genes.Protocols for in vitro differentiation of EPSCsinto BIC-lineage use the approach of

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Figure 19. Rapid clearance of Gd-MPO tagged NTERA2 cells from mouse brain, leaving an emptyspace at the injection site57.

recapitulating the induction ofbiological pathways generating thenormal stages of pancreasdevelopment by sequential additionof various growth factors, activin A,fibroblast growth factor and retinoicacid. ViaCyte (CA, USA) iscurrently conducting clinical trialsfor diabetics.

V. Cell Therapy Failures forCardiomyocytes inMyocardial Infarct

Considering the potential ofcell therapy after a catastrophic heartattack (myocardial infarct), multipleattempts were made during the lastdecades in animal models andpatients48,49,73,74. Stem cell technologies developed in USA,Europe and Japan are attempted in China, Korea and othercountries. Hyperexpectations from the paracrine effect orthe repair by regeneration of transplanted cardiomycytes(CMs, 20-200 million administered 30 transendocardially)resulted in terrible disappointments. In spite of multiplepublications, none of the results were reproducible and afew papers were finally retracted. Claims were made forincreasing regional myocardial contractility, increase ofejection fraction, low level of scar formation in multipleclinical trials after CM therapy. Majority of these studiesused cardiomyocytes derived from bone-marrow marrowstem cells. Recent studies also indicated potential benefitsfor ESC or iPSC-derived CMs in animal models.Intracoronary administration of MSCs showed a minorbenefit on the left ventricular ejection fraction50. However,these CM cells also beat at a slower rate than the healthymyocardial cells indicating the potential for arrhythmia aftertransplantation51.

VI. Cell therapy for Corneal Injury withLimbal Epithelial Cell Sheet

The limbal stem cells can be damaged by chemicalburns, microbiological infections, and autoimmune andgenetic disorders (aniridia) which that results in limbal stemcell deficiency (LSCD) and corneal opacity. Healthy limbalstem cells (LSC) maintain a healthy corneal epithelium andact as a barrier against conjunctival ingrowth onto thecornea by producing corneal epithelial cells. Thus, in totalLSCD, there are 360 conjunctival ingrowths. LSCD mayinduce eye irritation, low vision, superficial and deepcorneal neovascularization and fibrovascular pannus,

ulceration, scarring, and corneal perforation. Limbal tissuewas harvested from the fellow eye or a cadaver eye. Eyebiopsy tissues (1–4 mm2) are expanded on fibrin in cultureor human amniote cell membrane it generates a cell sheetto cover the entire cornea. Autologous LSC therapy resultsin long-term benefits52. Figure 16 shows the effectivecorneal layer with LSC therapy. Amniotic membrane wasfound to be non-inflammatory scaffold, since there is noscar formation post-fetal surgery.

VII. Photoreceptor and RPE Death in RetinalBlindness, Macular Edema and Cell TherapyProgress

About more than 30 cell types had been identified inthe retina. These cells initiate from neural precursor cellsand are layered in developing retina67 (Figure 15). VitaminA is an essential nutrient and is recycled all-trans to cis-configuration mediated by RPE-65 in the retinal pigmentepithelium (Figure 14). Retinitis pigmentosa refers to agroup of inherited eye diseases that cause progressive lossof photoreceptor cells in the retina. While RP can appearduring infancy, the first symptoms typically appear in earlyadulthood, beginning with night blindness. As the diseaseprogresses, affected individuals lose peripheral vision. Inlater stages, RP destroys photoreceptors in the macula,which is responsible for fine central vision. Mutations inat least 50 genes have been found to cause the disease,which affects about 1.5 million people worldwide.

Dr. M. Takahashi transplanted the first iPSC-derivedRPE cell sheet (1.3mm X4 mm) in a Japanese woman withAMD that was nonresponsive to anti-VEGF therapy64. No


