MESENCHYMAL STROMAL CELLS AND METHODS FOR OBTAINING MESENCHYMAL STROMAL CELLS FROM UMBILICAL CORD
20200339947 ยท 2020-10-29
Inventors
- Diego ARDIGO' (Parma, IT)
- Giovanni MILAZZO (Parma, IT)
- Massimo DOMINICI (Parma, IT)
- Alba MURGIA (Parma, IT)
Cpc classification
C12N2506/025
CHEMISTRY; METALLURGY
C12N5/0605
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to mesenchymal stromal cells and to an advantageous method for their isolation from umbilical cord. In particular, the cells are obtainable by a method comprising isolating the stromal Wharton's Jelly or a fraction thereof from an umbilical cord and subjecting the stromal Wharton's Jelly or fraction thereof to enzymatic treatment, thereby releasing at least one cell from the stromal Wharton's Jelly or fraction thereof. Preferably, cultivation of cells occurs under conditions allowing the expansion of the cell population. The cell population according to the present invention has proliferative properties and is of industrial interest for a variety of purposes.
Claims
1. A method for obtaining a mesenchymal stromal cell, the method comprising: (a) isolating the stromal Wharton's Jelly or a fraction thereof from an umbilical cord; and (b) subjecting the stromal Wharton's Jelly or fraction thereof to enzymatic treatment, thereby releasing at least one cell from the stromal Wharton's Jelly or fraction thereof.
2. The method according to claim 1, wherein the enzymatic treatment in (b) is followed by: (c) expanding said at least one cell.
3. The method according to claim 1, wherein (a) isolating the stromal Wharton's Jelly or a fraction thereof comprises physically removing the vessels and the perivascular area from the umbilical cord, and further comprises stripping out the vessels and the perivascular area from the umbilical cord.
4. (canceled)
5. The method according to claim 1, wherein (a) isolating the stromal Wharton's Jelly or a fraction thereof comprises physically separating the subamniotic stroma from the umbilical cord lining membrane, and further comprises separating the Wharton's Jelly from the umbilical cord lining membrane.
6. The method according to claim 5, wherein physically separating Wharton's Jelly from the umbilical cord lining membrane comprises scraping out the Wharton's Jelly.
7. The method according to claim 3, wherein the umbilical cord is segmented prior to physically removing the vessels and the perivascular area, and wherein the segmenting comprises segmenting perpendicular to the cord, and wherein each perpendicular segment further comprises a length of at least one or more of: (i) about 1 to about 4 cm; (ii) about 2 to about 3 cm; and (iii) about 2.5 cm, or any combination thereof.
8. (canceled)
9. The method according to claim 7, wherein segment(s) of vessels are removed from the segment(s) of the umbilical cord prior to the enzymatic treatment in (b).
10. The method according to claim 1, wherein the enzymatic treatment in (b) is a treatment comprising the enzyme collagenase, and further comprises: (i) mixing stromal Wharton's Jelly or a fraction thereof with a Digestion Buffer comprising collagenase, wherein the Digestion Buffer further comprises a specific activity of not more than 0.5 Wnsch U/ml; (ii) conducting the isolating in (a) and the enzymatic treatment in (b) together in a total time of less than 3 hours; (iii) subjecting the stromal Wharton's Jelly or fraction thereof to enzymatic treatment in the presence of platelet lysate (PL) or derivatives thereof, or (iv) any combination thereof.
11-13. (canceled)
14. The method according to claim 2, wherein the expanding in (c) comprises at least one cell expanded in a growth medium, wherein the growth medium comprises: (i) platelet lysate (PL) or derivatives thereof; (ii) human platelet lysate (hPL) or derivatives thereof; (iii) no added serum; (iv) no fetal bovine serum (FBS); (v) basal medium MSCBM CD, or (vi) any combination thereof.
15-16. (canceled)
17. A mesenchymal stromal cell (MSC), wherein said cell is characterized as follows: (a) the cell expresses APCDD1, and (b) the cell does not express 3G5.
18. (canceled)
19. The cell according to claim 17, wherein the cell: (i) is derived from the stromal Wharton's Jelly of the umbilical cord; (ii) is a human cell; (iii) displays one or more of HLA-A, HLA-B, and HLA-C; (iv) further expresses at least one of PPARG and FLVCR2; (v) does not display one or more of HLA-DP, HLA-DR, and HLA-DQ, or (vi) any combination thereof.
20-23. (canceled)
24. The cell according to claim 17, wherein the cell comprises: (i) a forward scattered light (FSC) value, determined by flow cytometry at an excitation wavelength 488 nm, is of 55 or less; (ii) an average surface area of not more than 3000 m.sup.2, or (iii) the combination thereof.
25-26. (canceled)
27. The cell according to claim 17, wherein the cell shows comprises: (i) 10% or less telomerase activity with respect to the positive control of the TeloTAGGG Telomerase PCR ELISA PLUS kit (considered as 100%); (ii) the ability to differentiate into various cell types, further comprising differentiation potentials for adipogenic, chondrogenic, and osteogenic; (iii) a mesenchymal stem cell, or (iv) any combination thereof.
28-30. (canceled)
31. The cell according to claim 17, wherein the cell is obtained by a method comprising: (a) isolating the stromal Wharton's Jelly or a fraction thereof from an umbilical cord; and (b) subjecting the stromal Wharton's Jelly or fraction thereof to enzymatic treatment, thereby releasing the cell from the stromal Wharton's Jelly or fraction thereof.
32. A population of cells, wherein the population comprises at least one cell as defined in claim 17.
33. (canceled)
34. The population according to claim 32, wherein all cells comprised in the cell population are autologous with respect to each other, and wherein the population further comprises: (i) at least 70% of the total number of cells are cells as defined in claim 17; (ii) an average FSC value of 55 or less; (iii) expression of APCDD1; (iv) the average surface area of the cells is between 1400 to 2000 m.sup.2, 1500 to 1900 m.sup.2, or 1600 to 1800 m.sup.2; (v) at least 70% of the total cells are viable; (vi) the population does not comprise at least one or more of perivascular cells and epithelial cells; (vii) a population obtainable by a method according to claim 3; (viii) a population comprising only human cells, or (ix) any combination thereof.
35-42. (canceled)
43. The method according to claim 1, wherein the cell obtained by the method is a mesenchymal stromal cell (MSC), wherein the cell comprises the following: (a) the cell expresses APCDD1, and (b) the cell does not express 3G5.
44. A cell obtainable by the method according to claim 1.
45. A cell population obtainable by the method according to claim 3.
46. The cell population according to claim 32, wherein the cell population comprises at least one or more of: (i) a frozen cell population; (ii) a cell bank; (iii) and a cell population in multiple aliquots in individual containers.
47-48. (canceled)
Description
EXAMPLES
Example 0: Materials and Methods Common to Various Aspects of the Invention
[0561] In all experimental examples, unless indicated otherwise, MSCBM CD or a basal medium substantially equivalent thereto was used. Exemplary media are described in the following.
