The birth of therapy withcultured cellsHoward Green

SummaryLong ago, I set out to solve a problem, but somethinghappened along the way: I was diverted by an unexpectedobservation. Thereafter, the direction of my research wasguided at each stage by increasing familiarity with theexperimental material and what could be done with it. Theresult was the birth of therapy with cultured keratino-cytes. Subsequent developments soon led to the for-mation of the company Biosurface Technology (latertaken over by the Genzyme Corporation), which providedautologous cultures for burn victims in many parts of theworld. Further progress by others led to new therapeuticapplications of cultured keratinocytes, such as treatmentof an ocular disease and gene therapy. Unfortunately,there have developed serious regulatory problems thatare a danger to future progress. As described in thisbrief history, the initial stages of development of celltherapy for the treatment of human disease were possibleonly because there was no restraint by committeesor governmental regulations. BioEssays 30:897–903,2008. � 2008 Wiley Periodicals, Inc.

The beginnings of keratinocyte cultivation

In 1974, I had no intention of studying therapy with cultured

cells or the treatment of human burns. At that time, current

thinking was that much could be learned about embryogenesis

from the study of cultivated murine teratomas. In general, this

hope was not fulfilled, but for me, studying cultures of a serially

transplantable teratoma derived by Leroy Stevens,(1) turned

out to be a very fruitful enterprise.

In the course of these studies, whose purpose had nothing

to do with what followed, my graduate student James

Rheinwald and I noticed that when the teratoma cells were

cultivated, some of them gave rise to interesting colonies of

epithelial appearance against a background of what appeared

to be fibroblasts. We attempted to obtain the epithelial cells in a

pure state, but we were unsuccessful because the isolated

cells grew very poorly. In order to support their growth, we

added lethally irradiated cells of the fibroblast line 3T3 that

had been made in my laboratory years earlier.(2) Under these

conditions, the epithelial cells grew quite nicely (Fig. 1). The

ability of 3T3 cells to support growth of an epithelial cell type

turned out to be specific for fibroblasts;(3,4) it was not simply

the result of increasing the cell density.

What was the nature of these epithelial cells that grew out of

the teratomal cultures? When sections through colonies were

examined by electron microscopy, they showed the presence

of desmosomes, keratohyalin granules and aggregated

tonofilaments.(4) These are features of the keratinocyte, the

principal cell type of stratified squamous epithelia, such as the

epidermis.

If, by adding 3T3 cell support, we could grow very well in

culture keratinocytes derived from a murine teratoma, could

we do the same thing with human keratinocytes, which I knew

had never been grown to an appreciable extent in cell culture?

We obtained a fragment of human skin, dissociated the

cells and put them into culture with 3T3 support.(5) Rapidly

growing colonies formed from single cells (Fig. 2). Successive

improvements in the cultivation made the keratinocyte the

most proliferative of all cultured human cell types.(6–11)

Starting with the cells of a small biopsy, we could now generate

in culture a number of epidermal cells sufficient to cover the

entire body of a human.

How to apply the cells?

We then developed a method of making a graft out of a

confluent sheet of cultured keratinocytes, using the enzyme

Dispase. This neutral protease has the property of breaking

the attachments of the cells to the surface of the dish without

affecting the junctions between adjacent cells. The confluent

sheet detaches intact.(12)

The next step was obviously to determine whether a

Dispase-detached sheet of human keratinocytes could engraft

on an animal.(13) We prepared such sheets, grafted them

to wounds prepared on athymic mice and showed that

they generated a human epidermis (Fig. 3). This was evident

from the greater thickness of the epidermis seen in histo-

logical sections and proven by reaction of that epidermis with

antibody specific for a human envelope precursor,(14) later

called involucrin.(15)

Having learned that we could grow vast amounts of such

cultures and that they could engraft successfully on a mouse,

we had to ask: could such a culture prepared from the residual

skin of a human whose epidermis had been extensively

destroyed by a burn be used to regenerate that epidermis?