adverse event of tumor formation or immune rejection wasobserved yet. Steroids or immuno-suppressive drugs, e.g.Cyclosporine or Sirolimus, were not necessary. Retinalthickness decreased suggesting reduction of retinal edemaand no recurrence of choroidal neo-vascularization (CNV)occurred. While the patient was progressively losing visionin the course of anti-VEGF treatment, stabilization wasachieved after RPE transplantation surgery. There are threecardinal observations found for successful therapy: Rapidselection of viable cell lines with appropriate morphologywith characteristic biomarkers, e.g. Rx, Pax6, Vsx2, Sox2using Q-PCR tests essential. Gentle handling of slowpipetting with a larger bore like that fickle-minded plateletswill reduce cell handling trauma during washing, pelletingand resuspension. RPE sheets were generated in Matrigelafter 20 passages (~60 days). Both patch (1.3 mm X4 mm)and single cell suspensions were evaluated. ESC-derivedRPEs are much easier than iPSCs. About 150-200thousands of RPEs were suggested to be adequate for RPEtherapy. Considering the exorbitant cost of derivingautologous RPE cells, Takahashi group are moving toallogenic transplantation, evaluating immune responses,genetic compatibility and HLA matching. Potential tumorformation dampened the pace of cell therapy.

Stem Cells Inc. used neural stem cells for treatinghuman blindness due to dry macular degeneration. In thedose escalation study (200,000 to 1 million cells), theysuggested higher visual acuity, lower level of geographicatrophy in subjects at 12 months after sub-retinal injectionof the treated eye vs the non-treated eye (Stem Cells Inc.,2015).

Ocata Therapeutics (Advanced Cell Technology) usedthe hESC-derived RPEs for treatments of dry maculardegeneration and Stargardt’s macular dystrophy. RPEs(50,000, 100,000 and 150,000, purity > 99%) were injectedin one eye. Visual acuity improved in most of the patients.The RPE cells are not integrated with PR cells to makefunctional light-captured visual activation. Only a few RPEcells out of a few hundred thousands are layered over theoriginal RPE layer as observed by the optical coherenttomography (OCT) imaging method. Long-term follow-upin a large number will be necessary for the validity of RPEtherapy in AMD patients75.

Therapy for retinal degeneration and blindness wasattempted with ESC- or iPSC- derived photoreceptors,mainly rod cells. Considering the potential toxicity fromrecycling retinal products between PRs and RPEs in thevisual cycle, PRs are very unstable and unsuitable for

therapy for retinal blindness. The alternative cell population,retinal ganglion cells may be an alternative which mightplay a regenerative role.

VIII. Cell Therapy Failures in Brain forStroke, Spinal Cord Injury andNeurodegeneration

Most of the drug development by Pharmaceuticalindustry (PHARMA) is in the area of psychiatry and notfor neurodegeneration. Blood-brain barrier (BBB) is anothermajor hurdle, since hydrophobic drugs that transportthrough BBB also have more side-effects due to multipleaccesses to multiple organs. Several neuronal cell lines wereevaluated in animal models. Neural progenitors slow downthe progression of Huntington’s disease53. Dopaminergicneurons evaluated for Parkinson’s disease54. iPSC-derivedcholinergic neurons were evaluated in rodent model ofregeneration of hippocampus55. Some PD patients derivedlong-term benefits from cell therapy56.

Single cell neuronal disease, e.g. Parkinson’s disease,where dopaminergic neurons are lost, is expected to betreated easily, at least in theory. Multiple regional neuronalcell therapies were attempted without much success, in spiteof claims in reputed journals. Intra-putamenal delivery ofimmortalized human fetal brain (CTX0E03) cells was madeinto rat putamen. NPCs were injected at four weeks aftermiddle cerebral artery occlusion57 inducing stroke.Transplanted hESC-derived NPCs delivered into the infarctcavity suggested low neurogenesis in SVZ in both youngadult and aged animals. However, only a few cells werefound at the injection sites and their neighborhood.Comparative genomics suggest that there are numerousdifferences in enhancers of animals with small and largebrains. By transplanting human DNA into mouse embryo,Wray recently indicated that human version of a regulatoryelement, called human accelerating region (HARE5), closeto a gene FZD8 increased mouse brain size by 12%,doubling neuronal density and folds via increase of neuralprecursor cells by shortening the‘ cell cycle time46.

Human fetal tissues from abortion clinic, althoughcontroversial, provide a unique access to developing tissuesafter and evaluated in multiple clinical investigations (PhaseI and II) of cell therapy, drug evaluation and mechanismof genetic diseases. Neurostem Inc. (USA) and ReNeuronLtd (UK) used fetal-derived immortalized neuronal cellsin SCI, ALS, stroke and limb-ischemia. TRANSEURO(UK) used fetal-derived dopaminergic neurons in PDpatients. Stem Cells Inc. used human fetal-derived neuralstem progenitors for therapy of lysosomal enzyme

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deficiency in advanced infantile or late-infantile ceroidlipofuscinosis (Batten’s disease) in children without adverseevents58. High doses of 1 billion cells into the brain werewell tolerated and survived till 2.5 years in these patients.In the genetic demyelination of Pelizaeus-Merzbacherdisease (PMD) patients59, neural progenitors were implantedinto the frontal lobe white matter in young PMD patients.Modest gains in neurological function and myelination wereobserved in these patients with sMRI and MRS (magneticresonance spectroscopy).