[0562] Basal Medium MSCBM CD and Growth Media Comprising the Same [0563] The basal medium MSCBM CD is commercially available from Lonza (Wakersville, Md., USA; Cat. No. 95062-688). According to the manufacturer, it is serum-free, optimized for multiple passage expansion of all types of human mesenchymal stem cells, supports multi-lineage differentiation and does not require an attachment matrix for plating of the cells. Basal media substantially equivalent to MSCBM CD are also suitable. [0564] MSCBM CD/GA is prepared as follows: [0565] 500 ml MSCBM CD (Lonza) [0566] 0.5 ml GA1000 (Lonza; according to the manufacturer, GA1000 contains 30 mg/ml Gentamycin and 15 g/ml Amphotericin) [0567] MSCBM CD/hPLunless diverging amounts or ratios are specifically indicatedcontains 5% (vol./vol.) human platelet lysate (hPL), and 2 U/ml heparin, which are both added to MSCBM CD (i.e. without the antibiotics Gentamycin and Amphotericin). The hPL is suitably prepared from a composition comprising between 3.810.sup.8 platelets/ml and 1.210.sup.9 platelets/ml, on average ca. 7.910.sup.8 platelets/ml); such platelet lysate is obtainable e.g. from the Polyclinic Blood Bank in Modena, Italy or any other supplier. [0568] MSCBM CD/GA/hPLunless diverging amounts or ratios are specifically indicatedcontains 5% (vol./vol.) human platelet lysate (hPL), and 2 U/ml heparin, which are both added to MSCBM CD/GA (i.e. with the antibiotics Gentamycin and Amphotericin).
[0569] Growth factors (other than those eventually comprised in the hPL and/or in the basal medium) are not added, unless explicitly stated otherwise.
[0570] The above media are particularly suitable for expanding the cells according to the present invention.
[0571] Cell Counting
[0572] For determination of the cell number, unless indicated otherwise, cells are counted by automated Cell Counters, such as particularly the NucleoCounter. If necessary, cells are enzymatically detached such as e.g. trypsinized, prior to cell counting. If counting of red blood cells is not desired, then the cell count is done after the cell population has been subjected to an ammonium chloride treatment; such treatment is known to lyse red blood cells. As a result, the total number of cells in a population, except red blood cells, can be determined. To that end, Ammonium Chloride Solution recommended for the lysis of red blood cells is commercially available from multiple providers (e.g. STEMCELL Technologies). Such solution can be added and used according to the manufacturer's instructions.
Example 1: Segmenting and Sectioning of the Umbilical Cord, and Obtaining Minced Stromal Wharton's Jelly
[0573] Obtain a human umbilical cord from a term birth. Accept the cord if the total weight is 40 g or more. Segment the cord by cutting perpendicular into ca. 2.5 cm long segments using a sharp, sterile scalpel. Rinse the cord segments with HEPES-Buffered Saline Solution (HBSS)+300 g/ml gentamycin and 0.15 g/ml amphotericin B for a total of 10 min. Use mechanical shaking to wash blood off the tissue.
[0574] For each so-obtained segment, proceed to obtain the stromal Wharton's Jelly section as follows: incise the cord lengthwise and remove the vessels with the immediately surrounding matrix of typically 3 mm (vascular-perivascular zone, also referred to as vascular-perivascular Wharton's JellyPVWJ) and discard. The removal should be carried out with care, in the sense that opening of the vessels and/or loss of vascular or perivascular cells should be avoided. Avoid pulling out the cord matrix.
[0575] Before collecting the matrix tissue, optically re-examine each segment of tissue to ensure that PVWJ has been completely removed. No vessels or fragments of vessels should remain in the segment.
[0576] Once the PVWJ has been removed from the umbilical cord segments, snip out pieces of matrix tissue from the umbilical cord segments using sharp scissors. Scrape the inside surface of the cord segments with a sharp scalpel to obtain the remaining matrix tissue. The thin amniotic epithelial membrane remaining after scraping is very carefully removed and discarded. The sub-amniotic (or mesenchymal) layer of the cord lining is included in the WJ that is isolated. This is achieved by carefully scraping tissue off the amniotic epithelium; it is important to be careful not to include pieces of the amniotic epithelial membrane when collecting said tissue. The so-obtained tissue is the pure stromal Wharton's Jelly (SWJ).
[0577] Mince the so-obtained (snipped and scraped) SWJ finely with sharp scissors and/or scalpel. Add HEPES-Buffered Saline Solution (HBSS) to the minced tissue. Pool the minced tissue in a Petri dish.
[0578] To extract SWJ, care is taken during extraction to avoid extracting cells from UC-blood, epithelial or endothelial cells and cells from the vascular structure. For control purposes (optional but recommendable, particularly when re-working this example for the first time), a marker for endothelial cells is CD31; a marker for epithelial cells is Epithelial cell adhesion molecule (EpCAM), a marker for perivascular cells is 3G5.
[0579] Steps of Example 1 are shown in
[0580] The method of the present invention as exemplified in Example 1 yields pure Wharton's Jelly through the complete sectional removal and elimination of the non-WJ components of the cord (such as umbilical cord lining membrane/amniotic membrane, vessels and blood), avoids extracting cells of UC-blood, epithelial or endothelial cells and cells from the vascular structure. This allows to obtain pure Wharton's Jelly tissue (WJ tissue). Owing to its origin, this specific WJ tissue is stromal WJ tissue.
Example 2A: Enzymatic Treatment
[0581] Material and Methods: WJ tissue obtained as described in Example 1 was used to isolate WJ-derived primary cells therefrom, as follows: Minced tissue/HBSS mixture was suspended in Digestion Buffer (see Table) at a ratio of 100 mg of minced tissue/HBSS mixture per 1 mL of Digestion Buffer (see Table below) in a 250 ml conical tube. The digestion buffer comprises an enzyme suitable to release the MSCs from the isolated tissue.
[0582] Table: Formulation of 100 ml Digestion Buffer (all Ingredients Sterile) [0583] 1 ml hPL, e.g. from the Polyclinic Blood Bank, Modena, Italy (prepared from a composition comprising between 3.810.sup.8 platelets/ml and 1.210.sup.9 platelets/ml, on average ca. 7.910.sup.8 platelets/ml) [0584] Heparin to a final concentration of 2 U/mL [0585] 1 ml sodium pyruvate (from a 100 mM stock) [0586] 36 l 1M CaCl.sub.2 [0587] Collagenase preparation NB4 or NB6 (both SERVA) to a final activity of 0.18 Wnsch U/ml Digestion Buffer [0588] Basal medium (commercial growth medium for mammalian cells, such as DMEM or MSCBM CD)ad 100 ml
[0589] Collagenase preparation from SERVA (NB4, NB6) was used, typically NB4. According to the manufacturer, collagenase preparation NB4/NB6 is a natural product from a collagenase-producing strain of Clostridium histolyticum, and is a collagenase preparation that is non-toxic, which contains collagenase and other proteases. According to the manufacturer, NB4 Standard Grade (17454.02 and 17454.01 (both SERVA)) is designed for dissociation of different tissues to isolate various cell types, e.g. chondrocytes, muscle cells, epithelial cells, skin fibroblasts, hepatocytes, adipocytes which are used for research purposes. According to the manufacturer, collagenase preparation NB4 can also be used as low cost alternative to collagenase preparation NB6 for procedure-establishing purposes. According to the manufacturer, collagenase preparation NB6 GMP Grade (NB6 GMP grade, 17454.02 (SERVA) is especially designed for the isolation of cells for some specific transplantation purposes. Throughout the experiments reported in the present disclosure, NB4 was used unless explicitly specified otherwise. According to SERVA, the manufacturer of the collagenase preparation used in this experiment, SERVA indicates collagenase activity in PZ units (PZ-U) according to Wnsch (synonymously termed Wnsch units herein).