Department of Cell Biology, Harvard Medical School, Harvard Medical

School, 240 Longwood Ave, Boston, MA 02115.

E-mail: hgreen@hms.harvard.edu

DOI 10.1002/bies.20797

Published online in Wiley InterScience (www.interscience.wiley.com).

BioEssays 30:897–903, � 2008 Wiley Periodicals, Inc. BioEssays 30.9 897

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The beginnings of human therapy

The first therapy of humans with third degree burns using

autologous cultures was performed on two patients at the

Peter Bent Brigham Hospital in 1980. The decision to carry

out this therapy was made by Dr. Nicholas O’Connor, Director

of the Burn Unit and myself. Before making that decision, we

discussed the possible risks, not with any committee (there

was none), but with individual colleagues who were located in

different institutions and whose judgment we respected. What

we heard from them supported our decision to proceed.

Small skin biopsies were taken from the patient, the

epidermal cells were cultivated in my laboratory and the

Dispase-detached grafts were applied to prepared wound

surfaces. The grafts generated epidermis on both patients.

One of the patients, a 61-year old man, had sustained partial

and full-thickness burns over 40% of his body surface. The

dorso-lateral surface of his left arm was excised to fascia

and numerous cultured grafts were applied. The patches

of regenerated epidermis resulting from the application of

circular (Petri dish) cultures are easily visible against

the background of the excised surface (Fig. 4). Successive

applications of such cultures resulted in complete coverage of

the arm.(16)

The next advance in the use of autologous cultures for

therapy of burns was the demonstration that large-scale use

of the method could be life saving.(17) Five- and six-year old

brothers in Wyoming sustained flame burns over 97 and

98 percent of their body surface. Of the burned areas, 83 and

89 percent were of third degree. Dr. John Remensnyder,

Director of the Shriners Burns Institute of Boston, informed me

that the two brothers had no chance of survival with conven-

tional treatment but if I would try to save them with cultured

cells, he would accept the brothers in transfer from Wyoming.

Although I was not prepared for the scale of the necessary

cultivation, I agreed and both brothers were grafted with

cultures of autologous epidermis. The application of the

cultures to the surface of the abdomen of one of the brothers

and the complete regeneration of the skin 14 weeks later is

shown in Fig. 4. Both brothers survived the terrible suffering of

the temporary skinless state and lived for over 20 years before

they died of complications not directly related to their burns.

Soon after, the regeneration of the skin resulting from

the application of cultured grafts was thoroughly studied by

Compton et al.(18) Prior to this work, some surgeons believed

that a healthy epidermis depends on the dermis and that if

the dermis is destroyed, grafting of epidermal cells alone

cannot produce a durable epidermis.(19) The work by Compton

et al. showed conclusively that when epidermal cultures were

transplanted to a wound bed lacking dermis, the quality of the

resulting epidermis did not deteriorate over the long term.

Moreover, a fully regenerated dermis developed beneath that

epidermis.

Compton et al. studied the regeneration of the skin in

twenty-one patients of age 4 months to 18 years grafted with

autologous epidermal cultures. A fully stratified epidermis

developed very quickly after grafting, including the granular

and cornified layers. After a much longer period, rete ridges

appeared at the dermo-epidermal junction of the regenerated

skin and became progressively more normal over suc-

ceeding years. The sub-epidermal connective tissue was

remodeled to produce papillary and reticular dermis, with fine

collagen fibers in the sub-epidermal region and thicker fibers

below. After one or two years, the number and size of the

anchoring fibrils closely resembled those of normal skin. All of

the characteristic features of the dermis continued to improve

Figure 1. Clones of XB cells, an epithelial cell line derived

from a murine teratoma, growing on a layer of lethally irradiated

3T3 cells.

Figure 2. Colonies of human epidermal keratinocytes

stained with rhodamine.

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898 BioEssays 30.9

with time. Five years after grafting, the dermis appeared

completely regenerated (Fig. 5).