Oligodendrocyte progenitor cells (OPCs) generateastrocytes and oligodendrocytes. Degeneratingoligonucleotides induce loss of insular myelin coat of axonsin multiple sclerosis, white matter stroke and cerebral palsy.Spinal cord injury also has similar pathology, Spinal nerveis like a tension cable and it shrinks soon after spinal cordinjury (SCI). After several years of evaluation, Geron finallystopped SCI repair therapy after four patients. Coordinateefforts used for skin injury and repair do not apply in SCItherapy. Chondroitin sulfate proteoglycans and a fewrepellent molecules and receptors inhibit cell growth, repairand attachment. Motor neuron diseases include amyotropiclateral sclerosis (ALS) and spinal muscular atrophy andastrocyte based therapies had been attempted60.

There is a significant interest in all industrial countriesfor understanding human brain. We have our own NIHbrain project as proposed by President Obama in April,2013 (BRAIN: Brain research through AdvancingInnovative Neurotechnologies). Blueprint was initiated forNIH funding in 2014 European countries and Japan(RIKEN: Marmoset model) have their own investments.Out of 28 Institutes and centers at NIH, about seveninstitutes (NIMH, NINDS, NIDA, NEI, NIDCD, NIAAA,and NIA) deal directly with brain and mental health issues.Other institutes also develop imaging tools, genetic databaseand drug screening technologies (NCATS, NIBIB, NIGMS,and NIEHS). Considering the large number of neurons inhuman brain, each connected with 10,000 synapses, brainsof insects and small animal models are preferred. In spiteof hyper-expectations, cell therapy used as single cellsuspension, do not work for preventing the retinal andneurodegeneration.

In spite of significant advances in understanding ofthe anatomy, cell biology of neurons and associatedsupporting cells, physiology, neuroimaging, genomics andchemistry of the brain, we still are struggling to resolvehow brain function correlates to mental health and whythis critical function declines irreversibly with aging.

Focused long-term funding will reveal the ultimate mysteryand translate into practical advances in computationalneuroscience, engineering of neuro- and retinal prosthetics;other discoveries from developmental biology will usherin the success from the recent frustrating cell therapyfailures in retinal and neurodegenerations47,69.

Autophagy dysfunction and subsequentneurodegeneration from toxic protein-aggregate deposit

Most human neurodegenerative diseases result fromthe extracellular and intra-cytoplasmic deposition of protein-aggregates and mitochondrial dysfunction. Initial phase ofneuroinflammation without counteraction results inneurodegeneration69. The roles of multiple cytokines ininflammation in tissue injury and wound-healing in the latephase are understood in the skin-injury model (Figure 8).The types of cytokines are different in different types ofneurodegeneration. Protein-aggregates are found in neuronsof patients, e.g. neurofibrillary tangles, Tau and extracellularamyloid of Alzheimer disease (AD), triplication of the ‘α’synuclein (SNCA) in Parkinson disease (PD), higherabundance of long (CAG)n trinucleotide expansionsencoding poly-glutamine (polyQ) tracts (Huntingtin) inHuntington disease (HD). By overexpression of theseprotein-aggregates, several mouse models were generatedfor understanding the disease mechanism, answer theclinically relevant questions and use these mice and theircell lines in drug development. Autophagy (self eating) isintended for the digestion and recycling of nutrients viaauto-phagosomes, which swallow the protein-aggregates,organelles and deliver them to the lysosome fordegradation. Autophagy actively removes these toxicproteins routinely and maintains neuronal health. However,autophagy dysfunction has also been implicated in thepathogenesis of various diseases61.