[0590] It is known that heparin is a suitable additive for platelet lysate-containing liquid compositions to avoid clogging (e.g. Lohmann et al., 2012, PLoS ONE, vol. 7e37839).
[0591] Digestion Buffers with higher collagenase activity (final concentration higher than 0.18 Wnsch U/ml) were also tested, initially based on collagenase activity that has previously been described for enzymatic digestion of adipose tissue, but the digestion in presence of higher collagenase activity was not advantageous (data not shown).
[0592] The minced tissue (Example 1) was placed into sterile tubes. Digestion buffer (see above) was added. These tubes containing minced tissue and Digestion Buffer (digest) were incubated at 371 C. for a digestion time interval of 3 hours with constant gentle agitation at 75 rpm on an orbital shaker for digestion. digest refers to the ensemble of tissue to be digested and digestion buffer.
[0593] At the end of the digestion time interval, the digest was diluted by adding 2 volumes of MSCBM CD, supplemented with 1% (v/v) human platelet lysate (hPL) and passed through two layers of one sterile gauze pad to separate from undigested tissue/partially digested tissue. The filtrate was termed digested mixture.
[0594] The digested mixture is centrifuged at 680g for 15 minutes using 250 ml centrifuge tubes. At the end of centrifugation, the supernatant is removed and the pellet (containing freshly-isolated mesenchymal stromal cells from the Wharton's Jelly of the umbilical cord) is re-suspended in 1-2 mL of MSCBM CD/GA/hPL (see Example 0).
[0595] Subsequently, the total cell number is determined. Most typically, the cells comprised in the digested mixture are not all MSCs:
Example 2B: First Culturing (P0 and Optionally P0+)
[0596] The re-suspended cells form Example 2A are plated in T-225 culture flasks (Falcon, a brand of Corning, Corning, N.Y., USA) in MSCBM CD/GA with 5% hPL (see Example 0; 50 mL medium per T-225 flask) to start the first in vitro culture of the cells originating from the stromal Wharton's Jelly. This cell culture is termed P0. The day that P0 is initiated is termed Day 0, and days are counted with ascending numbers thereafter. The cell density at the onset of P0 is 8.000 cells/cm.sup.2 or more. The cm.sup.2 refer to the surface of the flask which is at the bottom and covered by growth medium while the cells are being cultured in P0.
[0597] Feed cells every 3 days starting from Day 5 with fresh MSCBM CD/GA/hPL. Alternatively, it could also be possible to culture the cells during P0 in stacks, instead of flasks: in that case, 150 mL MSCBM CD/GA/hPL are used per 11 stack to start the first cell culture; however, unless explicitly mentioned otherwise, the P0 culture is performed in T-225 culture flasks.
[0598] The cells are cultured in P0 as described until the confluence of the cells is 80 to 90%. This is usually the case after a period of 5-12 days. Then, the cells are trypsinized. Cells are counted following trypsinization at the end of the passage.
[0599] The following acceptance criteria are assessed: 1 yield, 2 viability, 3 confluence, 4 morphology. Determination of acceptance criteria 1 and 2 occurs after trypsinization of the cells. The details are as follows.
TABLE-US-00003 Acceptance criterion After P0 and P0+ Test 1 Yield At least 8 million cells Cell counting after trypsinization 2 Viability More than 80% of cells viable Staining with 7-amino actinomycin D (7-AAD) 3 Confluence At least 80%, preferably 80 to Microscopic determination 90% confluent 4 Morphology Majority of cells small, spindle- Microscopic determination shaped and in defined colonies
[0600] In this experimental example, all four acceptance criteria must be fulfilled. In addition, in the morphological inspection, it is additionally visually inspected whether size and shape of the cells are relatively uniform. Preferably, this is the case.
[0601] In case any one or more of criteria 2, 3 or 4 are not fulfilled, the entire cell culture is discarded. It is the observation of the present inventors that criteria 2, 3 and 4 are normally fulfilled, and that discarding is hence not necessary. However, the possibility to discard in case of non-fulfillment guarantees that subsequent passages are only conducted if the acceptance criteria are fulfilled.
[0602] In case the total cell number after P0 is higher than 8 million cells (provided also acceptance criteria 2, 3 and 4 are fulfilled), proceed directly with Example 2B.
[0603] In case the total cell number after P0 is not higher than 8 million cells (provided however that acceptance criteria 2, 3 and 4 are fulfilled), an extra sub-cultivation step, termed P0+ or P0 plus is performed. (For the avoidance of doubt, throughout this document, it is specifically indicated in case certain cells were sub-cultivated in a step P0+; in other words, where no such specific indication is given, cells were passed from P0 directly to P1 (Example 2B)). The conditions of P0 correspond exactly to the conditions of P0, described above, except that the resuspended cells being subjected to P0+ originate from P0, not directly from Example 2A.
[0604] The cells are cultured in P0+ until confluence of the cells is 80 to 90%. Then, the cells are trypsinized. The above acceptance criteria are assessed: 1 yield, 2 viability, 3 confluence, 4 morphology. When all criteria are fulfilled, proceed with Example 2B. When one or more of the criteria are not fulfilled (including yield), discard the cells. In particular, the cells are discarded if the total cell number following P0+ is not at least 8 million cells.
[0605] Total cell numbers in this example are indicated based on an entire human umbilical cord as starting material (full scale run). If the amount of starting material is different (e.g. only half of a human umbilical cord), then the total amount of the cells for acceptance criterion 1 is adjusted arithmetically accordingly.
[0606] Depending inter alia on the yield of cells, which varies to some degree between different full-scale runs, also in view of the variation in the biological material from different individuals, the cumulative Population Doublings during P0 are subject to some variation.
Example 2C: Further Expansion (P1 and Subsequent Passages)
[0607] Trypsinized cells from P0 (or P0+, but only in case explicitly so described) in Example 2B, as described therein, were obtained and sub-cultured for one or more passages, as follows.
[0608] The first sub-culture is termed P1. At the end of P1, the cells are again trypsinized and sub-cultured. This sub-culturing/trypsinization cycle is done preferably for 4 passages (P1 counting as the first passage), although up to 12 passages have been done in some cases. Each passage is consecutively numbered, i.e. P1 follows after P0, P2 follows after P1, and so on. Unless specified otherwise, the cells were grown and sub-cultured until passage 4 (P4).
[0609] For each passage from P1 onwards, all culture plates have the same defined size. In particular, for each passage, the culture vessel was composed of a stack of CS5, CS10 or CS20 culture plates (Corning, Corning, N.Y., USA). For each passage from P1 onwards, the cells are grown in MSCBM CD with 5% hPL without antibiotics (see Example 0; 50 mL MSCBM CD/hPL per T-225 flask). Unless stated otherwise, antibiotics are discontinued in this example for all passages after P0; in other words, in this example MSCBM CD/hPL is used without addition of antibiotics for P1 and all subsequent passages.
[0610] The cell density at the onset of each passage is 2.500 cells/cm.sup.2.
