iPSC Therapy

What are IPSC cells?

The IPSC cells (AKA induced pluripotent stem cells) are hematopoietic stem cells. Their role is to give rise to other blood cell types. They are normally found in the bone marrow of (pelvis, femur, sternum), umbilical cord blood and peripheral blood. They are quiescent in nature which gives them the ability to survive in the hypoxic environment of the tissues they reside in. However, when they are “woken up,” they will have a high self-renewal capacity, multipotency and increased mobility.

How are IPSC Cells made?

RGCC uses magnetic beads and FACS (fluorescence-activated cell sorting) isolation methods to identify and isolate CD34 cells from peripheral blood. Cells are then cultivated with media and supplements that induce greatly the proliferation of your hematopoietic stem cells. Once their numbers are adequate, they are sent directly to the patients’ practitioner for infusion. They can be kept in LN (Liquid Nitrogen) tanks until requested. The thawing from LN needs to take place very fast (in just a few minutes) so that the iPSC loss will only be around 2.5 to 3%. This procedure is very well known for those already using autologous transplantation after.

The platform used is based on the fact that from these cells you may get several lines of differentiation by reprogramming. Therefore, they are able to be effective for more than just one tissue type (vascular, joint, etc.).

Quality control is performed on every step of the process until the final product is ready and safe for use. QC involves monitoring the cell culture for microbial contamination, and the cells themselves for specific marker expression.

Are these embryonic stem cells?

These are not originated from embryos but from each individual’s blood and therefore are NOT embryonic iPSCs.

Are the IPSC genetically altered?

The cells have had no genetic (DNA) tampering whatsoever and will have a document of sterility accompany each sample. You will be given the estimated number of iPSC’s received for each person. This is helpful to guide the practitioner as to how many cells are given especially in joint injection therapy.

What is the goal of IPSC?

• The goal is to replace damaged tissue.

Are IPSC useful for cancer?

IPSC’s have no direct anticancer effects. However, in many studies they have been used in combination with chemo (Mega Therapy) or as an adjuvant arm in immunotherapy.

IPSC’s can be harvested prior to chemotherapy and used post treatment to help replenish and repair. Their primary use is to replace damaged tissue.

Note: Only genetically engineered (DNA manipulated) IPSC’s have anticancer effect but they are not allowed to be used in the clinical field at this time.

What kind of conditions can be treated with IPSC?

• Non-cancer: Thalassemia, Sickle Cell Anemia, Aplastic Anemia, Fanconi Anemia, immune deficiency syndromes, Alzheimer’s, Parkinson’s, and Glaucoma.
• Repair of damaged tissue from previous diseases, medical conditions, degeneration, surgery, cancer treatments, etc.

Are there any contraindications to IPSC?

• Under 18 years old
• Arrhythmia
• Hypertension
• Dyspnea
• Active autoimmune diseases
• Active infections including Lyme or Lyme related coinfections
• Recent cytotoxic chemotherapy and/or radiotherapy
• Cachexia
• Scar tissue in the area of administration.

Are IPSC safe?

• IPSC’s are generally considered very safe.

Is there research on IPSC for IV protocols?

• A: Yes. See below:
  www.ncbi.nlm.nih.gov/pmc/articles/PMC5118044/
  www.ncbi.nlm.nih.gov/pmc/articles/PMC6332789/

What is the administration schedule of IPSC?

• 2 doses:
  • 1 dose - Day 0
  • 2nd dose - Day 14.

Important:

• These are live cells and need to be kept cold. Watch tracking closely to ensure the therapy will be arriving during office hours.
• Put into the refrigerator upon receipt. Do not freeze. The therapy must be administered by Day 7 at latest (Shipment from lab is on a Thursday (Day 1) administration must occur by Thursday of the following week (Day 7).
• After Day 8 the cells are no longer viable and cannot be administered.

What IS included in the IPSC package and what is the cost?

• Package Price: 4900 Euro
• Package Includes:
  • 2 doses of IPSC.

What pre-requisite tests are required to establish baseline for IPSC?