IX. Overcoming Repulsive Forces and Non-cooperative Chondroitin-sulfate Proteoglycans

For hundreds of years, the aged emperors, kings andnobility wanted to find the fountain of youth and regeneratetheir exhausted body parts, enhance organ function andimprove the quality of life with youthful contacts, or gettingcells and organs from younger generation. Indian surgeon,Susruta, the father of modern surgery (1200 BCE) did thefirst plastic surgery for nose repair. As the medical scienceand developed, wiser methods assisted in the simpler useof single cells, Mixtures of cells-ECM components alongwith growth factors were evaluated for tissue repair. Organtransplantation has almost been perfected; except there is


a large waiting list of patients for donor organs. ESCs andiPSCs provided excellent resources that are being evaluatedfor drug-screening. Transcriptome database suggested hownature refined the process of assembly and continuousremodeling for entry of life. However, as human beingshave to find our own way of repair and regeneration, sincenature did not design the exit policy properly. Recent studysuggests that as we age, our neural stem cells inhippocampus (HC) lose ER-membrane barrier and generatesdefective HC neurons. They do not help us in neither recentmemory preservation and nor their recollection62.

X. Growth of Stem Cells or DifferentiatedCells on Polymer Substrates

Stem cells are grown on a variety of natural andsynthetic polymers including Matrigel, mouse tumor derivedsubstrates (not suitable for human use), collagen-basedVitrigel, Poly D-lysine and sulfo-propyl polymers andpeptide-acrylates in chemically defined conditions. Rapiddifferentiation of ESCs and iPSCs posed a challenge forscientists to maintain them. The alkane-thiol self-assembledmonolayers or streptavidin proteins that are decorated withspecific sequences of peptides, the sticky residue ofarginine-glycine-aspartic acid (RGD), recombinant 36human laminin-511, a component of the natural hES-cellniche and a sequence from the heparin were found usefulfor maintaining them for several months.

XI. Patient-derived iPSCs and DifferentiatedCells for Drug Discovery: Diversion

The stem-cell-derived tissues are being extensivelyused for drug toxicity and other tests on human cells atpreclinical stages of development. Regenerative therapiesbased on stem cells, on the other hand, remain farther outon the horizon. Most major drug companies are involvedin both areas of research. Cellular Dynamics International(CDI), launched by pioneer James Thomson, UW Madison,USA and Cellartis, Switzerland, derived cardiomyocytes,hepatocytes and characterized them and is supplying themto Roche, Pfizer and GlaxoSmithKline. CDI uses iPSCs toderive cardiomyocytes on a large scale and a higher levelof purity (~99)%.

XII. Modeling and Estimation of DesiredNumber of Differentiated Cells for a SpecificVolume of Tissue Injury for Repair

At present, a random number of cells are injected forthe short-term estimation of cell-retention at injection sitesin solid organs and cell-viability. Ideally, we would like toestimate the volume of injury using MRI, preferably in

presence of a contrast medium. Assuming an approximatehalf-life, turn-over and a fixed number of cell-doublingtimes in tissue-culture (4-7 days), and a cell density ofone billion cells/gram of tissue, we could estimate thenumber of cells needed for filling the injury cavity in afixed period of expected repair time. However, thesemeasurements will be re-evaluated with more informationon wound-healing and ultimate tissue-repair (Figure 8).When I asked this question, I got a blank stare from thespeakers, who are invited for filling the vacancy of Directorof NIH Stem Cell Laboratory.

In the previous studies, we were successful in using111In-tagged platelets for the quantitation of the platelet turn-over and nuclear imaging studies for reducing thethromboembolic complications, mainly thromboticocclusion of vascular grafts and other cardiovascularprostheses and stroke (Figure 7 and 9). These studiessuggested their significant reduction with platelet-inhibitordrugs in animal models and patients. We used the 111In-tagged neural stem cells (NSCs) or NTERA2 cells forshort-term turn-over and SPECT-imaging studies. We haddeveloped the universal cell tagger, 153Gd-(mercaptopyridine N-oxide)3: [Gd-(MPO)3)] (Figure 10 and11). Since 153Gd- radionuclide will be used in traceramount, the toxicity is not an issue. At present, we hadoptimized the method of tagging less number of NSCs (10-12 million NSCs vs. billions for platelets and a few hundredmillion neutrophils), we used with 111In-(tropolone)3complex for clinical diagnosis of thrombosis and infection.

Neurodegenerative Diseases:No Good Therapy Options Yet!!!

Multiple sclerosis

Alzheimer’s disease

Parkinson’s disease

Neuropathies

Huntington’s disease

Amyotrophic lateral sclerosis

Clinical Trial (Geron Oligodendrocyte PC):Spinal Cord Injury (2-week after injury)

FDA Safety Issue: No tumor, No ectopic tissue,Viability.