[0611] After each passage cells are trypsinized. Cells are counted at the end of each passage, after trypsinization
[0612] As a function of the cell density at the time of inoculation, the ground surface of the cell culture vessel (which together define the total cell count at the beginning of the passage) and the total cell count at the end of the passage, the cumulative Population Doublings can be arithmetically determined.
[0613] In the experience of the inventors, the cumulative Population Doublings during P1 and P2 (total of P1 plus P2, but not including P0), as a very general rule, are normally in the range of 9-13.
[0614] In the experience of the inventors, the cumulative Population Doublings during P3 and P4 (total of P3 plus P4, but not including P0, P1 and P2), as a very general rule, are normally in the range of 6-10. Consequently, the cumulative Population Doublings during P1, P2, P3 and P4 (total of P1 plus P2 plus P3 plus P4, but not including P0), as a very general rule, are normally in the range of 15-23.
[0615] Optionallywhere specifically indicated hereincells were frozen after a specific passage, e.g. P2. The cells can be frozen, e.g. snap-frozen in liquid nitrogen, according to standard procedures. It is well possible to de-frost such frozen cells and to e.g. subject them to further culture passages after de-frosting. The freezing can occur either as entire batch or in aliquots. Freezing in aliquots, preferably in vials, is much preferred and was typically done in this example, unless specified otherwise.
[0616] When cells are frozen and thawed after a specific passage, the numbering of the passage continues after thawing. For example, when the cells are frozen after P2, the next passage after the thawing is then P3. Preferably, and unless otherwise indicated, also the counting of the cumulative Population Doublings continues after the thawing.
[0617] In a preferred embodiment, cells are aliquoted and frozen following P2. The so-obtained frozen aliquots are available for thawing individually at a desired point in time. The frozen cells, preferably in aliquots, can be referred to as Primary Cell Bank (PCB). In other words, it is not necessaryand typically not desiredto thaw all aliquots of the PCB at the same point of time. In contrast, the aliquoting and individual freezing of the aliquots provides the advantage that individual aliquots can be removed from freezing, thawed and subjected to further culturing (sub-culturing, further expansion) at individual, and individually desired points in time.
[0618] In a specific example of a full-scale run, 168 vials (each containing 2010.sup.6 cells, 4 ml content) were obtained after P2, beginning from one human umbilical cord as starting material. Each of the vials can be frozen, and individual vials can be thawed at individual time points for further sub-culturing, e.g. P3 and onwards. In one specific example of a full-scale run, one vial obtained and frozen after P2 is thawed and the cells contained therein are subjected to P3 and P4. In that specific example, after P4, 168 vials (each containing 2010.sup.6 cells, 4 ml content) were obtained.
[0619] The cells are cultured in each passage as described until the confluence of the cells is 80 to 90%. This is usually the case after a period of ca. 3-4 days. Then, the following acceptance criteria are preferably assessed: 1 yield, 2 viability, 3 confluence, 4 morphology. Determination of acceptance criteria 1 and 2 occurs after trypsinization of the cells. The details are as follows.
TABLE-US-00004 Acceptance After P1 and all passages criterion following P1 Test 1 Yield (full After P1: at least 225 10.sup.6 Cell counting after scale run) cells trypsinization After P2: at least 7 10.sup.9 cells (*) After P3: at least 250 10.sup.6 cells (*) After P4: at least 7 10.sup.9 cells (*) 2 Viability More than 80% of cells viable Staining with 7-amino actinomycin D (7-AAD) 3 Confluence At least 80%, preferably 80 to Microscopic determination 90% confluent 4 Morphology Majority of cells small, Microscopic determination spindle-shaped and in defined colonies (*) After P2 the cells can be frozen in aliquots to prepare a Primary Cell Bank (PCB). Cell numbers for P3 and P4 are indicated based on the scenario that P3 is initiated with one vial from the PCB, which contains 20 10.sup.6 cells.
[0620] It is important to note that the yields obtainable by expansion according to the present invention are very high compared to previously known methods for preparing and expanding mesenchymal stem cells.
[0621] In this example, all four acceptance criteria must be fulfilled: if one of the criteria is not fulfilled, the entire cell culture from the respective passage is discarded. It is the observation of the present inventors that all of criteria 1, 2, 3 and 4 are normally fulfilled, and that discarding is hence not necessary. However, the possibility to discard in case of non-fulfillment guarantees that subsequent passages are only conducted if all acceptance criteria are fulfilled.
[0622] In summary, Example 2 shows that the stromal Wharton's Jelly holds cells in an expansion-competent state. The stromal Wharton's Jelly cells were identified as a convenient source of potentially multi-potent stromal cells that can be maintained and expanded in culture.
COMPARATIVE EXAMPLES
[0623] For comparison purposes, cells were isolated as described by WO 2004/072273 A and Sarugaser et al., 2005, Stem Cells, vol. 23, p. 220-229, specifically as described in the following Comparative Examples 1, 2A, 2B and 2C.
Comparative Example 1
[0624] Vessels were isolated from human umbilical cord. The dissected vessels were sutured to create a loop, blocking the passage of fluid either into out of the vessel.
Comparative Example 2A
[0625] The loop (Comparative Example 1) was immediately placed into a 50 mL tube containing 1 mg/mL collagenase Sigma C-0130 solution in PBS (w/o Mg.sup.2+ and Ca.sup.2+) at 37 C. According to the literature, the collagenase Sigma C-0130 has a specific activity of 0.25-1.0 FALGPA units/mg solid. For information, FALGPA units are not directly arithmetically convertible into Wnsch units because the respective units refer to different assays with different substrates; however, there is some general experience in the art on how Wnsch units relate to FLGPA units. Based on that general experience it can be concluded that the total activity of collagenase used in Comparative Example 2 is much higher than the total activity of collagenase used in Example 2.
[0626] After 18-20 hours of incubation with collagenase, the digestion solution was stopped by adding 2 volumes of PBS and 1 mL of Fetal Bovine Serum (FBS). Thus, the total duration of collagenase treatment in Comparative Example 2 is much longer than the total duration of collagenase treatment used in Example 2. The digested samples were filtered to remove undigested tissue and centrifuged at 1,800 rpm (Beckham Coulter Allegra 25R) for 15 minutes. The cells were re-suspend in 75% -MEM medium, 15% defined FBS, 10% antibiotics. To lyse the blood cells, a solution of ammonium chloride (NH.sub.4Cl), potassium bicarbonate (KHCO.sub.3) and EDTA was used in a ratio of 1:3 v/v with the cellular suspension for 5-10 minutes on ice by gently agitation. After centrifugation at 500g for 10 minutes, the cells were re-suspend and counted by Trypan Blue (0.4%) dye.
Comparative Example 2B
[0627] The perivascular-Wharton's Jelly-derived cells (PVWJ) obtained as described in Comparative Example 2A were seeded at density of 6,000-8,000 cells/cm.sup.2 and incubated at 37 C. in -MEM (without nucleosides, Life Technologies, Waltham, Mass., USA, #22561-021) supplemented with 15% defined FBS (HyClone, GE Healthcare, USA, # SH30070.03) and antibiotics in a 5% CO.sub.2, 95% humidity incubator to start the culture P0.
[0628] The media was exchanged for the first time after 5-6 days, than was refreshed every 2 days. After 6-10 days of culture the cells reached confluence. At end of P0, after a period of 6-10 days, the cells reached confluence. They were then trypsinized. The total cell number was determined with Trypan Blue dye (0.4%).