• Recommended to have confirmation that patient does not have an active infection (CRP >3)
• Active infections –(includes active Lyme and coinfections) - Indicators:
  • WBC >10,000
  • Lymph > 20%
  • CRP >3.0
• Recommended to have confirmation that your patient is not highly inflamed:
• High inflammation - Indicators:
  • CRP>3.0)
  • Sed Rate >29 mm
  • AGP- a1-acid glycoprotein >110
  • ANC <2500 (indicator of immune insufficiency)
  • Hemoglobin <8.5
• Recommended to have confirmation that patient does not have active auto immune condition

What is the recommended follow up for IPSC?

Clinical monitoring - depending on the regenerating tissue or organ:

• Anatomical diagnostic, CT, MRIs, US etc.
• Functional testing, LFT, enzyme activity, eGFR etc.
• Direct cellular or molecular monitoring. Detect the level and the activity of progenitor cells of the damaged tissue.

How much blood is required for IPSC?

• Dose 1 and 2 of IPSC (Draw 75-90 ml) 3 vials.

What pre-medications are required for IPSC?

PRE-MEDS: Administer pre-medications prior to each dose of the THERAPY.

For IV:

• Mandatory: 4 mg dexamethasone I.V. in a 20-50 ml rapid drip saline solution or slow bolus push.
• Recommended: H1 Inhibitor – IV or oral.

For Joints: 1 – 2% Lidocaine prior to injection.

What needs to be avoided prior to the IPSC?

• Avoid other therapies for 14 days prior to IPSC blood draw and actual administration.
• Recent cytotoxic chemotherapy and/or radiotherapy – allow a window of 2-3 weeks
• Medications like cytokines/hormone therapy (specifically Estrogen and HGH)

What needs to be avoided after IPSC?

• Avoid other therapies for 14 days after the administration of the IPSC
• Recent cytotoxic chemotherapy and/or radiotherapy – allow a window of 2-3 weeks
• Medications like cytokines/hormone therapy (specifically Estrogen and HGH)

Are there any possible adverse reactions with IPSC?

The adverse reactions are not considered life threatening (NCI grade 1) – expect high blood pressure (last couple of days), a kind of arrhythmia (not ventricular fibrillation), a small degree of bronchospasm; may develop symptoms of photophobia, vomiting (if pressure builds up in the CNS) – can monitor blood pressure.

• Cardiovascular (hypertension, arrhythmia etc.) (47.6%)
• Pulmonary (dyspnea, hypoxia) (13.6%)
• Constitutional (3.4%)
• Neurologic (1%)

How to Administer ISPC

IV Application

• Bolus mode of application (for any condition of damaged tissue (with the exception of CNS)
• Premedication of: 4 mg of dexamethasone: helps maintain vessel wall integrity
• Parallel infusions: Lactated Ringer’s or Normal Saline for any IV application.

1. Gently pull the liquid from the crimp vial into the appropriate size syringe using a (a must) 21 G x 2-inch needle. The total volume of liquid is ~ 5-6 ml. Best approach is to draw the cells out by leaving the crimped vial sitting right side up, otherwise you may leave a lot of liquid (& stem cells) in the rippled underside of the rubber top.
2. Suspend the syringe into a 250 ml infusion bag of Lactated Ringer’s or Normal Saline, for all IV applications.
3. Apply the infusion as a rapid drip lasting about 30 to 60 minutes.Check for development of fever the next 6 to 12 hours. If the patient develops fever, try to counteract the fever with NSAID.
4. Check for development of fever the next 6 to 12 hours. If the patient develops fever, try to counteract the fever with NSAID.

Joint Injections

• Follow sterile technique in ALL cases of joint injections! Ultrasound-guided can be very helpful and often more effective
• For experienced clinicians in joint injection therapy - follow your protocols regarding needle size, etc.
• Premedication of 4 mg of dexamethasone is recommended – (at doctor’s discretion)
• Premedication of joint with 1% - 2% lidocaine in order to counteract the discomfort, especially when ultrasound-guided into deeper and/or larger joints.

Process: Gently pull liquid from the crimp vial into the appropriate size syringe using a 21 G x 2” needle. The total volume of liquid is 6 ml. Best practices is to draw the cells out by leaving the crimp vial sitting right side up, otherwise you may leave a lot of liquid & stem cells in the sealed rippled underside of the rubber top. Change the -needle of the syringe to a thinner 22 G-27 G depending on need. Use approximately 0.25-0.5 ml to each joint, your call. Try to apply the injections to multiple areas of the larger joints if not using ultrasound guided.