Desperate patients: Cautious optimism: ofcourse some good news of therapy outcome

VOL. 82, NOS. 9–10 291

We achieved our long-term goal of tagging NSCs at highefficiency and maintained their viability aftertransplantation, thus promoting the potential human celltherapy studies (4-10). 153Gd-radionuclide without carriermay also be useful for long-term follow-up of a variety ofcells in patients. The stable Gd used as Gd-DTPA complexis a mixture of several stable isotopes. However, Gd istoxic to cells and tissues at higher concentration, if it isnot excreted rapidly in patients, which needs a healthykidney function.

Until now, the major concerns of stem cell therapyare low efficiency of induction, their incompletedifferentiation, limited capacity of large scale production,their early death resulting in loss of endocrine growth-benefits, loss of potential repair of injured or degeneratedcells and potential tumorigenicity from undifferentiatedcells. However, recent studies suggest that the cell therapyin animal models and patients results in marginal benefitsfrom poor cell-integration in host tissue, low viability andtheir rapid decline49. For the expected benefits fromparacrine growth factors and repair of injured ordegenerated tissues, the transplanted cells must grow, grownew axons, dendrites and blood vessels and multiplysufficiently to replace injured or degenerate tissues ofinterest. They must be active in exocytosis and be amember of integrated neuronal network. Although wedifferentiated the NTERA-2 cells to neurons with retinoicacid, the residual undifferentiated NTERA-2 cells inducedbrain tumors when they were injected intracranially intonon-obese diabetes and severe combined immunodeficientmice (NOD-SCID).

Nuclear imaging studies and staining methodssuggested low abundance of a few cells and their tracksfor a period of a few days to a few months after delivery.Cells tagged with optical reporter probes, magnetic contrastmaterial of iron-oxide nanoparticles and 111In-tagged cells(half-life = 67 hours) demonstrated the localization,retention and limited migration of the transplanted cells.At present, in the absence of method of metrology of cell-viability and a long-term follow-up of transplanted cells,we do not have a data base of their life-time after injection.Since these cells decline rapidly, we used a radionuclideof shorter half-life, e.g. 111In-radionuclide with the tissue-penetrating radiation of 171 and 245 keV gamma rays. Weoptimized the conditions of NSC-tagging with 111In-(tropolone)3. We collaborated with NCI Molecular ImagingProgram for NSC-tagging, injected them in the neuralcortex under stereotactic guidance and imaged them at the

NIH Clinical Center Imaging facility (Figure 11 and 17).They followed my approach for developing the 89Zr-oxineand used the PET-imaging for fate-mapping the bone-marrow derived cells (3).

We did some preliminary imaging studies in nudemice demonstrating the migration of 111In-radionuclide fromthe site of injection on the right cerebral cortex to the leftcortex and a small percent to the rest of the body. Thesestudies suggested that these tagged cells are not useful forevaluation of cell-turnover in solid organs.

XIII. NSC Labeling with In-111 Tropolone andGd-153 Mercaptopyridine N-Oxide

Cell labeling method originally developed for highcell number was modified significantly to label a fewmillion neural stem cells. Tagging efficiency decrease withhigher dilution, increase with higher cell density asexpected. Due to cell-toxicity, we optimized the tagging at3-4 microgram of tropolone. In the absence of many cells,most of the sticky 111In-(tropolone)3 or 111In-(oxine)3,attaches to the wall of incubation tubes and cells must bewashed 2-3 times with ACD-saline to get rid of the taggedcells from free 111In and tagged cells must be transferredto a separate tube for measurement with a dose calibrator(Capintec Inc., NJ). The parameters of NSC-tagging wereoptimized by multiple incubation experiments. We neededonly 6 microgram of tropolone for tagging 10-20 millioncells. The tagging efficiency was 20%. We made thestandard by withdrawing 10 μl with the Hamilton syringe,which was also used for intracranial injection understereotactic guidance (Kopf Instruments, GE).

XIV. Cell Therapy Puzzles

Why host organs do not integrate the transplantedcells? They are true for young and healthy host organsand degenerated mouse retina models. Is there an “Organbarrier for transplanted cell integration”: OBTCI. RNA-Seq database of mouse retina indicates that developingretina express high level of integrins, extracellular matrixproteins (ECMPs), matrix degrading/modifying enzymes,required growth factors and cell adhesion molecules in theembryonic E11 to post-natal P28 days72. However, theseintegrins of transplanted cells do not anchor to the abundantECMPs or take the interface with the RPE cells in theretina. Hence, the unpolarized cells with limited resourcedie quickly in both healthy and degenerate organs. Thereare enough cell therapy challenges for the new generationof young scientists!


