Clinical Applications of Mesenchymal Stem Cell Therapy

Dr R R Sharma MDProfessor

Department of Transfusion MedicinePostgraduate Institute of Medical Education and Research,

Chandigarh, India

What are Stem Cells?

Stem Cells are extraordinary because:

• They can divide and make identical copies of

themselves over and over again (Self-Renewal)

• Remain Unspecialized with no ‘specific’

function or become . . . .

• Specialized (Differentiated) with the potential

to produce over 200 different types of cells in

the body.

Stem Cells are like magnets

Stem Cell

Society ResearchInstitutions

Investors Public/Private

Stem Cell Technology biotechs

Pharmaceutical Industry

Equipment Providers

Cryobiologists

Regenerative Medicine

Media

Academic leaders

Basis of “Stemness”

Asymmetric Division

Daughter stem cell Daughter transit-amplifyingor intermediate cell

Repopulates stem cell pool

Proliferative, Migratory andDifferentiating capabilities

Intermediate cell progenitors

Mature tissue

Maintenance of “Stemness”

The length of the telomere determines the ability of the stem cell to keep from differentiating and aging.

Thus, stem cells contain a lot of telomerase.

Telomeres contain highly conserved G-rich repeats

Sources of Stem Cells

Totipotent : Zygote, Morula

Pluripotent : Embryonal stem cells (inner cell mass)

: Amniotic epithelical cells

: Fetal stem cells: Umbilical cord stem cells: Adult stem cells

Multipotent : Fetal stem cells: Umbilical cord stem cells: Adult stem cells

- bone marrow, peripheral blood, brain , eyes , heart, lungs , kidneys, GIT, pancreas, liver, fat, breast, ovaries, prostate, testes

Hematopoietic Stem CellsBone marrow (1% to 3%)Mobilized peripheral blood (0.01% to 1%)Umbilical cord blood

Mesenchymal Stem/stromal CellsBone marrow (0.0001% to 0.01%)Umbilical cord blood (0.0001% to 0.0003%)Placenta (better source)

Tissue specific

Adult Stem Cells

Mesenchymal Stem /stromal Cells

• 1st described in 1968 (Friedenstein)

– Adherent, clonogenic, fibroblastic marrow cells

• Multiple sources

• ISCT definition (2006)

– Plastic adherence

– CD73, CD90, CD105 (+); lineage markers (-)

– In vitro differentiation to bone, fat, cartilage

– Lack of HLA-Class II Antigens(Immunopriviliged )

Adult MesenchymalStem Cell (MSC)

Chondrocyte MyoblastFusion

StromalFibroblast

Tenoblast PreadipocyteOsteoblast

Mesengenesis

BONE STROMA TENDONCARTILAGE MUSCLE ADIPOSE

The global landscape of stem cell clinical trials

Matthew D Li, Harold Atkins Regen. Med. (2014) 9(1), 27–39

ClinicalTrials.gov & WHO’s International Clinical Trials Registry

Matthew D Li, Harold Atkins Regen. Med. (2014) 9(1), 27–39

Goal of stem cell therapy• Regeneration (n=916) • Cell therapy (nonregenerative) (n=126) • Gene therapy (n=96) • Stem cell collection/mobilization (n=30) • Bioscaffold (n=15)• Immunotherapy (n=13)

Stem cell type • Hematopoietic (whole marrow, CD34+, D133+ or mononuclear fractions) (n=432) •Mesenchymal (n=432)• Endothelial progenitor cells (n=69)•Other (n=69)• Neural (n=22) •Unspecified (20) •Limbal (16)• Embryonic (6) •Cardiac (6)

Principle disease/condition targeted •Cardiovascular disease (n=278)•Neurological disease (n=169)• Cancer (n=97)•Liver disease (n=67)•Bone condition (n=65))•Other (n=56)• Immunodeficiency and other nonmalignant

hematologic conditions (n=49)•Gastrointestinal disease (n=46)•Cartilage disease (n=45)•Systemic rheumatological disease (n=45)•Diabetes (n=43)• Eye disease(n=39)•Skin condition (n=19)•Organ transplant-associated (n=18) •Lung disease (n=15)•Kidney condition(n=8)

Database of clinical trials (n = 4749)Non-novel (n = 3961)

Novel (n = 1058)

Matthew D Li, Harold Atkins Regen. Med. (2014) 9(1), 27–39

stem cell-related therapy and product development are likely to be an $8.5 billion global market by 2016

Therapeutic Potential of Mesenchymal Stem/stromal Cells

1. Tissue repair and regeneration as they differentiate into many tissues

2. Immunomodulation

3. Enhancement of HSC engraftment

4 .Remarkable expansion after ex-vivo culture, even with platelet lysate and maintain genetic stability

Biological Properties of MSCs

Paracrine Mechanisms of Mesenchymal Stem Cell-Based Therapy Cell Transplantation, Vol. 23, pp. 1045–1059, 2014

Instant blood mediated inflammatory reaction (IBMIR)

MSC engraftment, antagonizing mechanisms, and differential priming of therapeutic effects

TIME IN HOURS

Clinical Applications( www.clinicaltrials.gov ) June 2015 (453 Clinical Trials)

20 %

19%

18%

15%

13%

15%

Source of MSC’S( www.clinicaltrials.gov ) June 2015 (453 Clinical Trials)

