Duncan Ross Ph.D.Ph.D. Immunology and Biochemistry

Stem Cell TransplantationUniversity of Miami

This principls of cell transplantation have been known for over 60 years. If you know how to successfully transplant cord blood, you certainly know how to

unsuccessfully transplant it.

Shlomchik 2007

AMD-100

Stem Cell Transplantation

AMD-100GM-CSFG-CSF

Bone Marrow Transplantation

Day 0

DAY -4

Day -4

AHCT/BMT: THE ORIGINAL STEM CELL TRANSPLANT (since 1957)

A Murine Model of MHC Matched Myeloablative Minor Antigen Mismatched T cell Replete AHCT

0 hrs

Recipient: C3H.SW H2b

10.5 Gy (Day 0)MYELOABLATIVE 1050Gy(45.2+Ly9.1+)

Donor: B6 H2b

2.3x106 CD4+&CD8+ (45.2+,Ly9.1-) B Cell Depleted Splenocytes

6x106 TCDBM (45.1+Ly9.1-)

MHC “Matched”

Recipient: BALB.B H2b

7.5 Gy (Day 0)MYELOABLATIVE 750 Gy (45.2+Ly9.1+)

HOW WE MEASURE INFLAMMATION IN MOUSE MODELS

REGULATORY T CELLS CAN AMELIORATE CLINICAL GVHD IN A MIHA MISMATCHED TRANSPLANT MODEL

1 Treg:1 Tcon

B6->C3H.SW 10 .5Gy Same DaySingle Dose

6x106 TCDBM2.3x106 CD4+&CD8+ Tcells+ 1 or 2x106 Treg

PTC PTC

0 3 4 5

MESENCHYMAL STEM CELL (MSC)THE GOLDEN GOOSE

Identified by Friedenstein in 70’s, expanded by Caplan in 80’sMSC have been shown to suppress immune responses, enhance

neovascularization, and initiate fibroblast and keratinocyte growth

HGF FGF IGF IL-10 PDG-ETGF-βGrowth Anti-Inflammation

CD90+CD105+CD73+

MESENCHYMAL STEM CELLS ARE PERICYTESPROVIDING INSTANT ACCESS TO VEINS AND ARTERIES - THE SECRETORY HIGHWAY

MESENCHYMAL STEM CELLS CAN AMELIORATE CLINICAL GVHD IN A MIHA MISMATCHED

TRANSPLANT MODEL

A STEM CELL STRATEGY: WOUND HEALING

CELLS APPLIED IN A MATRIX IMPROVE HEALING

MORE CELLS =IMPROVED WOUND HEALING

CELL DISADVANTAGES

• Cell persistence is limited (Days)• Autologous cells require time to prepare –

unsuitable for burns• Third party cells would lead to rejection

(privileged?)• Cells are not FDA approved (unless they are IND

approved)

SOLUTION: EXOSOMES (CELL SECRETIONS)

• Cell-Cell communication is mediated by ~100nm lipid vesicles comprising both proteinsand mRNA signals that are secreted by stem cells

• mRNA allows neighboring cells to produce required proteins without the need to initiate DNA translation = FAST

Anderson 2016

A BUDDING INTEREST

Components of a cell Components of an exosome

DNAMessenger RNAMicro RNAProtein

(targeted)Messenger RNAMicro RNAProtein

Exosomes are not small cells

MSC mRNA

PROTEIN

miRNA

Fibroblast

EXOSOME SECRETION AND UPTAKEDNA

Cytoplasm

Nucleus

IGF-1

MSC

DNA

mRNA

miRNA FibroblastNucleus

Cytoplasm

EXOSOME MEDIATED PROTEIN EXPRESSION

PR

x

IGFRIGF-1

EXOSOME UPTAKE GFP LABELED EXOSOME UPTAKE BY HMEC

Human Mammary Epithelial Cells

EXOSOMES: OPPORTUNITY

• Can be sterile filtered as they are smaller than bacteria

• Tested for sterility and endotoxin levels• Can be concentrated• Cannot be rejected by a third party• Can be frozen

STEM CELL SECRETIONSSECRETIONS ALONE SHOW BENEFIT IN SOME MODELS

6 months cigarette smokePlus 2 months MSC treatment

MSC SECRETIONS ALONE

EXOSOMES CAN BE MORE EFFECTIVE

One cell may persist for 3-7 days outside of its environment on a patient’s wound (joint,

lung) and will manufacture 3-7x amount of factors (growth, inflammation)1 2 3 4 5 6 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Secretions (exosomes) from a cell growing in the laboratory can be collected indefinitely *and manipulated

7x

25x

A FORTUNATE EXPERIMENT - Halloween 2014 • Dr. Ross was present at an event with the following

patient who inflicted 2nd degree burns on himself by throwing water on a gasoline fire.