Comparative Example 2C
[0629] Trypsinized cells obtained from Comparative Example 1B were obtained and sub-cultured, as follows. From culture passage 1 (P1) onwards, the cells were seeded at density of 3,500-4,000 cells/cm.sup.2 in -MEM with 15% defined FBS. Antibiotics should be discontinued after P0; in other words, growth medium is used without addition of antibiotics. The cells are termed perivascular Wharton's Jelly-derived cells (PVWJ). Perivascular Wharton's Jelly (PVWJ)-derived mesenchymal stromal cells can also be reffered to as perivascular zone-derived mesenchymal stromal cells. Unless specified otherwise, the cells were grown and sub-cultured until passage 4 (P4).
[0630] Optionallybut only where specifically indicatedcells were frozen after a specific passage, e.g. P2. It is well possible to thaw (de-frost) such frozen cells and to e.g. subject them to further culture passages after de-frosting.
Example 3: Analysis of Cells after Digestion
[0631] Cells obtained as described in Example 2A (i.e. after the digestion step, either with or without platelet lysate (PL)) are termed stromal-Wharton's Jelly derived cells or SWJ. These are compared to PVWJ-derived control cells (PVWJ, comparative Example 2A). SWJ and PVWJ were stained with 7-Amino-actinomycin-D (7AAD)-staining (Via Probe; BD Biosciences; used according to the manufacturer's instructions) to detect apoptosis by FACS analysis. Unstained sample was acquired to detect the sample auto-fluorescence and set the photomultiplying tubes (PMT; FACS sensors) for all the considered channels. Compensation settings were established by acquiring single color stained tubes. Collected data were analyzed by Diva software (BD Biosciences). Statistics by t-test. Results are shown in
[0632] The FACS analysis of freshly isolated samples revealed that, for both NB4-collagenase preparation-based digestion protocols (with and without platelet lysate (PL), see
[0633] Further, by comparing the use of platelet lysate (yes/no) and not observing a significant difference regarding the percentage of AAD7-positive cells (
Example 4A: Analysis of Physical Properties of SWJ-Derived Cells Isolated According to the Present Invention in Comparison with PVWJ-Derived Cells
[0634] Next, the inventors aimed at evaluating the total cell number and the physical properties of SWJ-derived cells isolated according to the present invention (Example 1 and Example 2A) versus the PVWJ-derived cells from the protocol according to Comparative Example 1 and Comparative Example 2A). This comparative study was based on the evaluation of collagenase efficiency and physical parameters (by FACS) of respective freshly isolated cells.
[0635] First, regarding the total number of freshly isolated cells, the inventors obtained more PVWJ-derived cells by the method according to Comparative Example 1 than they obtained SWJ-derived cells by the method according to the present invention as described in Example 1.
[0636] Second, the analysis of physical parameters by flow cytometry, namely forward-scattered light (FSC) and side-scattered light (SSC), revealed that the SWJ-derived cells have a lower FSC and SSC in comparison to the PVWJ-derived cells isolated by the method according to Comparative Example 1. Results are indicated in the following table and visualized in
TABLE-US-00005 TABLE SSC and FSC as experimentally determined Cell FSC SSC PVWJ-derived cells Mean of six samples 75.42166667 64.65666667 according to ST-DEV 18.66791951 15.1647779 Comparative Example 2A SEM 7.75 6.19 SWJ-derived cells Mean of four samples 46.1925 50.795 according to ST-DEV 9.660766986 21.28367273 Comparative Example 2A SEM 5.58 12.29 t-test 0.025231608 0.296671217
[0637] On average, the SWJ-derived cells have a smaller cell volume (determined by FSC) with a lower internal complexity (determined by SSC) than the PVWJ-derived cells. The difference in FSC is statistically significant. In particular the SWJ-derived cells (according to the present invention, n=4) have a lower FSC and SSC in comparison to the PVWJ-derived cells isolated by the method according to Comparative Examples 1 and 2A (PVWJ, n=6). This flow cytometry readout indicates that the SWJ-derived cells isolated by the method according to the present invention are characterized by more homogeneous small cells with low cytoplasmic complexity, whereas the PVWJ-derived cells are characterized by a heterogeneous population with larger average volume and greater cytoplasmic complexity. Homogeneous in this context means that substantially all cells are much alike or similar. Smaller volume and lower internal complexity are both features that indicate stemness and performance (see e.g. Wagner et al., 2008, PLoS One, 2008, 3: e2213). In general, cells with a more complex internal structure appear more shiny/brighter in the side scatter analysis, owing to the internal structures.
Example 4B: Morphological Phenotype of Freshly Isolated Cells: PVWJ Cells are Different from the Cells Isolated According to the Present Invention
[0638] The morphology of SWJ cells and PVWJ cells was compared. To ensure a high degree of comparability, one single cord was divided in two parts: one part was prepared with the method described in Examples 1, 2A and 2B, the other part was prepared according to Comparative Examples 1, 2A and 2B, in both cases however, the cells were analyzed specifically two or three days post-seeding culture passage 1 (P1), (original magnification 100).
[0639] The inventors found that cells derived from pure stromal Wharton's Jelly according to the present invention (SWJ) are homogeneous, small and spindle shaped (
Example 4C: Colony Formation of Freshly Isolated Cells: PVWJ Cells are Different from the Cells Isolated According to the Present Invention
[0640] Next, the colony-formation potential of the cells according to the present invention was investigated. This was done as follows: SWJ were obtained as described in Examples 1 and 2A, PVWJ were obtained as described in Comparative Example 1. Then, using a sterile funnel, the cell-containing extract is filtered through two layers of one sterile gauze pad to remove undigested tissue. Centrifuge at 680g for 15 minutes using 250 ml centrifuge tubes. Discard the supernatant (pour off or aspirate using low vacuum). Re-suspend the pellet in 1-2 mL of MSCBM CD/GA/hPL (see Example 0). Plate the re-suspended cells in 3T225 flasks or 11-Stack in MSCBM CD/GA/hPL medium (see Example 0; 50 mL medium per T-225 flask or 150 mL per 11 stack) to start the P0 culture. Do not move the P0 culture for the first 5 days after seeding. Change medium after 5 days. Feed every 3 days thereafter with MSCBM CD/GA/hPL. Harvest P0 cells on day 12 with 3 U/mL trypsin and neutralize with MSCBM+1% hPL. Results are shown in
Example 5: Comparison of SWJ-Derived Cells According to the Present Invention Vs. Bone Marrow-Derived Cells (BM)
[0641] Bone marrow derived MSC (BM-MSC, obtained as described by Grisendi et al., Cytotherapy, 2010, vol., 12, p. 466-477, were seeded at density of 6,000 cell/cm.sup.2. SWJ-derived cells according to the present invention (Examples 1, 2A, 2B and 2C, specifically cultured until passage 5 (P5), were seeded at density of 2,800 cell/cm.sup.2. After 2-3 days of culture they were sub-confluent, and five representative images were acquired for both cell types. In particular, the plastic-adherent MSC were visualized with Axioskop-Observer-1 microscope (Zeiss) using a 10 objective, equipped with Axiocam camera. The images were acquired and processed by AxioVision 4.8.2 software to evaluate the cell area. (Moore et al., Exp. Eye Res. 2013, vol. 115, p. 178-188; Zeilbeck et al., PLoS One., 2014; vol. 9(4), e95546).