Similar results were obtained after grafting of a five year old

burn patient with epidermal cultures prepared in the laboratory

of Dr. Yann Barrandon, located at that time in Paris.(20,21)

For this treatment, the cultured grafts were prepared on a

detachable fibrin matrix, instead of by the older Dispase

method. I believe that this has been the only important

improvement in the preparation of autologous human kerati-

nocytes for grafting. The presence of 3T3 cells is still required,

but the fibrin-supported cultures have advantages over

Dispase-detached cultures for the treatment of burns.(21)

The time required to produce graftable cultures is shortened.

Fibrin-supported cultures are easier to prepare and easier to

use by the surgeon. Moreover, a fibrin substrate has been

essential for the grafting of human limbal cells.(22) The method

was developed entirely in France and Italy; to my knowledge, it

is not being used in the United States.

Studies of the stem cells of cultured

epidermal keratinocytes

At the time the early therapy with cultured keratinocytes

was carried out, there was not yet an understanding of the

necessity for the presence of stem cells in the cultured graft. A

Figure 3. Human epidermis generated on an athymic mouse 108 days after application of a cultured human epidermal sheet. A: Note

thickness of generated human epidermis compared with mouse epidermis. Only the human epidermis stains with a specific antibody

to involucrin. B,C: Comparison of B with C shows that involucrin is present in only the outer, terminally differentiating layers of the

human epidermis generated from the culture. The thick stratum corneum, typical of the human but not of the mouse, is also shown in C. The

numbers and arrows in B and C indicate identical positions in cell layers stained for involucrin (B) and those revealed by

phase microscopy (C).

Figure 4. Left, regions of engraftment of Petri dish cultures of autologous epidermal cells to an excised surface of first human treated.

Center, abdomen of one of the brothers treated several years later by application of now rectangular grafts to abdominal surface excised to

muscle fascia; right, 14 weeks later, when the surface was covered with thick confluent epidermis.

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BioEssays 30.9 899

method of analysis of proliferative capacity was developed a

few years later (23). Single cells were isolated from a strain

of keratinocytes and each cell was inoculated into a dish

containing supporting lethally irradiated 3T3 cells. Seven days

later, each growing colony produced by a founding cell was

isolated and trypsinized. The cells were transferred to

duplicate indicator dishes, again containing supporting 3T3

cells. Twelve days later, the cultures were fixed and stained.

From the appearance of the colonies in the indicator dishes,

we identified three clonal types of founding cells and classified

them as holoclones, meroclones or paraclones (Fig. 6). The

holoclone has the greatest proliferative capacity, giving rise to

fewer than 5% of terminal colonies. The paraclone has the

least proliferative capacity, as all the colonies abort and

terminally differentiate. The intermediate meroclone is a broad

category with considerable proliferative potential but less than

the holoclone. The evolution of clonal type is holoclone or

stem cell!meroclone! paraclone. Interest in stem cells on

the part of both scientists and lay public was destined to grow

enormously during subsequent years.

We also noted the decline in stem cell population with

age. In contrast to cultures derived from newborns, in which

28–31 percent of clones were holoclones, by age 64 the value

dropped to 3 percent and by age 78, to zero; only meroclones

and paraclones remained. It was shown later that the number

of holoclones in a cultured graft could be determined from the

number of cells possessing the DNp63 isoform a, since the

two were demonstrated to be closely correlated in humans of

different ages.(24)

Unanticipated but beneficial consequences

When scientific discovery opens up a new field of research, it is

impossible to anticipate where it may lead. I now wish to turn to

some unanticipated consequences of the discoveries that

I have just described.

We knew from early experiments on cultivating keratino-

cytes of all stratified squamous epithelia (esophageal, oral,

vaginal etc.) that corneal keratinocytes grew poorly on

subcultivation. The reason for this was elucidated in an

extremely important article from the laboratory of T.T. Sun.(25)

From an analysis of the keratins of corneal cells and the

surrounding limbal cells, as well as other data, the authors

postulated that the stem cells of the cornea were located in the

limbus, which surrounds the cornea and separates it from the

conjunctiva (Fig. 7).