• Normal cell expansion work by Dr. Ross had produced a large amount of stem cell secretionswhich were frozen in the laboratory for future use for inflammatory diseases (COPD, MS).

• Dr. Ross was able to provide these secretions to the patient within 12 hours of the burn

Day 0 Day 1

2nd degree burn victim

2nd degree burn victim

Day 7 6 months

Between 200-300proteins are secreted within exosomesranging from growth factors to anti-inflammatory cytokines

• Adult MSC secrete ~110• Placental Mesenchymal Stem Cells secrete over 300 growth

factors

WHAT MAKES OLD NEW AGAIN

AMNIO vs EXOA Tale of Two Secretory Regen Products

The secret is in the growth factors

“The key message that I want everyone here to understand is that regenerative medicine is not a one shot deal. Whether you are going to use BMAC, Adipose, Amniotic fluid, PRP (etc) you have to recognize that this is not a one shot deal. You can’t just inject this all in at once and say good day. You have to progressively apply your tools”

- Arnold Caplan Ph.D. TOBI 2017

EXOSOME COMPONENTSAMNIO VS PEXO

Amniotic fluid and exosomes are anti-inflammatory, but in differentand complimentary ways

ANTIIL-1

TNF Temporary

PermanentTGF-β3 converts Inflammatory T cells to Regulatory T cells

AMNIO

PLACENTALEXOSOMES

EXOSOME COMPONENTSAMNIO VS PEXO

Amniotic fluid and exosomes are pro-stimulatory, but in different andcomplimentary ways

AMNIO

SCF

EXOSOME GROWTH FACTORS

P EXOSOMES ALONE SIX WEEK POST

Insulin-Like Growth Factor-1 Sustains Stem Cell–Mediated Renal RepairBarbara Imberti,* Marina Morigi,* Susanna Tomasoni,* Cinzia Rota,* Daniela Corna,* Lorena Longaretti,* Daniela Rottoli,* Federica Valsecchi,* Ariela Benigni,* Jun Wang,* Mauro Abbate,* Carla Zoja,* and Giuseppe Remuzzi*†

*“Mario Negri” Institute for Pharmacological Research and †Unit of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Bergamo, Italy

MECHANISM OF OLD VS NEWRescue of drug induced kidney damage

Studies in rats with ischemia-reperfusion injury have suggestedthat the renoprotective action ofMSC is due to their ability toproduce high levels of growthfactors such as vascularendothelial growth factor,hepatocyte growth factor, IGF-1and inducing antiapoptoticsignals.17

IGF-1 was implicated as an important mediator ofcardiac and kidney regeneration in models of AKI.24–28

MSC REPAIR RENAL DAMAGE VIA EXOSOME STEM CELL EXOSOME MEDIATED TRANSFER OF IGF-1

Control Drug Repair

STEM CELLS AND DEVELOPMENTVolume 22, Number 5, 2013© Mary Ann Liebert, Inc.DOI: 10.1089/scd.2012.0266

Transfer of Growth Factor Receptor mRNA Via Exosomes Unravels the Regenerative Effect of Mesenchymal Stem Cells

Susanna Tomasoni,1 Lorena Longaretti,1 Cinzia Rota,1 Marina Morigi,1 Sara Conti,1 Elisa Gotti,2 Chiara Capelli,2 Martino Introna,2 Giuseppe Remuzzi,1,3 and Ariela Benigni1

Results

• We learned a few things: 1) IGF-1 is required for Proximal tubule epithelial cell regeneration 2) IGF-1 is not present in exosomes derived from the cells they worked with 3)IGF-1 Receptor was transferred and could be found in cells that did not express it when incubated with the MSC exosomes

SEEMS PRETTY CLEAR…HOWEVER

• Human (h) MSC were obtained from BM aspirates collected from adult

subjects (30–40 years old) within the Hematology Division,

• Azienda Ospedaliera, Ospedali Riuniti di Bergamo

Let’s corroborate their findings.

FURTHER CORROBORATION

• The previous results were from ~40 yr old adult bone marrow. In the adult, MSC are required to maintain immune function NOT to cause growth.

• What about a child?