[0642] Results are shown in
Example 6: Cell Surface Markers of Freshly Isolated Cells
[0643] Materials and Methods: Three umbilical cords were subjected to two different methods (state of the art according to Comparative Examples 1 and 2A, PVWJ vs. cells according to the present invention, see Examples 1 and 2A, SWJ.
[0644] Immediately after isolation, the PVWJ/SWJ-derived cells were counted and re-suspended in 1 mL of PBS 1 in fluorescence-activated cell sorting (FACS) analysis polypropylene tubes (200,000 cells/tube). The cells were incubated in 100 L blocking buffer containing Dulbecco's modified Eagle medium (DMEM), 10% fetal bovine serum (FBS), 0.1 M sodium azide and 66.6 mg/mL human immunoglobulin G for 20 minutes on ice. Subsequently they were washed in 1 ml of 1PBS and centrifuged at 300 g for 5 minutes.
[0645] Samples were subsequently stained for 30 min on ice with primary antibodies in 90 L PBS with 0.5% bovine serum albumin (BSA; Sigma) and analyzed with FACSAria III flow cytometer (BD Biosciences) equipped by two air-cooled lasers at 488 and 633-nm wavelengths. For indirect staining (for anti-GD2 only), a secondary rat anti-mouse Allophycocyanin (APC)-conjugated antibody (BD Pharmingen) was used. 7-Amino-actinomycin-D (7AAD)-staining (ViaProbe; BD Biosciences; used according to the manufacturer's instructions) was evaluated by flow cytometry to detect apoptosis. Unstained sample was acquired to detect the sample auto-fluorescence and set the PMT for all the considered channels; fluorescent cross talk is controlled by compensation adjustment. Compensation settings were established by acquiring single color-stained tubes. Collected data were analyzed by Diva software (BD Biosciences).
[0646] Results: The immuno-phenotypic analysis of freshly isolated samples revealed that, for both isolation methods, the inventors identified CD105, CD73 and CD90, which are typical surface antigen expression of MSCs. Similarly, platelet-derived growth factor receptor 13 (CD140b) was detected after both isolation methods. HLA-DR was not detected in both samples, however PVWJ-derived cells were found to express 3G5, while SWJ-derived cells did not, indicating that the method according to the present invention does not lead to isolation of perivascular cells, in accordance with the origin of the cells in the stromal Wharton's Jelly. Relative display of some surface markers is shown in
[0647] The cell surface markers were also analyzed after prolonged culture of the cells. Cell cultures originating from two independent isolations and at different cumulative population doublings were analyzed. The following was found: There are no significant differences in the display of CD105, CD90 and CD73 (positive markers of MSC) among different isolations of cells. CD105, CD90 and CD73 are also uniformly high at different cumulative Population Doublings (cPD). To the contrary, CD45, CD14, CD34, CD19 and HLA-DR (known to be negative markers of MSC) are expressed at very low levels in cells from both isolations and at different cumulative Population Doublings (see following Table).
TABLE-US-00006 TABLE phenotypic characterization by flow cytometry (% positive cells). CD45 CD73 HL-DR CD14 CD34 CD90 CD105 CD19 Batch 1 3.8 98.9 0.6 0.0 1.1 99.2 99.1 0.5 after 8 cPD Batch 1 4.1 99.1 0.7 0.2 1.7 99.2 99.1 0.8 after 16 cPD Batch 2 1.4 99.2 0.3 0.1 2.6 99.2 99.1 0.2 after 8 cPD Batch 2 0.8 99.1 0.2 0.7 4.2 99.1 99.10 0.1 after 16 cPD cPD = cumulative Population Doublings
[0648] These data show that the conditions of expanding stromal WJ-derived cells in the method according to the present invention are able to generate populations of pure stromal WJ-derived cells at high yield with a stable phenotype over several cumulative Population Doublings. Therefore, the cells of the invention are suitable for the creation of a Cell Bank.
Example 7: Cell Surface Markers on Expanded Cells
[0649] Materials and Methods: In order to characterize surface antigens on PVWJ-derived cells and SWJ-derived cells, a FACS array was performed from cells contained in a Cell Bank, prepared from the same umbilical cord according to different methods (Examples 1 and 2A, 2B and 2C vs. Comparative Examples 1 and 2A, 2B and 2C). The cells were de-frosted at P2 (Example 2C/Comparative Example 2C) and expanded until P4 (Example 2C/Comparative Example 2C) to perform FACS analyses. After detachment with a cell detachment solution of proteolytic and collagenolytic enzymes (Accutase, Thermo Fisher) the cells were centrifuged 500g for 10 minutes. The pellet was re-suspend in culture media (MSCBM CD/hPL for SWJ or -MEM with 15% defined FBS for PVWJ) and counted by Trypan Blue 0.4% dye. The cells were washed in 1PBS and centrifuged at 300g for 5 minutes. The cells were resuspend in BD Pharmingen Stain Buffer+EDTA and 250,000 cells/well are plated in 96-well plates. The flow cytometry screening was conducted according to the Human Cell Surface Marker Panel datasheet (BD Lyoplate, BD Biosciences) according to the Manufacturer's instructions. Results are shown in
[0650] It was specifically tested whether freshly isolated cells according to the present invention (Examples 1, 2A) express CD46, CD55, and CD59. As shown in
Example 8: Gene Expression of Cells According to the Present Invention as Determined by Quantitative Reverse Transscriptase PCR (qRT-PCR)
[0651] Materials and methods: Cells obtained according to the present invention (Example 2C), in particular: after passage 4, were used for this analysis. Total cellular RNA was isolated using a single-step method with TRIzol (Life Technologies) according to the manufacturer's instructions. First-strand complementary cDNA was synthesized from 1 g of total RNA using a revertAid H minus first-strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer's instructions. The single strand cDNA was quantified by spectrophotometer (Beckman Coulter DU 730) in order to use 10 ng of cDNA in each Real-Time PCR well.
[0652] The quantitative real-time PCR technique was performed by Applied Biosystems StepOne Real-Time PCR System and the Fast SYBR Green Master Mix reagent. The quantification of gene expression for each target gene and reference gene was performed in separate tubes. Forward and reverse primers were designed using IDT PrimerQuest (available online at http://eu.idtdna.com/PrimerQuest/Home/Index), ensuring they spanned an intron sequence to be specific for mRNA rather than genomic DNA. The relative expression level of the target gene was normalized to the expression level of the endogenous reference -actin gene.
[0653] Results are shown in
[0654] Remarkably however, the expression pattern of these genes differs remarkably from PVWJ-derived cells isolated according to the state of the art. In particular, APCDD1 is not expressed at all in PVWJ-derived cells. This is concluded from the finding that no amplification of the APCDD1 gene was observed at all in the sample from PVWJ-derived cells. The inventors conclude that by RealTime-PCR APCDD1 expression is detectable only in SWJ-derived cells according to the present invention, but not detectable in PVWJ-derived cells. The expression of APCDD1 thus clearly distinguishes the cells of the present invention from PVWJ-derived cells.