Chemical burns of the eye, if they destroy the limbal stem

cells, can lead to a very nasty condition of inflammation,

pain and loss of vision. The limbus can be restored by grafts

from the healthy eye to the affected one.(26) This requires large

transfers of limbal tissue from the healthy eye. But limbal

stem cells belong to the same family as epidermal keratino-

cytes and can therefore be cultivated in the same way,

starting with a tiny biopsy. Thiswas first shown in the laboratory

of J. Rheinwald.(27)

A few years later it was demonstrated in the laboratory of

M. De Luca and G. Pellegrini, formerly in Venice and now in

Modena, that limbal cultures could be used therapeutically.

They took a 1–2 mm biopsy from the limbal region of the

healthy eye, grew the cells in culture, prepared a graft and

applied it to the suitably prepared injured eye.(28) Later, they

introduced the use of fibrin-supported limbal cultures and,

in collaboration with numerous ophthalmologists,(22) they

obtained relief of symptoms in 80% of over 100 patients. With

subsequent repair of deeper injury, vision could be totally

restored (Fig. 7).

Figure 5. The regenerated epidermis appears normal

with regularly spaced rete ridges (stars). The subepithelial

connective tissue is bilayered, with finer collagen bundles

superficially and coarser collagen bundles beneath, together

with normal vascular architecture (From Compton CC, Gill JM,

Regauer J, Gallico GG, O’Connor NE. 1989. Lab Invest 60:

600–612 with permission).

Figure 6. Colonies produced on indicator dishes by each of

the three founding clones. The holoclone gives rise predomi-

nantly to large colonies with smooth perimeters. The paraclone

gives rise to small colonies, with irregular perimeters, contain-

ing markers of terminal differentiation. The meroclone gives

rise to large and small colonies, many of which have irregular

perimeters.

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900 BioEssays 30.9

Another unanticipated consequence of the work on

cultured keratinocytes was their use for gene therapy. This

discovery was another contribution of the laboratory of M. De

Luca and G. Pellegrini.(29) They studied a severe genetically

determined blistering disease of the skin—Junctional Epider-

molysis Bullosa. The patient that they studied was a double

heterozygote containing a frame shift/single point mutation in

the gene for Laminin 5-b3, which links basal epidermal cells to

the basement membrane.

They cultivated epidermal cells of the patient and trans-

duced them with a retroviral vector bearing a full-length Lam5-

b3 cDNA under control of the Moloney virus LTR. They then

removed some affected skin and replaced it with grafts of

transduced cells (all treatment with virus was ex vivo). As

shown in Fig. 8, the disease was cured.

Strangulation by regulation

Returning now to the beginning of this story, I remind you how

the decision to graft humans with cultured cells was made

in 1980 by Dr. Nicholas O’Connor and myself. By 1992, I was

unable to do any experiment on a mouse without approval by

a committee. Since that time, there has been continuous

increase in what has been called Regulation but might better

be called Strangulation. By this I mean that the ever-increasing

regulation threatens experimental work on new therapies with

cultured cells. I may summarize the situation in this way: if the

present regulatory climate in the United States had existed in

1980, I would never have pursued the development of therapy

with cultured cells.