Elucidating the Secretion Proteome of Human Embryonic Stem Cell-derived Mesenchymal Stem Cells*□S

Siu Kwan Sze‡, Dominique P. V. de Kleijn§¶, Ruenn Chai Lai , Eileen Khia Way Tan§, Hui Zhao , Keng Suan Yeo§, Teck Yew Low**, Qizhou Lian§, Chuen Neng Lee‡‡, Wayne Mitchell§§, Reida Menshawe El Oakley‡‡, and Sai-Kiang Lim§¶¶

IGF-1 PROTEIN IS IDENTIFIED IN EXOSOMES OF EMBRYONIC MSC

Emrbyonic~placental stem cells secrete far more protein and mRNA transcripts than adult cells. Many of these transcripts are regulatory (e.g. Histone Deacetyl Transferase) and cause downstream epigenetic changes that impart a lasting regeneration of cells.

WHAT ABOUT CORD BLOOD?

THANK YOU

Duncan Ross Ph.D.305.454.STEM (7836)info@duncanross.net

Prepared by Duncan Ross Ph.D. – founder Kimera Labs

‘I don’t know what anybody else does, I just know what I do’

– Douglas Spiel M.D. Medical Director Kimera Labs Inc.

What is Amnio2?• Amnio2 is an advanced form of amniotic fluid that is both

scientifically more potent and meets newly released FDA regulatory guidance towards a ‘361’ product. The laboratory processes were recently inspected by the FDA (Nov 2017) and no further regulatory action was requested.

• Amnio2 is processed from FRESH amniotic fluid and frozen once prior to shipment and assignment of expiration date. Many Amnio fluids have spent years in the freezer before being bottled and shipped and the expiration date is erroneously assigned from the bottling date.

• Amnio2 is acellular amniotic fluid – there are no cells included in the final product.

Why is it called Amnio2?• The ‘2’ in Amnio2 denotes a 2 fold greater amount of amniotic

fluid volume per vial than some competitors/what competitors would need to create to be scientifically sound. Cell preservation protocols are written for cells, not whole fluid.

Competitors(ideally)

1cc 1cc50%~20% DMSO/albumin

or 50% glycerol

50% Amniotic fluid

100%

Am

niot

ic F

luid

How is Amnio2 made?• Amniotic fluid is collected from elective C-section donations and shipped

immediately to our processing facility in Miami. The fluid is then processed to remove live cells and vernix, the waxy substance that coats the newborn and can cause embolisms if introduced into the bloodstream.

• 80% of deaths in childbirth are attributed to amniotic fluid entering the bloodstream ending in a vernix caused embolism.

• The fluid is then packaged and frozen, NOT CRYOPRESERVED, at -80o Celsius until delivered overnight to the clinic.

• Cryopreservation implies the use of a cryopreservant (DMSO) which we do not need to include, as there are no cells to begin with.

• Because of this, Amnio2 can be stored in a clinic’s regular freezer for a period up to 6 months, or in a <=-80oc freezer for much longer periods. A year or more.

Vernix is a waxy element: after step one processing, it is still apparent and requires further processing to remove completely

What does acellular mean? The cells are the magic dust, right?

Acellular means there are no cells in the Amnio2 product. Stem cells present in an amniotic fluid treatment presupposes the presence of the cell when administered to the human. We will try toclarify that question in the coming slides.

What about the stem cells in typical commercial amniotic fluid?• The functionality of stem cells in amniotic fluid, as sold today, is a myth• Literature refers to uses of amniotic fluid collected early in gestation and

obtained via amniocentesis• Research shows that 250cc of fresh C-section delivered amniotic fluid,

when introduced immediately into culture, only yields approximately 40 stem cells. That means there is 0.16 stem cells per 1cc of the competitor’s amniotic fluid.

• In order to preserve that 0.16 stem cells, one must add 50% DMSO/Serum mix (cryopreservant) by volume thereby halving the amount of amniotic fluid present in 1cc. That now means there is 0.08 stem cells per 1cc. If a product has less DMSO than that, the cells will not survive thaw as they will crack open when frozen. An individual manufacture might say they use less DMSO, but can they provide studies showing the growth of cells after thaw?

• Further, DMSO is lethal to cells at room temperature, so by the time the clinician thaws the vial to inject it, the 0.08 stem cells will be killed.

• Lastly, DMSO may cause an allergic reaction.

But I was told you can see millions of cells under a microscope?