[0655] While earlier literature has been uncertain on whether or not nonperivascular MSCs are distinct from perivascular MSCs (e.g. Davies et al., Stem Cells Translational Medicine, 2017, vol. 6, p. 1620-1630), this example now provides proof that stromal Wharton's Jelly derived cells obtained according to the present invention are distinct from perivascular MSCs.
Example 9: Proliferative Potential of the Cells According to the Present Invention Vs. Reference Cells
[0656] Materials and Methods: Cell Culture: Stromal Wharton's Jelly-derived cells (SWJ) according to the present invention, obtained as described in Examples 1, 2A, 2B and 2C after P1, or perivascular Wharton's Jelly-derived cells (PVWJ), obtained according to Comparative Examples 1, 2A, 2B and 2C, both cell populations obtained from the same umbilical cord, were seeded MSCBM CD with 5% hPL (see Example 0) without growth factors and placed in a 37 C., 5% CO.sub.2 incubator with 95% humidity. Three days after plating, and every 3 days thereafter, cultures were re-fed. On day 4 or 5 of each passage cells were trypsinized, and counted.
[0657] Cell Counting: the cells were counted using 0.4% Trypan blue dye in a Burker chamber and the cumulative population doubling (CPD) was calculated as described above.
[0658] Results are shown in
[0659] SWJ-derived cells have a high proliferative potential, which is maintained for at least 9 passages in the culture conditions used (growth medium MSCBM CD with 5% hPL). Under these culture conditions, as experimentally determined with two independent isolates of SWJ-derived cells, the SWJ-derived cells will reach senescence between passage 10 and passage 12, as shown in the following Table.
TABLE-US-00007 TABLE growth of SVWJ according to the invention Passage Isolation 1 Isolation 2 1 5.89 4.99 Population 2 5.00 5.47 Doublings per 3 5.04 4.76 passage 4 5-02 4.24 5 4.58 5.03 6 4.95 5.30 7 5.40 4.69 8 5.22 4.63 9 4.92 4.54 10 4.83 1.81 11 4.58 n/a 12 3.27 n/a Total 58.70 45.44 Cumulative Population Doublings
[0660] This example adds additional evidence that the conditions of isolating and expanding stromal Wharton's Jelly-derived cells (SWJ-derived cells) according to the present invention allows to generate large cell populations of SWJ-derived cells. This Example also adds evidence that the SWJ-derived cells are different from PVWJ-derived and have a higher proliferative potential.
[0661] The high proliferative potential and population doubling (PD) per passage increases the yield of the cell bank and/or reduces the time for production of a cell bank, respectively, compared with reference MSCs, such as PVWJ-derived cells. This is advantageous from an industrial point of view.
Example 10: Telomerase Activity
[0662] For this assay 200,000-500,000 Wharton's Jelly-derived stromal cells (SWJ) according to the present invention were aliquoted in a 1 mL Eppendorf tube and then centrifuged at 1,200 rpm (Beckham Coulter Allegra 25 R) for 10 minutes at +4 C. The supernatant was removed and the cells were re-suspended in 1PBS. After a second centrifuge step, the dry pellets were stored at 80 C. To perform the assay, the TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche Diagnostics) was used according to the manufacturer's instructions. Results are shown in
Example 11: Senescence
[0663] Material and Methods: Wharton's Jelly-derived stromal cells (SWJ) according to the present invention after trypsinization (for cell origin see Examples 1 and 2A; for passages see Examples 2B and 2C) following passage 6 (P6), passage 8 (P8) or passage 11 (P11), passage 12 (P12), were seeded in six multi-well plates at a density of 3,000 cells/cm.sup.2 in 3 ml of culture media (DMEM with 5% hPL or MSCBM CD with 5% hPL) and cultured for 4-5 days at 37 C. in 5% CO.sub.2, 95% humidity incubator, in triplicates unless otherwise specified. Then, the cells were washed in 1 phosphate-buffered saline (PBS) and fixed for 10 minutes at room temperature in 2% formaldehyde/0.2% glutaraldehyde. The culture plate was rinsed two times in PBS 1 and 1 mL of fresh -Gal stain solution (1 mg of 5-bromo-4-chloro-3-indolyl-D-galactoside/100 L of dimethylformamide, 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl.sub.2) was added (used according to the manufacturer's instructions).
[0664] The cells were incubated over night at 37 C. and the color was developed with the light of an inverted microscope as described in the instructions provided by the manufacturer (Cell Signaling Technology). The positive stained cells appeared blue-green in the cytoplasm. Stained samples were washed in PBS 1 and visualized by 2.5 objective using an inverted microscope (Zeiss). The positive stained cells (per field) were quantified by one observer and at least 8 fields of view were examined for each donor (unless otherwise specified).
[0665] Results: At early culture passages (P6-P8) only a few cells were weakly positive for -galactosidase staining (data not shown), indicating that, even at very low density of seeding, SWJ-derived cells according to the present invention have a tendency to be marginally senescent. Without wishing to be bound to any theory, it is possible that the platelet lysate comprises supplements which are advantageous for growth of the cells ex vivo, e.g. growth factors.
Example 12: Osteogenic Differentiation Potential
[0666] Description of the Method: SWJ at passage 5 (Example 2C) were induced to differentiate into the osteogenic lineage. Briefly, SWJ were seeded at density of 10,000 cell/cm.sup.2 in MSCBM CD with 5% hPL in 6-multiwell (6-MW) plates. These 6-MW plates were pre-coated with 0.1% gelatine solution. After 3-4 days' equilibration, the maintenance medium was exchanged and replaced by osteogenic medium. The osteogenic medium was composed of MSCBM CD with 5% hPL, supplemented with 10 mM beta-glycerophosphate, 0.1 mM ascorbic acid-2-phosphate and 10 nM Dexamethasone, plus additionally of 100 ng/mL rhBMP-2 (bone morphogenic protein 2) from the 7.sup.th day of induction onwards. Confirmation of osteogenic differentiation was performed through von Kossa staining (so that bone matrix is labelled dark) combined with Real-Time qPCR analysis (described below).
[0667] After 2 weeks, the cultures under osteogenic medium (cf. above) and parallel-control cultures (grown in parallel in MSCBM CD plus 5% hPL, i.e. a medium which does not induce osteogenesis) were fixed and stained to identify the organic calcium deposits. For von Kossa staining, samples were fixed in ice-cold methanol for 2 min, then rinsed twice in distilled water, and incubated under a ultraviolet (UV) lamp with silver nitrate in water for 30 min. After two further washes in distilled water, 10 high-power fields were observed using 10 magnification (Axioskop-Observer-1 inverted microscope; Zeiss). Digitally recorded images were analysed by ImageJ software (National Institute of Health, USA) selectively quantifying dark positively stained areas of organic calcium deposits. The experiments were performed in triplicates.
[0668] Results: Results are shown in
Example 13: Adipogenic Differentiation Potential
[0669] Description of the Method: For adipogenic differentiation, SWJ cells (Examples 1, 2A, 2B, 2C) at culture passage 5 were seeded at 10,000 cell/cm.sup.2 in MSCBM CD with 5% hPL in 6 multiwell (6-MW) plates and, after 3 days, the medium was replaced by adipogenic induction medium. The adipogenic medium was composed of MSCBM CD with 5% hPL, supplemented with 1 M dexamethasone, 60 M indomethacin, 10 pH insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10% rabbit serum, 5% horse serum, 1% L-glutamine and 1% penicillin/streptomycin. The control cells were cultured with MSCBM CD with 5% hPL. Cells were seeded in technical duplicates for each condition.