The American regulatory authorities have classified cul-

tured epidermal grafts as xenografts (grafts derived from a

non-human species) because 3T3 (mouse) cells are used to

support the multiplication of the autologous keratinocytes. It

has been shown that, by the time the graft is prepared,

there are very few remaining 3T3 cells. More importantly,

all the 3T3 cells are lethally irradiated and could not possibly

engraft. In addition, in 1990, our 3T3 cultures were exhaus-

tively tested and shown to be free of pathogens. After the

cultured autologous epidermal cells had been made available

to patients for 10 years, the regulatory authorities decided that

they must determine that the product does not pose an

unreasonable or significant risk of illness or injury. Of course,

no consideration was given to the fact that, since the birth of

therapy with cultured epidermal cells in my laboratories

twenty seven years ago and after its extensive use in the US,

France and Italy, no example could be cited of a harmful

effect resulting from the use of these ‘‘xeno’’ grafts. Such

considerations apparently did not influence the regulatory

authorities. What if, they said, even a tiny number of irradiated

3T3 cells remain on the surface of the grafted person so that

they could support the growth of a mouse virus? All this is the

result of a fevered collective imagination.

Another consequence of this exaggerated concern over

hypothetical dangers is a tendency to move the field toward

other methods of cultivation that do not require 3T3 cells.

Actually, after many years of use of 3T3-supported cultures for

preparing grafts, a medium containing bovine products, but not

3T3 cells, was invented for cultivation of human keratinocytes.

It is a useful method for many experiments but the proliferative

capacity of the keratinocytes is not nearly as well preserved

as in 3T3-supported cells. As I have described above, in the

preparation of autologous grafts, it is essential to preserve the

stem cells to the maximum degree possible. For this reason,

the 3T3 cells cannot as yet be dispensed with. It is a disservice

Figure 7. A: From Schermer A, Galvin S, Sun TT. 1986. J Cell

Biol 103:49–62with permission;B: before treatment;C: 5 years

after graft of limbal culture and 4 years after corneal transplant

to remove stromal scarring (De Luca et al.).(24)

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BioEssays 30.9 901

to the field to press for a change, where no suitable alternative

exists.

Recently, several changes have taken place in the re-

gulatory situation in various countries. At the end of 2002,

Tego Science, in Seoul, Korea received Marketing Approval for

their autologous cultured epidermal cells (Holoderm) for the

treatment of burns and nevi, and for scar revision.

On October 3, 2007, the Japanese Ministry of Healthcare,

Labor and Welfare publicly announced its regulatory approval for

cultured autologous epidermal grafts for the treatment of burns

by the Japan Tissue Engineering Company (J-TEC) in Aichi,

Japan.

On October 29, 2007, after twenty years of ‘unapproved’

use, the Genzyme Corporation in Boston, Massachusetts,

unexpectedly received FDA Marketing Approval for its cultured

autologous epidermal grafts (Epicel) for the treatment of burns.

Conclusion

I have described how the research that gave birth to therapy

with cultured cells was unplanned and was guided by the

new possibilities that developed at each stage of the work.

From the use of cultured keratinocytes for the treatment of

burns, practical applications were extended in the laboratory

of Professors De Luca and Pellegrini to the unanticipated

therapeutic use of cultured limbal cells to repair corneal

defects and the use of epidermal keratinocytes for gene

therapy of inherited blistering diseases. Many investigators

throughout the world are now attempting to use cultured cells

for other therapeutic purposes, some more realistically than

others. One group I would specially like to mention is that of

Dr. Anders Lindahl and his collaborators in Gothenburg, who

developed a therapeutic use for cultured chondrocytes.(30)

The use of such cells for therapy of cranio-facial deformities

Figure 8. A: Untreated region. Arrows indicate blisters. B: Excisions followed by application of grafts. C: 8 days after grafting. D: 60 days

after grafting. Hatched line in D corresponds to hatched line in A. (From Mavilio F, Pellegrini G, Ferrari S, Di Nunzio F, Di Iorio E, et al. 2006.

Nat Med 12:1397–1402 with permission.)

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902 BioEssays 30.9

has also been successful.(31) With respect to dangers, there

can be no progress in therapy without some risk, but regulatory

committees have not dealt realistically with the problem. It

should be the primary responsibility of those scientists and

clinicians who know the subject best to evaluate that risk, in

consultation with individual colleagues whose opinions they

respect.

Acknowledgments

Based in part on a lecture to the European Academy of

Sciences in Brussels, September 12, 2007.

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