• The cells visible under a microscope are in fact the mother’s epithelial cells. These cells will both be rejected and cause inflammation due the damage they receive in handling

Problem 1: Allogeneic (non-self) cell rejection

• In order to transplant a kidney, a donor cell must not only be matched to the recipient but the recipient must be immunosuppressed. Without immunosuppression, every kidney transplanted would be rejected, even a matched kidney. This is due to minor transplant antigens.

• To this end, any skin graft from a third party without immunosuppression will be rejected.

• The cells in amniotic fluid are mostly ‘skin’ cells with less than one stem cell per ml (cc). They will be rejected within hours-days.

The premise of the use of amniotic fluid, cord blood cells etc, as sold today, is that it contains stem cells that are protected from rejection. This is simply not the case!

ALLOGENEIC CELL REJECTIONThe following experiment demonstrates a similar situation (splenocytes) of the persistence of third party cells when injected into an unrelated individual as occurs when amniotic fluid is used in a typical patient. The cells are there on the first day and gone by day 3 after injection.

Elkin et al Am J Blood Res. 2011; 1(2):

Cells from a twin still At Day 3

Cells from a stranger Visible at day 3

WHAT DOES THE LITERATURE SAY?

While many groups refer to the utility of stem cells in amniotic

fluid, the literature cited generally refers to pre-term cells obtained

during amniocentesis.

Other reports look at cells prior to cryopreservation, plated within 3-

4 hours after child birth at an academic institution

AMNIO STEM CELL ARTICLE TAKEAWAYS

• Moreghabi et al. Recovered 0.4 LITERS of amniotic fluid. They state that translates to 7 million nucleated cells per LITER (one never recovers a liter of amniotic fluid, so they actually recovered 2.8 million cells total, avg)

• Of these cells, 420 formed colonies. Meaning 420 had the potential to be stem cells, while a smaller percentage showed stem cell surface markers.

• That’s less than one stem cell per ml, and this is PRIOR to cryopreservation. Similar results have been given at the AATB and other published works. Often, there are no colonies recovered, 40, or up to 1000. This is still a low number.

PROBLEM 2: WHAT DOES THE FDA HAVE TO SAY ABOUT AMNIO?

• Recent FDA guidance documents have clarified that the transplantation of unrelated cells of any kind cause a product to be termed a 351 product and therefore requires a biologics license to be acquired prior to use. 361 products can be used in the clinic as a HCT/P product with no issues at this time

• Kimera labs and the Amnio2 product production schema were recently inspected by the FDA and no regulatory follow up was taken.

• We at Kimera understood the FDA’s concern with the transplant of DNA containing cells that will ultimately be rejected and therefore our product has been in compliance since we first began manufacturing amniotic tissue. This why the FDA did not cite us during their recent inspection.

PROBLEM 3: WHAT IS CRYOPRESERVATION AND HOW DO YOU FREEZE CELLS?

• Cryopreservation is the process of COOLING cells in the presence of a cryopreservant that can preclude the formation of ice crystals inside the cells, thereby protecting them and allowing them to be reanimated.

• Cryopreservation is therefore a component of the following

TIME, TEMPERATURE, AND CRYOPRESERVANT (DMSO, GLYCEROL, etc)

DO YOU LIKE FRESH ORANGE JUICE OR JUICE FROM CONCENTRATE?

• If we agree that even acellular Amniotic Fluid consists ofprotein and RNA then each freeze thaw cycle diminishes the quality of the product. We should also agree that unless proteins are stored in liquid nitrogen, degradation of the product will occur whether frozen or not.

• In the past month we have been offered collected but not processed (not yet vialed) amniotic fluid from a year old birth and a two year old birth (we refused both)

• This means, that whereas we place a one year shelf life on our product, if a competing lab gives you a one year shelf life, it may already be two years past that shelf life (if stored at -80oc)

ROOM TEMPERATURE AMNIO

Some amnio providers sell a room temperature liquid AMNIO product. When we saw this, we told the distributor this was impossible. We then spent $7000 to prove it. Room temperature Amnio (left column) came back with zero growth factors across the board when compared to Kimera amnio and Kimera exosomes.