In Wave Mode Application: CNS Target Only

• Premedication of: 4 mg of dexamethasone: helps maintain vessel wall integrity
• Parallel infusions: Mannitol (dose determined for individual patient) application for the CNS damage only.

1. Pull the liquid from the crimp vial into the appropriate size syringe using a (a must) 21 G x 2-inch needle. The total volume of liquid is ~ 6 ml. We prefer to draw the cells out by leaving the crimp vial sitting right side up, otherwise you may leave a lot of liquid (& stem cells) in the sealed rippled underside of the rubber top. The reason for the 21 G x 2-inch needle
2. Connect the syringe into the 3-way and apply 1 ml and after that switch the 3-way to the mannitol infusion and allow to drip in for 10-15 minutes
3. Gently swirl the syringe so that the liquid inside the syringe will be homogenous again
4. Repeat step 2.
5. Continue with another 15 minutes with mannitol infusion
6. Repeat steps 2-3 until you apply all 6 ml and then allow the infusion of mannitol to continue until done
7. Ask the patient to observe their temperature for the next 48 hours for any increase.

IV Technique – CNS Target Only

1. Start with 150 ml of NS
2. Add 6 grams (1/2 bottle) of mannitol
3. Drip for 30 minutes
4. Have a second 150 ml bag of saline ready to hang when mannitol is finished
5. As soon as the mannitol is done - connect the 2nd 150 ml of saline to the IV line and flush line with about 10-15 ml of saline
6. Then through the 2nd access port on the IV line:
7. First stop the saline after the flush
8. Slowly push (tap) only 1 ml of IPSC’s at a time
9. Restart the saline and run slow for 10 minutes
10. Repeat this process until all remaining stem cells are given
11. The complete process should take about 1 hour or slightly longer.

Note: These instructions are not intended to replace appropriate training for the administration of stem cells by the physician.

FAQ – ISPC

Why did IPSC change from one dose to two doses?

The reason for the new requirement of two doses of the IPSC cells is due to the need to ensure that the required differentiation of stem cells actually occurs. Dr. Papasotiriou states that it is assumed by most doctors that this will occur automatically however, in reality, this is not always the case. In 10-12% of the time there is a risk that the patient can develop a benign tumor. Although benign, this tumor can still cause symptoms for the patient.

Additionally, although the two doses are ordered at the same time, they will not be shipped at the same time. The second dose will ship in time for the second dose to be given at two weeks. The second dose is the one that will help ensure that the differentiation actually occurs.

Q: How does IPSC compare to HUC, HSC, CD34+ve?

A: To reply to your questions concerning the autologous stems cells we have to clarify the following: Stem cells are classified as follows:

Totipotent stem cells: They are able to differentiate to any kind of tissue without limitations and no barriers. They have limitless capacity of replication, and the telomeres are exact copies in every cell division without shortening (they have the complete ability to use the enzyme telomerase to replicate 100% of the telomeres). Stem cells from specific lines of cells, HSCT (Hematopoietic stem cell transplantation), Somites, etc. These cells are able to generate a specific kind of tissue cells, but no other tissues. They have an increased life span but their telomerase -hTERT- have a lower than 100% catalytic activity and eventually the telomeres will shorten, and these stem cells will die in time. Essentially, these are finite differentiated cells with a specific limit of replication, life span and the telomeres are shortened with every cell division. We have to classify cells of interest. The umbilical cord derived stem cells are closer to the second category and therefore their telomerase is deficient, and the telomeres are shortened from one generation to the next, hence the length of telomeres becomes important. This is the reason we identify them as CD34+ve only cells (more like HSC). In our case the IPSC are reprogrammed from more differentiated to less differentiated stem cells without genetic engineering (NO DNA manipulation). Therefore, the final cells are totipotent stem cells, and their hTERT has 100% efficiency and the telomeres do not shorten from one generation to the next. Hence, the comparison between umbilical cord derived stem cells vs induced stem cells (IPSC) derived from the same patient is more favourable than the second kind of cells for the following reasons: 1. They are limitless in life span, 2. They are identical to the HLA (Human leukocyte antigen) from each individual patient. 3. They are much higher in number after processing. I hope that I’ve manage to give you more insight concerning this topic, but in case you have further questions please do not hesitate to ask.

Best regards, Ioannis Papasotiriou, MD, PhD, C.Cy.