[0670] After 10 days the cells were stained with the stain Oil Red 0 (a commercially available stain for adipocytes) to confirm the differentiation, as follows: the cells were washed briefly with PBS 1, fixed with vapors of 37% formaldehyde for 10 minutes and then washed with water for 2 minutes. Staining was obtained by adding an Oil Red 0 solution (10 mg/mL Oil Red 0 in ethanol 70% and acetone) for 3 minutes into the wells. The cells were counterstained by Harris Hematoxylin (Bio-Optica, Milan, Italy) for 3 minutes. After treatment, the differentiated cells and controls were visualized by microscopical observations (Axio Observer A1 with Axiocam MRCS color camera and Axiovision 4.82 software; all Zeiss).
[0671] Results are shown in
Example 14: Chondrogenic Differentiation Potential
[0672] Description of the Method: For chondrogenic differentiation, 200,000-500,000 SWJ (Examples 1, 2A, 2B, 2C) at culture passage P5 were aliquoted in a 15 mL tube and centrifuged at 1,200 rpm (Beckham Coulter Allegra 25R) for 10 minutes. Cells were maintained in the pellet in growth medium at 37 C. with the plug opened. After 2 days of incubation the growth medium was substituted with the induction medium, composed of MSCBM CD with 5% human platelet lysate (hPL), supplemented with: 500 ng/mL rhBMP-6, 10 ng/mL transforming growing factor-, 50 mg/mL ITS+Premix (containing insulin 6.25 g/mL, transferrin 6.25 g/mL, selenic acid 6.25 ng/mL, BSA 1.25 mg/mL and linoleic acid 5.35 g/mL), 100 nM dexamethasone, 0.2 mM L-ascorbic acid-2-phosphate, 100 g/mL sodium pyruvate, 40 g/mL proline. The medium was exchanged every three days. Before and after each medium exchange, tubes were centrifuged at 1,200 rpm for 10 minutes as described above. During the incubation period, cells remain as pellet with the tube plugs opened. Induction lasted 21 days, and specimens were fixed for 1 hour in 10% formaldehyde and then dehydrated by serial passages into ethanol at increasing concentrations, from 70% (v/v) to 100%. Samples were then included into paraffin blocks and cut in slices onto microscope slides for Alcian Blue staining. Slides were de-paraffinized with the Histo-C cleaning agent and rehydrated through passages into a decreasing concentration alcoholic ladder (from 100% ethanol to 70% ethanol) (v/v). Sample sections were then incubated with a 0.5 mg/mL hyaluronidase in buffer phosphate solution (8 g/L NaCl, 2 g/L NaH.sub.2PO.sub.4, and 0.3 g/L Na.sub.2HPO.sub.4) with 10 mg/mL BSA. Slides were washed in water for 5 minutes and then immersed in a 3% (v/v) acetic acid solution for few seconds. The sections were stained with 10 mg/mL Alcian Blue solution (a commercial dye) in 3% acetic acid (pH 2.5), remained there 30 minutes, and after a washing step in water, were counterstained for 5 minutes with nuclear Fast Red solution (a commercial dye).
[0673] Results are shown in
BRIEF DESCRIPTION OF THE FIGURES
[0674]
[0675]
[0676] |.Math.|: Perivascular area of about 2.00 mm including WJ+pericyte+endothelial cells.
[0677] Materials and methods: A human umbilical cord specimen was formalin fixed and paraffin embedded. Sections were isolated and cut in 5 m sections for further staining. The distinct staining in the different areas representing the stromal Wharton's Jelly and the perivascular area is apparent. The perivascular area has a more complex histo-morphological pattern with vessels (V), perivascular cells (PV) bordering the WJ areas. The complexity is also related to the different cell type represented in the perivascular region. On the contrary, the stromal Wharton's Jelly appears histologically as a relatively more uniform area with more homogeneous cellular elements dispersed into the matrix of the stromal tissue. Isolated cells express markers that are commonly found in MSC populations ex vivo, such as CD10, CD140b, GD2, CD73 (not shown in this Figure). The immunohistochemical staining pattern for these markers also differs between the two regions. In
[0678]
[0679]
[0681] A: Apoptotic cells: effect of platelet lysate and comparison to a conventional method. Regarding the number of freshly isolated cells, the inventors obtained more cells (PVWJ) from the protocol according to Comparative Example 1, (4.151.2810.sup.6) than from the protocol according to the present invention (1.490.3510.sup.6). The graph represents the mean and the standard error of the mean (SEM) of apoptotic cells in three digestions (n=3). 30.6%9.6% of total isolated PVWJ-derived cells according to Comparative Example 1 were positive for 7AAD (
[0682] B: FACS analysis of freshly isolated cell samples. The 4 FACS plots represent physical parameters (right) and the 7AAD positivity (i.e. apoptosis, on the left) of SWJ-derived cells (top) PVWJ-derived cells (bottom), respectively. In the graphs on the right, living cells are found left of the vertical line.
[0683] C: Graphical representation of total cell number, as determined by flow cytometry (
[0684] D: Graphical representation of percentage of apoptotic cells, as determined by flow cytometry (
[0685]
[0686] A: Physical parameters determined by flow cytometry, i.e. Forward Scatter (FSC) and Side Scatter (SSC) of freshly isolated cells from an umbilical cord are represented (FACS plot on the left). The graph represents the mean and the SEM of physical parameters. The SWJ-derived cell population isolated (according to Examples 1 and 2A) is characterized by significantly smaller FSC values than the PVWJ-derived cell population isolated (according to Comparative Examples 1 and 2A; p=0.02 by T-Test). SSC: SWJ derived cells have lower cytoplasmic complexity than cells from PVWJ. For details see Example 4A.
[0687] B: Morphology studies at culture passage 1 (n=3). After expansion, the SWJ-derived cells (Example 2C) were characterized by small and spindle shape cell population, instead PVWJ-derived cells (according to Comparative 2C) were characterized by a large shape and prominent cytoskeleton. The two pictures represent the morphology of SWJ-derived cells (left) versus PVWJ-derived cells (right) two- or three days post-seeding in culture passage 1 (P1) in a representative sample (original magnification 100).
[0688] C: Colony formation of the SWJ-derived cells and PVWJ-derived cells. For details see Example 4C. Cells were stained with crystal violet before taking the picture shown at the bottom. Cultures of SWJ-derived cell populations are characterized by consisting of cells with a small surface area; these cells tend to build colonies, while cultures of PVWJ-derived cell populations are characterized by consisting of cells with different surface areas, also including large cells. Thus, the PVWJ-derived cells appear more homogeneous; homogeneous in this context means that substantially all cells appear much alike or similar. The larger PVWJ-derived cells tend to grow relatively slower (data not shown).
[0689]
[0690]
[0691] B: The graph represents the mean percentage of cells displaying specific surface markers, in freshly isolated cells from umbilical cords by two different methods (Comparative Example 1, PVWJ vs. cells according to the present invention, SWJ).
[0692]
[0693] APCDD1 expression was not detected in control cells.
[0694]
[0695]
[0696]
[0697]
[0698]
[0699]