Popular methods of cryopreservationTwo methods can be used. Each diminishes the volume of amniotic fluid

• Glycerol

PROBLEM 4: DMSOWhen DMSO is used in cell cryopreservation in the laboratory, is removed as QUICKLY as possible. In fact, the DMSO cannot meet the cells at room temperature or it will kill the cells. Further, if not slowly diluted out, it can cause cell rupture due to osmotic pressure. It is very difficult to do this and certainly these procedures are not followed in the clinic.Further there are side effects associated with DMSO• Allergy. DMSO can induce a release of histamine. Common allergic reactions

include flushing, rash, and edema.• Gastrointestinal. Affecting the limbic-hypothalamic pathways, DMSO can result in

symptoms like nausea, abdominal pain, and emesis.• Renal. The incidence of kidney-related ARs is comparatively low, but includes

symptoms like hemoglobinuria, proteinuria, and urine incontinence.• Cardiovascular. Symptoms include hypertension, arrhythmias, tachycardia, shock,

cardiac arrest, and seizure.• Neurological. Symptoms include bilateral thalamic infarction, blurred vision, severe

encephalopathy, cognition, muscle weakness, and numbness.• Hepatic. Symptoms include progressive jaundice.• Source http://www.bloodjournal.org/content/bloodjournal/75/3/781.full.pdf?sso-

checked=true

PROBLEM 5: CRYOPRESERVATION TIME AND TEMPERATURE

• The speed with which you freeze a solution of cells is as important as the solution you freeze it in. Unless a cell’s temperature is lowered at exactly 1-10 degrees celcius per minute, ice crystals will form outside the cell or the cell will burst.

• If amniotic fluid is frozen together as one big bolus, there is no chance of obtaining this freeze rate, thus the cells will be damaged before they are even packaged.

DRY

ICE

& E

THAN

OL

Cells must be frozen at a controlled rate to avoid damage. Individual vials must be surrounded by ethanol and frozen at exactly -80o Celcius.

FREEZING CELLS too slowly will cause ice

crystals to form

OUTSIDE the cells, crushing them (as seen

to the left).

If cells are frozen too quickly,

they will explode. Freezing must take place

at a rate that allows water to leave cells

without damaging them

Suffers from none of the aforementioned issues

How you think Amniotic fluid is processed

• Processed and Vialed within 24-48 hours of birth

• No need for cryopreservation• Can be stored at -20 in a normal

freezer for up to 6 months. One year in colder freezers.

• FDA COMPLIANT

COMPETITOR• Possibly frozen up to two years

before packaging• Must include a volume reducing

cryopreservant that may be toxic to humans or cells

• Contains cells that will be rejected• Not FDA compliant under new

regulations

Well then why does regular Amnio work? and why does Amnio2 work?

• While we have explained the myths about cell requirements in regular amnio, there is a great bit of anecdotal evidence that non-Amnio2 amniotic fluid works in pain and tissue regeneration. We do not dispute this. We have all also heard of patients experiencing pain from regular amnio injections, along with the patients who benefit.

• The functionality of regular amniotic fluid can be attributed to the growth factors found in the fluid. Amnio2 has these same growth factors, but up to TWICE as many, and more carefully preserved. Therefore, one can consider Amnio2 to be twice as effective.

• Amnio2 further benefits from its ease of transportation and storage due to that fact that it retains its function at regular freezing temperatures.

Amniotic fluid

Amniotic fluid is anti-inflammatory

ANTIIL-1

TNF Temporary

AMNIO

Il-1 and TNF are major inflammatory molecules. Amniotic fluid binds these cytokines and neutralizes them.

Amniotic Fluid

Amniotic fluid is pro-stimulatory, much like PRP, and contains many of the same factors, without the negative inflammatory effects of PRP

Platelet derived growth factorTGFB1 Cartilage growthHepatocyte Growth FactorStem Cell Factor (Hair regrowth)VEGF (Vasculature growth)

Is Amnio2 better than PRP?

• Amniotic fluid is similar to PRP in the types of growth factors it contains.

• Unlike PRP, which causes inflammation, Amnio2 can dampen inflammation. It’s easy to believe. You wouldn’t want to stop inflammation in a cut/wound would you? But you would around a child. Platelets plug wounds.

• PRP contains bacterial DNA derived from mitochondria that spills out into the environment and causes inflammation, again reinforcing the function of PRP in healing due to a wound. There is no way to centrifuge around this/prepare the PRP to be anti-inflammatory.

• The annual TOBI conference acknowledged that PRP causes inflammation and described methods to ameliorate it, all of which included differential centrifugation.

Oh, and one more thing…(this might be the most important selling point for a surgeon)

• Amniotic fluid is extremely anti-microbial.• This is due to defensins, which are proteins

that break down bacterial cell walls, essentially poking holes in the membrane.

• These proteins are also found in a mother’s milk.

• Defensin ‘copies’ are the new hope to counteract bacterial antibiotic resistance and synthetic versions are in clinical trials (Think MRSA-defensins work against it).