XV. Cell Therapy Challenges to Meet PatientHyper-expectations

Heterogenous cell populations in an organized tissueform a dynamic and unique pattern of cell-cell and cell-matrix interactions mediated by mechanical force. Radialmigration of neuronal progenitors and tangential migrationof interneurons, their early symmetric and late asymmetricdivision into neurons, glial cells, astrocytes, result in aclose-knit network and leave no provision for integrationof transplanted cells. The fibrous proteins, e.g. myosins,actins and tubulins along with cell-repellent proteoglycansmaintain this resistant framework and make little provisionfor access to transplanted cells (OBTCI). Additions ofmatrix-degrading enzymes, growth factors and/or platelet-derived vesicles, e.g. alpha-granules, are inadequate for PR-integration in mouse retina. We have to revert the tissue inan embryonic state before the initiation of cell therapy.Certain cell types are very susceptible to early death. In acomposite layer, some cells make promoters along withinhibitors, which must be inactivated during co-culture.Neuronal cell types in excitatory states with higherglutamate must be slowed down with GABA typeneurotransmitters for long-term survival. Dr. Brockessuggested that initial proliferation of progenitor cells oftendepends on the nerve supply, whereas morphogenesisreflects the generation of positional disparity betweenadjacent cells: the principle of intercalation63. If the nervesare removed from the amputated arm of star-fish, they donot regenerate. With a reasonable developmental modelsystem, variable parameters may be evaluated for theultimate Eureka moment!

Cautions for Clinicians, Scientists, Patients andCare-givers: There are “Stem Cell Bazaars” in Caribbeanisland countries, Mexico, Latin America and many countriesin Asia. There many dishonest entrepreneurs who areexploiting the gullible victims in all parts of this planet.One of my colleagues took his mother to a Caribbean islandfor neural cell therapy after a severe stroke for a heftycharge of $25,000. After the first trip, I persuaded thefamily for stopping the crude therapy. I suggested that sheeven might be infected with poor cell-handling and otherdeceiving unpredictable maneuvers. I met a mother of aMS patient, who took two lakh rupees (~$3000) for neuralcell therapy in Mumbai. Even in USA, there are manyunlicensed clinics that are using stromal vascular fraction(SVF) from fatty tissue collected by liposuction. Theyderive SVF for transplantation in victims of Parkinson’sdisease, muscular dystrophy and heart failure. Patients are

charged $5,000 to $50,000 for each study considered asFDA-approved 351 or 361 products65. Since the FDA rulesare ambiguous, these clinics in USA are operating withoutregulation. We must be cautious and vigilant in these newfrontiers sufficiently to avoid any setback from theadventure of cell therapies. I am writing this article foreducating me and the future customers not to be victimized.They are not ready yet for myocardial infarct, stroke,diabetes, retinal and neurodegeneration. Considering thehyper-expectations generated by clinicians and scientistsabout false expectations of cell therapy, there was a warningpublished recently by the senior scientists66.

Acknowledgements

I highly appreciated the support of Dr. AnandSwaroop (NEI, NIH), Dr. R. Puri and Dr. S. Bauer (FDA,HHS) for letting me work as a Special Volunteer and anORISE Fellow in their laboratories. Dr. G. Ma and Dr. J.Catalano supplied the cultured cells and assisted in opticalimaging of mouse brain. Dr. A. Ali assisted in optimizingcell-tagging with iron-oxide nanoparticles, In-111 and Gd-153 tagged neural stem cells and N-TERA-2 cells. Dr. M.Lizak assisted me during MRI in mouse models.

Disclaimer: This article represents only my personalviews and does not represent the view of any of the federalagencies: FDA, NIH, I worked in cell therapy during thelast decade.

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Websites: Cell Therapy ApplicationsAll NIH Clinical Trials: http://www.clinicaltrials.gov/

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http://nihrecord.nih.gov/newsletters/2015/05_22_2015/story2.htm

NIH Brain-mapping Blueprint Initiative in Progress: http://www.NeuroscienceBlueprint.nih.gov

http://stemcells.nih.gov/info/basics/basics4.asp 46

http://nihrecord.nih.gov/newsletters/2015/05_22_2015/story2.htm

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