MSCs Isolation Bone MarrowAspiration Isolation Culturing

Final ProductAdministration

MSC Isolation from Bone Marrow

40-50 ml BM Aspiration PSIS under LA

MNC separation by Ficoll density gradient

Cell seeding (5x104 MNC/cm2 to 1.7 x 105 cells/cm2) at different conc. in – MEM (with & without Fetal bovine

serum)

50% of Medium & Non-adherent cells will be replaced with fresh medium

Procedure repeated @ 3 days until 70-80% confluency in the adherent cells

Cell replating (1-5) x 103 MSC’s/cm2 (in different conc.) to (1-5)x105 MSC’s/cm2

QC assessment

• Cell counts – Automated cell counters

• Viability – Trypan blue dye exclusion/Acridine orange/Propidium iodide

( ≥70%)

• Immunophenotyping - CD105>75%, CD73>85%, CD-90>85%

CD-34 & 45 (<5%)

• Trilineage differentiation assay - Osteo, chondro and adipogenic

lineages

• Aerobic/Anaerobic bacterial cultures, fungal cultures

• Mycoplasma testing

• Endotoxin Assay <5 EU/kg/dose

• Dose of MSC’s-- 1X106 cells /Kg

Day 8 (10X) Day 16 (10X) Day 24 (10X)

hMSC

Adipocyte differentiation

Control (10X) After differentiation10X 20X

Osteocyte differentiation

Control (10X) After differentiation10X 20X

Before differentiation (10X)

Control undifferentiated (40X) Differentiated MSC

Cardiomyocytes differentiation

Actinin (40X) Troponin (40x)

Negative Positive

CD34-PE

CD45-FITC

CD105-FITC

CD90-PE

CD73-PE

Fig: A- Unstained(US) MSC

Fig: B- MSC showing positive for Stemness marker

Fig: C- MSC negative for Hematopoietic lineage marker

Characterization of MSC

BA

90%

97%

97%US

US

Effect Of Mesenchymal Stem Cells On The T Cell Repertoire Of Kidney Transplant Patients

Results: T cell proliferation is markedly reduced with MSC therapy at various time points post transplantation. T-Cells that generally progress through six divisions using CFSE dilution assay were arrested following two divisions at 90 days post transplantation.

Trans-differentiation of cultured Mesenchymal Stem Cell into islets cells

Results: Evaluated the therapeutic effects of MSC (2x106/kg) infused intravenously to streptozotocin induced diabetic wistar rats, two times at day 7 and 21 of diabetic induction, reported decreased sugar levels.

Autologous Bone Marrow Mononuclear Cells In Idiopathic Membranous Nephropathy

Results: Autologous MNC cell infused to patients with biopsy proven IMN showed a transitory reduction in proteinuria and improvement in serum albumin in treatment refractory IMN.

MSCs Clinical Studies Done at Stem Cell Research Facility

PGIMER,Chandigarh

Indian J Med Res 142, July 2015, pp 63-71 DOI:10.4103/0971-5916.162116

Adult mesenchymal stem cells and chronic burn wound epithelialization

Results: Autologous bone marrow derived mesenchymal stem cell transplantedto, two areas similar in size and depth of burns, followed by split skin grafting.The test wounds healed completely at an earlier date compared to the controlwounds in six patients.

Mesenchymal stem cell and Osteoarthritis

Results: Intra-articular injection of autologous MSCs can be considered apotential treatment of early osteoarthritis knee which relieves pain, stiffness,improves physical functions and improves the articular cartilage integrity torelief treatment of OA.

Autologous mesenchymal stem cells and meniscal tear of knee joint

Results: Autologous MSC infused to patients showed a transitory reduction in painand improvement in meniscal tear.

Human dental pulp stem cells (DPSCs), umbilical cord, blood

Techniques used: isolation of dental pulp, follicle and apical papilla, expansion, characterization and differentiation to adipocyte and osteocytes.

Challenges in Clinical Medicine

1. What SCs to use?

2. At what stage of the disease will therapy be

most effective?

3. What dose, which site?

4. Single or multiple doses?

5. Regulatory Framework

Donor Related Issues

• Requirements similar to other tissue based products(Allogeneic Bone Marrow donors)

• Stringent Donor screening for infectious & genetic disease testing (one donor-multiple recipients)

• Age of Donor(10-fold birth teenage &10-fold teenage -elderly

Stem cell based therapeutics large scale manufacturing

• Regulatory requirements

biological license application

Establishment of safety and efficacy

• Investigational new drug application

• Compliance with GMP/GTP

Stem cell use in patients, other than that for hematopoietic stem cell reconstitution for approved indications, is investigational at present. Accordingly, any stem cell use in patients must only be done within the purview of an approved and monitored clinical trial with the intent to advance science and medicine, and not offering it as therapy. In accordance with this stringent definition, every use of stem cells in patients outside an approved clinical trial shall be considered as malpractice.

Regulatory Issues

Regulatory Issues

• Institutional committee on Stem cell research (ICSCR)&Institutional Ethics Committee (IEC)

• National Apex Committee for Stem Cell Research and Therapy (NAC-SCRT)

• All Pre-clinical/Clinical trials SCR must be registered with ICMR Clinical Trial Registry

Future Directions in MSC Research

• Need to identify an ideal source

• Optimization Culture, characterization and

cryopreservation Techniques

• Understanding of the biology of Paracrine effects

• Defining appropriate cell dose and frequency

• Appropriate Donor Selection

• Working as per the regulatory frame work

Churning of the Ocean …….

For the Nectar of Immortality

Matthew D Li, Harold Atkins Regen. Med. (2014) 9(1), 27–39